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Veterinarni Medicina, 58, 2013 (3): 113–185 Review Article

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Antiviral agents targeting the influenza virus: a review and publication analysis

L. Eyer, K. Hruska

Veterinary Research Institute, Brno, Czech Republic

ABSTRACT: Influenza is a serious infectious disease, which is life-threatening especially in children, seniors and immunocompromised patients. In addition to vaccination, the development of new anti-influenza agents represents a crucial defence strategy to combat seasonal and pandemic influenza strains. At present most attention is paid to the development of inhibitors of influenza neuraminidase, which has been established as a key drug target for the prophylaxis and treatment of influenza infections. However, the emergence of drug-resistant influenza vari-ants highlights the need of continuously innovative strategies for the development of new drugs with improved antiviral effects, higher safety and increased tolerability. In this review article, an analysis of publications describ-ing anti-influenza agents indexed in the Web of Science® database has been carried out. The most important publications are presented in tables and are characterised by several key words, abstracts and references. The presented publications have been sorted according to five basic criteria: (i) review articles, (ii) design, synthesis and evaluation of new anti-influenza drugs, (iii) major classes of anti-influenza drugs, (iv) combination therapy of influenza infections and (v) influenza drug resistance. The design of this review article allows us to offer a complex overview of known antiviral agents targeting influenza viruses, facilitates easy and rapid orientation in numerous publications written on this subject, and aids the gathering of required data.

Keywords: influenza; virus; antiviral; agents; drug; resistance; therapy; structure-based drug design

Contents

1. Introduction1.1. Database used as the source of information

Table 1.1. Analysis of publications: search profiles and numbers of results retrieved1.2. Basic analysis of publications on antiviral agents targeting the influenza virus

Figure 1. The number of publications on anti-influenza agents during the period from 1990 to 2012Table 1.2. Analysis of publications: TopTen authors, institutions and countries according to the number

of published papers (Web of Science®, publications in total 14 458)2. Review articles

Table 2. Review articles3. Design, synthesis and evaluation of new anti-influenza drugs

Table 3. Design, synthesis and evaluation of new anti-influenza drugs4. Major classes of anti-influenza drugs

Figure 2. Life-cycle of the influenza virus (according to Beigel and Bray 2008)Figure 3. Structure of the influenza virus (according to Ludwig et al. 2003) Figure 4. Chemical structures of the most important anti-influenza drugs

4.1. Inhibitors of haemagglutininTable 4.1. Inhibitors of haemagglutinin

Supported by the Ministry of Education, Youth and Sports, Czech Republic (AdmireVet; Grant No. CZ 1.05/2.1.00/01.0006-ED 0006/01/01) and the Ministry of Agriculture of the Czech Republic (Grant No. MZe 0002716202).

Review Article Veterinarni Medicina, 58, 2013 (3): 113–185

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1. InTRoduCTIon

Influenza is considered to be one of the life-threatening infectious diseases. In some countries seasonal influenza affects annually up to 40% of the population and 500 million people die from it worldwide every year. New highly-virulent in-fluenza strains can arise unexpectedly to cause world-wide pandemics with markedly increased morbidity and mortality, such as the “avian flu” in 1997 and “swine flu” in 2009. At present, the development of antiviral drugs represents a crucial strategy in the control and prevention of seasonal and pandemic influenza infections. Antiviral drugs can overcome the limitations of vaccination strate-gies, such as the time-consuming vaccine design, insufficient protection for immunocompromised patients and the unpredictable antigenic changes in influenza strains which render vaccination inef-fective. In general, the anti-influenza agents can be divided into two basic groups, i.e., synthetic analogues of biomolecules required during virus infection and substances derived from natural plant extracts. With regard to the considerable genetic and antigenic variability of the influenza virus, research has been predominantly focused on broad-spectrum antiviral drugs, which are effective against a large variety of influenza strains. The de-velopment of antivirals targeting host-cell proteins, which play an important role in viral replication, has also gathered pace recently. Moreover, combi-nation therapy based on the application of two or more different antivirals represents a promising approach to combat influenza infections.

The aim of this review is to offer an overview of known antiviral agents targeting influenza viruses and to discuss their characteristic properties, modes of action and advantages or limitations of their ther-apeutic use. Publications which contain key informa-tion concerning the issues of anti-influenza agents are in this review presented in tables and character-ised with descriptive words, full or shortened ab-stracts and relevant references. The text in the tables contains several format imperfections, which exist in the Web of Science® database and are caused by transmission and copying of data between various information sources. The loss of cursive typeface in Latin names in titles of references and in abstracts is such an example. The presented publications have been classified according to five basic criteria: (i) re-view articles, (ii) design, synthesis and evaluation of new anti-influenza drugs, (iii) major classes of anti-influenza drugs, (iv) combination therapy of in-fluenza infections and (v) influenza drug resistance. This review article should facilitate orientation in numerous publications dealing with anti-influenza drugs and enable rapid and easy search for specific data, information and protocols. The previous re-views in this format have been well received (Vass et al. 2008; Eyer and Hruska 2012; Hruska and Kaevska 2012; Hruska and Franek 2012).

1.1. database used as the source of information

The publications were retrieved from the Web of Science® database using the general search profile:

4.2. M2 ion channel blockersTable 4.2. M2 ion channel blockers

4.3. Inhibitors of viral RNA polymeraseTable 4.3. Inhibitors of viral RNA polymerase

4.4. Inhibitors of neuraminidaseTable 4.4. Inhibitors of neuraminidase

4.5. Host cell factor targetingTable 4.5. Host cell factor targeting

4.6. Other anti-influenza agentsTable 4.6. Other anti-influenza agents

5. Combination therapy of influenza infectionsTable 5. Combination therapy of influenza infections

6. Influenza drug resistanceTable 6. Influenza drug resistance

7. Acknowledgement8. References


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Table 1.1. Analysis of publications: search profiles and numbers of results retrieved

Timespan All years 2010–2012General search profileTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*)Results 14 458 4 673Review articles cited in Table 2 17design, synthesis and evaluation of new anti-influenza drugsTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (design OR synthesis OR evaluation)Results 3083 1048Cited in Table 3 19Haemagglutinin inhibitorsTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (hemagglutinin OR haemagglutinin)Results 2616 742Cited in Table 4.1 18M2 ion channel blockersTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (adamantane* OR amantadine OR rimantadine)Results 897 249Cited in Table 4.2 15Inhibitors of viral RnA polymeraseTopic= (influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND polymerase*Results 824 344Cited in Table 4.3 22Inhibitors of neuraminidaseTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND neuraminidase*Results 2658 930Cited in Table 4.4 21Host cell factor targetingTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (“host factor*” OR “host protein*”)Results 43 22Cited in Table 4.5 14other anti-influenza agentsTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND (nucleoprotein* OR “non-structural protein” OR siRNA* OR plant* OR herbal* OR antibod*)Results 3899 1382Cited in Table 4.6 16Combination therapy of influenza infectionsTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND “combination therapy”Results 102 38Cited in Table 5 11Influenza drug resistanceTopic=(influenza OR flu) AND (antivir* OR virostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*) AND resistanceResults 1757 700Cited in Table 6 19


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Topic = (influenza OR flu) AND (antivir* OR vi-rostatic* OR drug* OR “anti-influenza” OR “anti-flu” OR inhibit*), Timespan = all years. A total of 14 458 publications were obtained, demonstrating the huge number of papers on the subject pub-lished during the period from 1945 to 2012. To ease orientation through the numerous publications, the results were subsequently refined using more specific search profiles as described in Table 1.1. A selection of the most important publications has been based on reading abstracts and hundreds of available full papers.

1.2. Basic analysis of publications on antiviral agents targeting the influenza virus

The Web of Science® utilities were employed for search result analysis. Regarding the high num-ber of retrieved publications, attention has been predominantly paid to papers published in the pe-riod 2010 to 2012; however, some older key papers are also included in the tables. As is evident from Figure 1, publication activity on the topic has con-tinuously increased since the early 1990s. In the last three years, 4673 papers were published, with 1529 papers in 2012. The oldest papers were published in the late 1940s and describe the research on the first influenza haemagglutinin inhibitors in pigs. In 1960s, the antiviral effects of the first ion channel blockers were reported. The latest publications are focused on computer-aided structure-based design of new neuraminidase and viral polymerase inhibi-tors to combat pandemic influenza strains. Of all the published papers, original research articles pre-vail (82.7%). Our analysis shows that 2938 institu-tions from 112 countries are concerned with the

Table 1.2. Analysis of publications: TopTen authors, institutions and countries according to the number of published papers (Web of Science®, publications in total 14 458)

Item Number of publications

Authors (12 596 in total)

Garcia-Sastre A 156

Webster RG 112

Suzuki Y 103

Hayden FG 95

Kawaoka Y 85

Gubareva LV 74

Suzuki T 66

Palese P 62

Kochs G 61

Li Y 61

Institutions (2938 in total)

Centers for Disease Control and Prevention 307

Harvard University 197

Chinese Academy of Sciences 175

Icahn School of Medicine at Mount Sinai, New York 165

National Institute of Allergy and Infectious Diseases 164

University of Virginia 155

University of Wisconsin 150

University of Washington 149

University of Oxford 133

Emory University 125

Countries (112 in total)

USA 5695

Japan 1344

England 1169

People’s Republic of China 1097

Germany 963

Canada 710

France 653

Australia 637

Italy 561

Netherlands 484

0200400600800

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Figure 1. The number of publications on anti-influenza agents during the period from 1990 to 2012


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The designed structures that are predicted to show high affinity to the viral target are then syn-thesised using various chemical procedures (Shie et al. 2011; Rawat et al. 2012) and their anti-influ-enza effects are evaluated using standardised in vitro screening methods. These methods include biochemical assays (Hung et al. 2012) and cell-based antiviral screens (Schmidtke et al. 2001), such as the plaque reduction assay to monitor viral replication efficiency, dye-uptake assays for measuring virus cytopathic effect, and yield-re-duction assays for quantification of specific virus antigens. Through the combination of bioinfor-matic approaches, applicative robotics and min-iaturisation strategies, most of these techniques can be adapted to high-throughput screen for-mats, which are applicable for testing large drug libraries containing millions of unique chemical structures (Dai et al. 2012). As an alternative to experimental screening methods, virtual (com-putational) screening can be used to select the desired chemical structure from large molecular databases (Yamada et al. 2012).

Besides in vitro tests, potential influenza virus inhibitors are further studied using animal models, such as the ferret, laboratory mouse, and chicken (Yoshimoto et al. 2000; Sauerbrei et al. 2006). Such in vivo studies play a crucial role in the preclini-cal evaluation of new drugs. The final step in the development of new medications is represented by clinical trials, which focus particularly on clinical pharmacokinetic studies and evaluation of antiviral efficacy, safety and tolerability in human volunteers (He et al. 1999; Cao et al. 2012). For key publica-tions describing the design, synthesis and evalua-tion of anti-influenza drugs see Table 3.

4. Major classes of anti-influenza drugs

The anti-influenza drugs are usually classified ac-cording to their target in the viral life-cycle, which is schematically depicted in Figure 2. Such antiviral molecules are particularly used as inhibitors of the following processes: attachment of the virus to host cell receptors, endocytosis and fusion of viral and cell membranes, replication and transcription of the viral genome, synthesis of viral proteins, as-sembly of the viral progeny and release of the new virions into the outside environment. The following paragraphs are focused on the description of basic classes of influenza virus inhibitors.

subject of antiviral agents targeting the influenza virus. The authors, institutions and countries which show the highest publication activities (TopTen) are listed in Table 1.2.

2. Review articles

Review articles represent 9.2% of all publications retrieved from the Web of Science® database us-ing the above mentioned search profile. In Table 2, seventeen key review articles are presented and characterised with a few descriptive words (left column), abstract (in the middle), and source ref-erence (right column). These papers offer overall reviews on all important topics, especially regard-ing structure-based drug design techniques (Du et al. 2012), chemical synthesis of antiviral drugs (Gong and Xu 2008; Marra et al. 2008), biochemi-cal and cell-based antiviral screen assays (Tisdale 2000; Atkins et al. 2012), animal models for study of influenza virus inhibitors (Sidwell and Smee 2000), structure, chemical properties and modes of ac-tion of the major groups of anti-influenza drugs (Gong et al. 2009; Chintakrindi et al. 2012; Grienke et al. 2012), influenza virus-neutralising antibod-ies (Martinez et al. 2009), natural products with antiviral activity (Grienke et al. 2012), combina-tion therapy of influenza infections (Kaihatsu and Barnard 2012) and mechanisms of influenza drug resistance (Pizzorno et al. 2011). All the topics are also discussed in detail in the original papers pre-sented in Tables 3 to 6.

3. design, synthesis and evaluation of new anti-influenza drugs

The first anti-influenza drugs were usually iden-tified using large-scale screening methods or by chance and their chemical structure and modes of action were not completely understood (Davies et al. 1964). In contrast, the current development of new antivirals is based on detailed knowledge of the X-ray crystallography-derived structure of influenza proteins as drug targets. Such an inven-tive process of finding new drugs, which is termed structure-based drug design, involves the devel-opment of organic molecules or macromolecular scaffolds that are complementary in shape and charge to potential ligand-binding pockets of the viral target protein (Kim et al. 1997; Lv et al. 2011).


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(Figure 4), have been licensed for influenza control and are commercially available under the trade-marks Symmetrel® and Flumadine®, respectively (Galvao et al. 2012). The adamantanes are relatively cheap, highly stable in storage and show strong anti-influenza activity at micromolar concentra-tions. At present, the application of adamantanes for prevention and treatment of influenza infec-tions is, however, not recommended because of the rapid emergence of drug-resistant virus vari-ants that retain full virulence and transmissibility (Bright et al. 2005; Barr et al. 2008). Moreover, seri-ous gastrointestinal and neurological side effects were observed in patients undergoing adamantane therapy (Galvao et al. 2012). Another disadvantage of adamantanes is their strong specificity against influenza A strains only (Rosenberg and Casarotto 2010). Selected publications describing the struc-ture, chemical properties and application of ada-mantanes, adamantane-derivatives and other M2 ion channel blockers are presented in Table 4.2.

4.3. Inhibitors of viral RnA polymerase

Transcription and replication of the influenza virus genome is carried out by the influenza RNA polymerase holoenzyme, which is characterised by two catalytic activities. Polymerase activity is

needed for the elongation of nascent RNA chains, whereas endonuclease activity is essential for cleavage of the 5’-capped primer sequence of the host mRNA. The cap is the terminal 7-methyl-guanosin bound through a triphosphate group to the host mRNA. This “cap snatching” process is needed for the initiation of viral RNA transcrip-tion (Lv et al. 2011). Influenza RNA polymerase is an extremely suitable target for the develop-ment of new broad-specific antivirals because of its highly conserved structure among influenza strains. It is thought that the influenza polymerase plays a crucial role in virus adaptation to human-to-human transmission and, consequently, in the formation of pandemic influenza variants (Miotto et al. 2008; Boivin et al. 2010; Aggarwal et al. 2011; Ping et al. 2011).

Two basic classes of RNA polymerase inhibitors have been described based on different mecha-nisms of action. The first group is represented by nucleoside analogues for the blocking of viral RNA chain elongation (Tisdale et al. 1995). A typ-ical member of this group is favipiravir (T-705, Figure 4), which is an inhibitor of influenza A, B and C strains, including variants resistant to amantadine or oseltamivir. This compound is currently in the stage of clinical testing (Furuta et al. 2005). Other nucleoside analogs with anti-influenza activity include ribavirin (Virazole®)

Figure 4. Chemical structures of the most important anti-influenza drugs; (a) amantadine, (b) riman-tadine, (c) favipiravir (T-705), (d) ribavirin, (e) viramidine, (f ) zan-amivir, (g) oseltamivir

(a) (b)

(c) (d) (e)

(f ) (g)


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and its derivative viramidine, originaly licensed for treatment of hepatitis C infections (Figure 4). Their application is, however, sometimes con-nected with the development of haemolytic anae-mia (Sidwell et al. 2005). The second class of antiviral molecules targeting the influenza poly-merase is represented by compounds which block the endonuclease and cap-binding domains of the polymerase holoenzyme. These antivirals include cap analogues (Lv et al. 2011), short capped oligo-nucleotides (Tado et al. 2001), and small organic compounds, such as 4-substitued 2,4-phenylbu-tanoic acid (Hastings et al. 1996) and flutimide isolated from the fungus Delitschia confertaspora (Tomassini et al. 1996). For the representative publications concerning viral RNA polymerase inhibitors, see Table 4.3.

4.4. Inhibitors of neuraminidase

Neuraminidase, also referred to as sialidase, is an antigenic glycoprotein anchored in the surface en-velope of the influenza virions, which hydrolytically cleaves the terminal sialic acid from the host cell receptors (Figure 3). Thus, it plays a crucial role in the release of viral progeny from the membranes of infected cells, prevents self-aggregation of virions and facilitates the movement of the infectious viral particles in the mucus of the respiratory epithelia (Matrosovich et al. 2004; Suzuki et al. 2005). Influenza neuraminidase has been established as a key drug target for the prophylaxis and treatment of influenza infections, predominantly for the following reasons: Firstly, the structure of the influenza neuraminidase active site is highly conserved between influenza A and B strains, making neuraminidase an attractive target for the development of broad-spectrum in-hibitors (Yen et al. 2006). Secondly, resistance to neuraminidase inhibitors develops less commonly than to other anti-influenza drugs. Nevertheless, the intensive application of neuraminidase inhibitors for influenza treatment results in a permanently in-creasing number of drug-resistant strains (Garcia et al. 2009). Thirdly, in contrast to adamantanes, neuraminidase inhibitors are mostly well tolerated in patients under therapy (Cao et al. 2012). Finally, neuraminidase protein is a freely accessible target for antiviral molecules with an extracellular mode of action.

The development of neuraminidase inhibi-tors started in the middle 1970s, when the first

structural analogues of sialic acid were described and denoted as DANA (2-deoxy-2,3-didehydro-N-acetyl neuraminic acid) and its trifluoroacetyl derivative FANA (Schulman and Palese 1975). At present, several licensed anti-influenza medica-tions are available on the market, most notably the inhalant zanamivir with the trademark Releza®, and the orally administered oseltamivir (Tamiflu®) having excellent bioavailability and relatively long half-life in vivo (He et al. 1999; Greengard et al. 2000) (Figure 4). In response to the emergence of some oseltamivir-resistant influenza strains, peramivir and laninamivir have been recently developed (Bantia et al. 2006; Kubo et al. 2010). New-generation neuraminidase inhibitors are cur-rently under investigation, e.g., multimeric forms of zanamivir (Watson et al. 2004), dual-targeted bifunctional antivirals (Liu et al. 2012), and several herbal remedies, such as flavonols, alkaloids and saponins (Jeong et al. 2009). The key publications on the structure, synthesis and therapeutic appli-cation of neuraminidase inhibitors are presented in Table 4.4.

4.5. Host cell factor targeting

Many human host cell molecules play a crucial role in influenza virus propagation and, there-fore, represent promising targets for the design of new generation inhibitors of the virus-cell interaction. Muller et al. (2012) describes in his review 35 cellular factors essential for influenza virus infection for which 57 inhibitors with ap-parent anti-influenza activity are available. The most intensively studied are the compounds which effectively inhibit intracellular signalling cascades with a resulting negative influence on the establishment of viral infection (Nacken et al. 2012). Studies have also focused on inhibi-tors of vacuolar proton-ATPase which render the viral M2 ion channels inactive (Guinea and Carrasco 1994), inhibitors of cellular proteases which block the proteolytic activation of haemag-glutinin (Zhirnov et al. 2011), and blockers of the cellular ubiquitin-proteasome system (Dudek et al. 2010). Although the development of host fac-tor inhibitors is a promising research strategy to limit the emergence of drug-resistant mutants, their possible toxic side-effects in vivo need to be carefully studied. Selected papers on the topic are presented in Table 4.5.


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4.6. other anti-influenza agents

During the last decades, a large variety of chemical compounds with anti-influenza activity have been investigated. Several examples of such novel drugs are the inhibitors of viral nucleoprotein (Hung et al. 2012), blockers of influenza non-structural pro-teins (Basu et al. 2009) and short interfering oligo-nucleotides (siRNAs) used for viral RNA silencing (Stewart et al. 2011). Using large-scale screening techniques, new antiviral molecules which show significant anti-influenza effects have been identi-fied; however, their chemical structure and mecha-nism of action remains unknown. These include, for instance, natural substances isolated from plants in chemical and pharmaceutical studies (He et al. 2012; Jiao et al. 2012). Another important group of prospective therapeutics are monoclonal antibod-ies and recombinant antibody fragments with high virus-neutralising activities (Hanson et al. 2006; Wei et al. 2011). Sixteen publications describing alterna-tive anti-influenza agents are presented in Table 4.6.

5. Combination therapy of influenza infections

Recent in vitro and in vivo studies have demon-strated that the simultaneous application of two or more anti-influenza drugs with different modes of action, e.g. oseltamivir and amantadine, results in increased virus inhibition and enhanced thera-peutic efficiency (Masihi et al. 2007). Similar find-ings were made with the combination of influenza virus inhibitors and immunomodulatory agents, especially corticosteroids (Zheng et al. 2008; Quispe-Laime et al. 2010). These observations are in accordance with the hypothesis that the applied drugs exert additive or synergistic anti-influenza effects in the infected cells. Such a combination regimen enables a reduction in the concentration of the individual inhibitory drugs, resulting in de-creased drug toxicity and a reduced risk of antivi-ral resistance emergence in seasonal and pandemic influenza viruses (Govorkova et al. 2004; Smee et al. 2010; Nguyen et al. 2012). The principals of the combination therapy can in the future become a crucial strategy not only in the treatment of influ-enza infections, but also in the therapy of other se-rious viral, bacterial and parasitic diseases. Selected publications, which discuss combination antiviral therapy, are presented in Table 5.

6. Influenza drug resistance

As with all antimicrobials, propagation of vi-ruses in the presence of antiviral drugs increases the selection pressure for mutations in the viral target proteins, which results in the induction of virus drug resistance. As an example, adamantane-resistant strains are typically characterised by a single substitution in the transmembrane region of the M2 ion channel (Saito et al. 2003; Shiraishi et al. 2003). On the other hand, resistance to neu-raminidase inhibitors can result from mutations in the neuraminidase active cavity, but also from amino acid substitutions on the molecular surface of the neuraminidase protein (Yen et al. 2006; Du et al. 2010). It is noteworthy that resistance to adamantanes is acquired rapidly and by a high number of virus strains (Bright et al. 2005). In contrast to adamantane resistance, neuraminidase inhibitor resistance has developed over a longer time period and occurs with a relatively lower fre-quency (Garcia et al. 2009). This may be due to the fact that some mutations significantly affect viral infectivity and ability to replicate in the host cell. While amantadine-resistant strains do not show growth or virulence impairment (Sweet et al. 1991), influenza variants resistant to oseltamivir exhibit reduced neuraminidase activity and viral fitness in vitro (Yen et al. 2006), and decreased transmissibility in ferret models (Herlocher et al. 2004). The increasing emergence of drug-resistant influenza strains highlights the need to search continuously for innovative strategies for the de-velopment of new drugs with improved antiviral effects, higher safety and better tolerability. For key publications describing the mechanisms of viral drug resistance, as well as prevalence and clinical impact of drug resistant influenza strains see Table 6.

7. Acknowledgement

We wish to thank A. Durisova (Veterinary Research Institute, Brno) for her excellent graphical service.

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Received: 2013–03–25Accepted after corrections: 2013–04–05

Corresponding Author:

Mgr. Ludek Eyer, Ph.D., Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech RepublicTel. +420 533 331 911, E-mail: [email protected]


130

Tabl

e 2.

Rev

iew

art

icle

s

Stru

ctur

e-ba

sed

drug

de

sign

Neu

ram

inid

ase

inhi

bito

rs

Dru

g re

sist

ance

Rece

nt e

mer

genc

e of

influ

enza

A H

5N1

and

H1N

1 st

rain

s ha

s he

ight

ened

con

cern

, esp

ecia

lly a

s a

resu

lt of

thei

r dr

ug re

sist

ance

. The

life

cyc

le o

f inf

luen

za v

irus

es h

as b

een

wel

l stu

died

and

nea

rly

all t

he v

iral

pro

tein

s ar

e be

com

-in

g po

tent

ial t

hera

peut

ic ta

rget

s. In

this

revi

ew, w

e pr

esen

t an

over

view

of r

ecen

t pro

gres

s in

str

uctu

re-b

ased

ant

i-in

fluen

za d

rug

desi

gn, p

ayin

g cl

ose

atte

ntio

n to

the

incr

easi

ng ro

le o

f com

puta

tion

and

stra

tegi

es fo

r ov

erco

min

g dr

ug

resi

stan

ce.

Du

et a

l. 20

12

Stru

ctur

e-ba

sed

drug

de

sign

Hae

mag

glut

inin

inhi

bito

rN

eura

min

idas

e in

hibi

tor

M2

inhi

bito

rPo

lym

eras

e in

hibi

tor

Kin

ase

inhi

bito

r

In th

is re

view

, we

will

dis

cuss

dru

g de

sign

bas

ed o

n pr

oven

and

pot

entia

l ant

i-in

fluen

za d

rug

targ

ets

incl

udin

g vi

ral

hem

aggl

utin

in (H

A),

neur

amin

idas

e (N

A),

M2

ion

chan

nel,

3P p

olym

eras

e co

mpl

ex, a

nd h

ost f

acto

rs s

uch

as k

inas

es.

We

have

sum

mar

ized

influ

enza

inhi

bito

rs b

ased

on

thei

r m

ode

of a

ctio

ns. F

or in

stan

ce, i

nclu

ded

are

desc

ript

ions

of

(1) i

nhib

itors

of H

A c

leav

age,

suc

h is

naf

amos

tat,

cam

osta

t, ga

bexa

te, e

psilo

n-am

inoc

apro

nic

acid

and

apr

otin

in,

(2) i

nhib

itors

of f

usio

n an

d en

try,

suc

h as

ben

zoqu

inon

es a

nd h

ydro

quir

ione

s, C

L 38

5319

, BM

Y-27

709,

sta

chyf

lin,

and

thei

r an

alog

ues,

(3) i

nhib

itors

of v

iral

RN

Ps/p

olym

eras

e/en

donu

clea

se, s

uch

as T

-705

, L-7

35,8

22, f

lutim

ide

and

thei

r an

alog

ues,

(4) i

nhib

itors

of M

EK, s

uch

as P

D 0

3259

01, C

I-10

40 a

nd A

RRY-

1428

86, a

nd (5

) inh

ibito

rs o

f NA

su

ch a

s D

AN

A, F

AN

A, z

anam

ivir,

and

ose

ltam

ivir,

etc

. Alth

ough

am

anta

dine

and

rim

anta

dine

are

not

reco

mm

ende

d fo

r tr

eatin

g in

fluen

za v

irus

infe

ctio

ns b

ecau

se o

f dru

g re

sist

ance

pro

blem

, the

se v

iral

M2

ion

chan

nel b

lock

ers

esta

b-lis

hed

a pr

oof-

of-c

once

pt th

at th

e en

docy

tosi

s of

vir

ion

into

hos

t cel

ls c

an b

e a

valid

dru

g ta

rget

bec

ause

M2

prot

ein

is in

volv

ed in

the

endo

cyto

sis

proc

ess.

The

influ

enza

pol

ymer

ase

com

plex

not

onl

y ca

taly

zes

RNA

pol

ymer

izat

ion

but

also

enc

odes

the

“cap

sna

tchi

ng” a

ctiv

ity. A

fter

bei

ng e

xpor

ted

from

the

nucl

eus

to th

e cy

topl

asm

, the

new

ly s

ynth

e-si

zed

vRN

Ps a

re a

ssem

bled

into

vir

ions

at t

he p

lasm

a m

embr

ane.

The

pro

geny

vir

ions

will

then

leav

e th

e ho

st c

ells

th

roug

h th

e ac

tion

of N

A. T

he s

trat

egie

s fo

r di

scov

ery

of s

mal

l mol

ecul

e in

hibi

tors

of i

nflu

enza

vir

us re

plic

atio

n ba

sed

on e

ach

part

icul

ar m

echa

nism

will

be

disc

usse

d. F

inal

ly, t

he le

sson

s le

arne

d fr

om th

e de

sign

of N

A in

hibi

tors

(NA

I) a

re

also

incl

uded

. Man

y ex

citin

g op

port

uniti

es a

wai

t the

cad

re o

f vir

olog

ists

, med

icin

al c

hem

ists

, and

pha

rmac

olog

ists

to

desi

gn n

ovel

influ

enza

dru

gs w

ith fa

vora

ble

phar

mac

olog

ical

and

pha

rmac

okin

etic

pro

pert

ies

to c

omba

t thi

s th

reat

en-

ing

infe

ctio

us d

isea

se.

Hsi

eh a

nd H

su

2007

Hig

h-th

roug

hput

scr

een

tech

niqu

esIn

trod

uctio

n: In

fluen

za a

ntiv

iral

hig

h-th

roug

hput

scr

eens

hav

e be

en e

xten

sive

, and

yet

no

appr

oved

influ

enza

ant

ivi-

rals

hav

e be

en id

entif

ied

thro

ugh

high

-thr

ough

put s

cree

ning

. Thi

s un

ders

core

s th

e id

ea th

at d

evel

opm

ent o

f suc

cess

-fu

l scr

eens

sho

uld

focu

s on

the

expl

oita

tion

of th

e un

derr

epre

sent

ed v

iral

targ

ets

and

nove

l, th

erap

eutic

hos

t tar

gets

. A

reas

cov

ered

: The

aut

hors

revi

ew c

onve

ntio

nal s

cree

ning

app

licat

ions

and

em

ergi

ng te

chno

logi

es w

ith th

e po

tent

ial

to e

nhan

ce in

fluen

za a

ntiv

iral

dis

cove

ry. R

eal-

wor

ld e

xam

ples

from

the

auth

ors’

wor

k in

bio

cont

aine

d en

viro

nmen

ts

are

also

pro

vide

d. F

utur

e in

nova

tions

are

dis

cuss

ed, i

nclu

ding

the

use

of ta

rget

ed li

brar

ies,

mul

tiple

xed

assa

ys, p

rox-

imity

-bas

ed e

ndpo

int m

etho

ds, n

on-l

abor

ator

y-ad

apte

d vi

rus

stra

ins,

and

pri

mar

y ce

lls, f

or im

med

iate

phy

siol

ogic

al

rele

vanc

e an

d tr

ansl

atio

nal a

pplic

atio

ns. E

xper

t opi

nion

: The

lack

of s

ucce

ssfu

l ant

i-in

fluen

za d

rug

disc

over

y us

ing

high

-thr

ough

put s

cree

ning

sho

uld

not d

eter

futu

re e

ffort

s. In

crea

sed

unde

rsta

ndin

g of

the

func

tions

of v

iral

targ

ets

and

host

-pat

hoge

n in

tera

ctio

ns h

as b

road

ened

the

targ

et re

serv

oir.

Futu

re s

cree

ning

effo

rts

shou

ld fo

cus

on id

entif

y-in

g ne

w d

rugs

aga

inst

une

xplo

ited

vira

l and

hos

t tar

gets

usi

ng c

urre

ntly

dev

elop

ed a

ssay

s, a

nd o

n th

e de

velo

pmen

t of

nove

l, in

nova

tive

assa

ys to

dis

cove

r ne

w d

rugs

with

nov

el m

echa

nism

s. In

nova

tive

scre

ens

mus

t be

desi

gned

to id

entif

y co

mpo

unds

that

spe

cific

ally

inhi

bit p

rote

in-p

rote

in o

r pr

otei

n-RN

A in

tera

ctio

ns o

r ot

her

viru

s/ho

st fa

ctor

inte

ract

ions

th

at a

re c

ruci

al fo

r vi

ral r

eplic

atio

n. F

inal

ly, t

he u

se o

f rec

ent v

iral

isol

ates

, inc

reas

ed b

ioco

ntai

nmen

t (fo

r hi

ghly

-pa

thog

enic

str

ains

), pr

imar

y ce

ll lin

es, a

nd ta

rget

ed c

ompo

und

libra

ries

mus

t con

verg

e in

eff

icie

nt h

igh-

thro

ughp

ut

prim

ary

scre

ens

to g

ener

ate

high

-con

tent

, phy

siol

ogic

ally

-rel

evan

t dat

a on

com

poun

ds w

ith ro

bust

ant

ivir

al a

ctiv

ity.

Atk

ins e

t al.

2012


131

Susc

epti

bilit

y m

onit

orin

gPl

aque

-red

uctio

n as

say

Yiel

d-re

duct

ion

assa

yD

ye-u

ptak

e as

say

Neu

ram

inid

ase

Hae

mag

glut

inin

With

the

clin

ical

dev

elop

men

t of a

nti-

vira

l age

nts,

mon

itori

ng fo

r the

con

tinue

d su

scep

tibili

ty o

f wild

-typ

e st

rain

s has

be

com

e im

port

ant i

n di

seas

e m

anag

emen

t. Va

riou

s met

hods

hav

e be

en u

sed

to m

onito

r vir

al su

scep

tibili

ty; t

he a

dvan

-ta

ges a

nd d

isad

vant

ages

of w

hich

dep

end

on th

e vi

rus,

the

targ

et a

nd th

e sc

ale

of th

e re

sear

ch b

eing

und

erta

ken.

The

plaq

ue-r

educ

tion

assa

y is

val

uabl

e fo

r mea

suri

ng su

scep

tibili

ty o

f mos

t vir

uses

but

is n

ot id

eal f

or la

rge-

scal

e m

onito

ring

. Yi

eld-

redu

ctio

n, m

easu

ring

spec

ific

viru

s ant

igen

s, a

nd d

ye-u

ptak

e as

says

, mea

suri

ng v

irus

cyt

opat

hic

effec

ts, a

re m

ore

suita

ble

for h

igh-

thro

ughp

ut re

quir

emen

ts, b

ut th

e IC

50 v

alue

(the

con

cent

ratio

n th

at in

hibi

ts 5

0% o

f vir

us) v

arie

s with

th

e vi

ral i

nocu

lum

. Sur

veill

ance

of i

nflue

nza

susc

eptib

ility

to ri

man

tadi

ne/a

man

tadi

ne in

the

clin

ic h

as p

redo

min

antly

us

ed E

IA-b

ased

ass

ays,

sinc

e pl

aqui

ng o

f infl

uenz

a cl

inic

al is

olat

es is

var

iabl

e. W

ith d

evel

opm

ent o

f the

influ

enza

NA

in

hibi

tors

it b

ecam

e ap

pare

nt th

at c

urre

nt c

ell-b

ased

ass

ays w

ere

unsu

itabl

e fo

r mon

itori

ng su

scep

tibili

ty to

this

new

cl

ass o

f dru

gs. V

aria

bilit

y m

ay re

sult

from

vir

us sp

read

dir

ectly

from

cel

l to

cell

in c

ultu

re b

y-pa

ssin

g th

e N

A fu

nctio

n.

Furt

herm

ore,

mut

atio

ns se

lect

ed in

the

HA

, whi

le n

ot a

ppar

ently

con

trib

utin

g to

phe

noty

pic

resi

stan

ce in

viv

o, m

ay

resu

lt in

cel

l-cul

ture

bas

ed re

sist

ance

, and

may

mas

k N

A re

sist

ance

in c

ell c

ultu

re b

y m

odify

ing

rece

ptor

-bin

ding

spec

i-fic

ity. O

ne im

port

ant d

istin

ctio

n be

twee

n N

A in

hibi

tors

and

oth

er a

ntiv

iral

enz

yme

inhi

bito

rs is

that

bot

h ta

rget

enz

yme

and

inhi

bito

r wor

k ex

trac

ellu

larl

y. N

A a

ssay

s are

ther

efor

e m

ost r

epre

sent

ativ

e of

the

in v

ivo

situ

atio

n fo

r mon

itori

ng su

s-ce

ptib

ility

, sup

port

ed b

y H

A se

quen

cing

. As t

he c

linic

al u

se o

f NA

inhi

bito

rs e

scal

ates

, a m

ajor

cha

nge

will

be

requ

ired

in

appr

oach

es u

sed

to m

onito

r sus

cept

ibili

ty o

f infl

uenz

a is

olat

es in

vir

olog

y la

bora

tori

es w

orld

-wid

e.

Tisd

ale

2000

Synt

heti

c st

rate

gies

Ose

ltam

ivir

pho

spha

teO

selta

miv

ir p

hosp

hate

(Tam

iflu)

is th

e on

ly o

rally

act

ive

anti-

influ

enza

dru

g th

at p

oten

tly in

hibi

t neu

ram

inid

ase.

The

rece

nt e

mer

genc

e of

avi

an fl

u, e

spec

ially

the

H5N

1 ty

pe, m

akes

the

situ

atio

n of

Tam

iflu

supp

ly a

nd d

eman

d in

crea

sing

ly

seri

ous.

Fur

ther

opt

imiz

atio

n of

the

curr

ent c

omm

erci

al a

ppro

ach

and

expl

orat

ion

of n

ew sy

nthe

tic ro

utes

are

urg

ent.

Her

e, d

iffer

ent s

ynth

etic

stra

tegi

es o

f ose

ltam

ivir

pho

spha

te a

re re

view

ed, i

nclu

ding

dis

cove

ry a

nd im

prov

ed sy

nthe

tic

rout

e fr

om (–

)-qu

inic

aci

d or

(–)-

shik

imic

aci

d, n

ew a

sym

met

ric

synt

hesi

s via

cat

alyt

ic d

esym

met

riza

tion

of a

mes

oazi

ri-

dine

(CD

MA

), D

iels

-Ald

er R

eact

ion

and

from

oth

er a

vaila

ble

mat

eria

ls.

Gon

g an

d X

u 20

08

Synt

heti

c st

rate

gies

Tetr

a- a

nd o

ctav

alen

t si

alos

ide

clus

ters

Tetr

a- a

nd o

ctav

alen

t sia

losi

de c

lust

ers w

ere

prep

ared

in g

ood

yiel

ds e

xplo

iting

for t

he fi

rst t

ime

the

mul

tiple

cop

per-

cata

lyze

d cy

cloa

dditi

on o

f a p

ropa

rgyl

thio

sial

osid

e w

ith c

alix

[4]a

rene

pol

yazi

des.

The

cycl

oadd

ucts

feat

ured

the

hydr

o-ly

tical

ly st

able

car

bon-

sulfu

r bon

d at

the

anom

eric

pos

ition

and

the

1,4-

disu

bstit

uted

tria

zole

ring

as t

he sp

acer

bet

wee

n th

e si

alic

aci

d m

oiet

ies a

nd th

e pl

atfo

rm. I

t was

dem

onst

rate

d th

at th

ese

unna

tura

l mot

ifs d

id n

ot h

ampe

r the

des

ired

bi

olog

ical

act

ivity

of t

he si

aloc

lust

ers.

In fa

ct, t

hey

wer

e ab

le to

inhi

bit,

at su

bmill

imol

ar c

once

ntra

tions

, the

hem

aggl

uti-

natio

n an

d th

e vi

ral i

nfec

tivity

med

iate

d bo

th b

y BK

and

influ

enza

A v

irus

es.

Mar

ra e

t al.

2008

Ant

ivir

al a

ctiv

ity

eval

uati

onIn

vitr

o an

tivir

al a

ssay

sA

nim

al m

odel

s

Eval

uatio

n of

pot

entia

l infl

uenz

a vi

rus i

nhib

itors

may

util

ize

mul

tiple

step

s. F

irst

wou

ld b

e to

det

erm

ine

if th

e vi

ral t

arge

t (e

.g. i

nflue

nza

viru

s neu

ram

inid

ase)

bei

ng fo

cuse

d up

on w

ill b

e in

hibi

ted

in th

e ap

prop

riat

e as

say.

Stan

dard

in v

itro

antiv

iral

ass

ays,

use

d ne

xt in

ant

ivir

al e

valu

atio

ns, m

ay u

tiliz

e in

hibi

tion

of v

iral

pla

ques

, vir

al c

ytop

athi

c eff

ect (

CPE

), an

d vi

ral h

emag

glut

inin

or o

ther

pro

tein

, with

inhi

bitio

n of

vir

al y

ield

use

d in

follo

w-u

p ev

alua

tions

. The

CPE

can

be

dete

rmin

ed v

isua

lly a

nd b

y dy

e up

take

, Ani

mal

mod

els u

sed

for s

tudy

of p

oten

tial i

nflue

nza

viru

s inh

ibito

rs in

clud

e th

e fe

rret

, the

labo

rato

ry m

ouse

, and

the

chic

ken,

with

a v

arie

ty o

f par

amet

ers u

sed

to in

dica

te th

e se

veri

ty o

f the

infe

ctio

n an

d its

inhi

bitio

n by

ther

apy.

Mul

tiple

par

amet

ers a

re re

com

men

ded

in a

ny in

viv

o an

tivir

al e

valu

atio

n. Th

e fe

rret

and

th

e m

ouse

infe

ctio

n m

odel

s hav

e be

en u

sefu

l in

stud

ying

the

deve

lopm

ent o

f dru

g re

sist

ance

and

the

rela

tive

viru

lenc

e of

dru

g-re

sist

ant v

irus

es. Th

e in

fluen

za m

ouse

mod

el h

as a

lso

been

of v

alue

for t

he e

valu

atio

n of

imm

unom

odul

atin

g eff

ects

of t

est c

ompo

unds

and

for t

he st

udy

of th

e ut

ility

of a

ntiv

iral

dru

gs fo

r use

aga

inst

influ

enza

vir

us in

fect

ions

in th

e im

mun

ocom

prom

ised

hos

t. In

con

side

ring

the

use

of a

ny a

nim

al m

odel

, spe

cies

diff

eren

ces i

n dr

ug p

harm

acol

ogy

and

met

abol

ism

mus

t be

take

n in

to a

ccou

nt. Th

is re

view

has

des

crib

ed th

e sy

stem

s whi

ch h

ave

been

use

d m

ost f

requ

ently

by

ant

ivir

al in

vest

igat

ors,

usi

ng, a

s exa

mpl

es, r

ecen

t stu

dies

with

the

clin

ical

ly a

ppro

ved

influ

enza

vir

us n

eura

min

idas

e in

hibi

tors

ose

ltam

ivir

and

zan

amiv

ir.

Sidw

ell a

nd S

mee

20

00


132

Ant

i-in

fluen

za a

gent

sIn

fluen

za is

a d

isea

se fo

r de

eply

affe

ctin

g m

illio

ns o

f peo

ple

ever

y ye

ar. R

ecen

tly, t

here

has

bee

n co

nsid

erab

le c

once

rn

rega

rdin

g th

e hi

ghly

pat

hoge

nic

H5N

1 av

ian

influ

enza

vir

us, a

nd it

s hu

man

pan

dem

ic p

oten

tial.

With

dev

elop

men

ts

in v

iral

bio

logy

, the

re a

re m

ore

nove

l ant

ivir

al s

trat

egie

s ta

rget

ing

thes

e vi

ruse

s. In

this

revi

ew, w

e w

ill d

iscu

ss s

ever

al

prov

en a

nd p

oten

tial a

nti-

influ

enza

targ

ets,

incl

udin

g vi

ral f

acto

rs (s

uch

as h

emag

glut

inin

(HA

), M

2 io

n ch

anne

l pro

-te

in, R

NA

-dep

ende

nt R

NA

pol

ymer

ase

(RdR

p), n

ucle

opro

tein

(NP)

, non

-str

uctu

ral p

rote

in (N

S) a

nd n

eura

min

idas

e (N

A))

and

hos

t fac

tors

(suc

h as

v-A

TPa

se, p

rote

ase,

inos

ine

mon

opho

spha

te d

ehyd

roge

nase

(IM

PDH

) and

intr

acel

lula

r si

gnal

ling

casc

ades

), an

d th

eir

rele

vant

inhi

bito

rs.

Gon

g et

al.

2009

neu

ram

inid

ase

Nat

ural

pro

duct

sSe

cond

ary

plan

t m

etab

olite

s

Influ

enza

neu

ram

inid

ase

(NA

), a

key

enzy

me

in v

iral

repl

icat

ion,

spr

ead,

and

pat

hoge

nesi

s, is

con

side

red

to b

e on

e of

th

e m

ost p

rom

isin

g ta

rget

s fo

r co

mba

ting

influ

enza

. Des

pite

the

subs

tant

ial m

edic

al p

oten

tial o

f NA

inhi

bito

rs (N

AIs

), on

ly th

ree

of th

ese

drug

s ar

e cu

rren

tly o

n th

e m

arke

t (za

nam

ivir,

ose

ltam

ivir,

and

per

amiv

ir).

Mor

eove

r, su

dden

ch

ange

s in

NA

I sus

cept

ibili

ty re

veal

ed th

e ur

gent

nee

d in

the

disc

over

y/id

entif

icat

ion

of n

ovel

inhi

bito

rs. N

atur

e of

fers

an

abu

ndan

ce o

f bio

synt

hesi

zed

com

poun

ds c

ompr

isin

g ch

emic

al s

caffo

lds

of h

igh

dive

rsity

, whi

ch p

rese

nt a

n in

finite

po

ol o

f che

mic

al e

ntiti

es fo

r ta

rget

-ori

ente

d dr

ug d

isco

very

in th

e ba

ttle

aga

inst

this

hig

hly

cont

agio

us p

atho

gen.

Thi

s re

view

illu

min

ates

the

incr

easi

ng re

sear

ch e

ffort

s of

the

past

dec

ade

(200

0–20

11),

focu

sing

on

the

stru

ctur

e, fu

nctio

n an

d dr

ugga

bilit

y of

influ

enza

NA

, as

wel

l as

its in

hibi

tion

by n

atur

al p

rodu

cts.

Fol

low

ing

a cr

itica

l dis

cuss

ion

of p

ubli-

catio

ns d

escr

ibin

g so

me

150

seco

ndar

y pl

ant m

etab

olite

s te

sted

for

thei

r in

hibi

tory

pot

entia

l aga

inst

influ

enza

NA

, the

im

pact

of t

hree

diff

eren

t str

ateg

ies

to id

entif

y an

d de

velo

p no

vel N

AIs

is p

rese

nted

: (i)

bioa

ctiv

ity s

cree

ning

of h

erba

l ex

trac

ts, (

ii) e

xplo

itatio

n of

em

piri

cal k

now

ledg

e, a

nd (i

ii) c

ompu

tatio

nal a

ppro

ache

s. T

his

wor

k ad

dres

ses

the

late

st

deve

lopm

ents

in th

eore

tical

and

exp

erim

enta

l res

earc

h on

pro

pert

ies

of N

A th

at a

re a

nd w

ill b

e dr

ivin

g an

ti-in

fluen

za

drug

dev

elop

men

t now

and

in th

e ne

ar fu

ture

.

Gri

enke

et a

l. 20

12

neu

ram

inid

ase

inhi

bito

rsN

eura

min

idas

e cr

ysta

l st

ruct

ure

150-

and

430

-loop

regi

on

Neu

ram

inid

ase

inhi

bito

rs s

uch

as o

selta

miv

ir a

nd z

anam

ivir

hav

e be

en w

idel

y us

ed in

the

trea

tmen

t and

hav

e ga

ined

re

mar

kabl

e su

cces

s. A

lthou

gh, t

hey

are

effe

ctiv

e in

pre

vent

ion

of in

fluen

za; t

he c

once

rn fo

r dr

ug re

sist

ance

stil

l re

mai

ns a

que

stio

n. R

ecen

tly, t

he a

vaila

bilit

y of

cry

stal

str

uctu

res

of th

e en

zym

e ga

ve a

new

tren

d to

the

stru

ctur

e ba

sed

drug

des

igni

ng o

f neu

ram

inid

ase

inhi

bito

rs. T

he a

rtic

le re

view

s a

deta

iled

unde

rsta

ndin

g of

the

stru

ctur

al fe

a-tu

res

with

in n

eura

min

idas

e en

zym

e w

hich

turn

outs

to b

e cr

ucia

l for

futu

re d

rug

deve

lopm

ent.

In d

epth

ana

lysi

s fo

r th

e ne

wly

pro

pose

d sp

ots

with

in th

e 15

0 an

d 43

0-lo

op re

gion

s in

N1

mak

es it

dis

tingu

isha

ble

amon

g th

e su

btyp

es. F

urth

er

we

have

dis

cuss

ed th

e va

riou

s co

mpu

tatio

nal s

tudi

es c

arri

ed o

ut in

opt

imiz

ing

the

desi

gnin

g of

neu

ram

inid

ase

inhi

bi-

tors

ther

eby

prov

idin

g ne

w c

lues

to m

odify

the

curr

ently

ava

ilabl

e dr

ugs.

Chi

ntak

rind

i et

al. 2

012

Cel

lula

r dr

ug ta

rget

sT

his

revi

ew s

umm

ariz

es c

urre

nt k

now

ledg

e on

influ

enza

A v

irus

repl

icat

ion

with

a fo

cus

on e

mer

ging

cel

lula

r dr

ug

targ

ets.

Inte

rest

ingl

y, fo

r m

any

of th

ese

targ

ets,

com

poun

ds fo

r w

hich

saf

ety

test

ing

has

been

car

ried

out

in h

uman

s ar

e av

aila

ble.

It is

pos

sibl

e th

at s

ome

of th

ese

com

poun

ds, s

uch

as in

hibi

tors

of h

eat s

hock

pro

tein

90,

pro

teas

ome,

impo

r-tin

0/5

or

prot

ein

kina

se C

, will

be

used

for

trea

tmen

t of I

AV in

fect

ions

aft

er c

aref

ul e

valu

atio

n in

hum

an p

rim

ary

cells

an

d se

vere

ly il

l flu

pat

ient

s.

Mul

ler e

t al.

2012

Influ

enza

neu

tral

izin

g an

tibo

dies

The

hum

an a

ntib

ody

resp

onse

to in

fluen

za v

irus

infe

ctio

n pl

ays

a pr

otec

tive

role

aga

inst

re-i

nfec

tion,

yet

litt

le m

olec

u-la

r de

tail

is a

vaila

ble

rega

rdin

g ho

w h

uman

ant

ibod

ies,

whe

n ch

arac

teri

zed

at th

e m

onoc

lona

l lev

el, n

eutr

aliz

e th

is

impo

rtan

t hum

an p

atho

gen.

Rec

ent s

tudi

es, u

sing

a d

iver

se a

rray

of s

trat

egie

s, h

ave

isol

ated

and

cha

ract

eriz

ed h

uman

an

ti-vi

rus

neut

raliz

ing

antib

odie

s an

d sh

ed li

ght n

ot o

nly

on th

e sp

ecifi

city

and

ori

gin

of th

ese

antib

odie

s bu

t on

thei

r po

tent

ial f

or th

erap

eutic

use

aga

inst

influ

enza

vir

us in

fect

ion.

Mar

tinez

et a

l. 20

09


133

Trad

itio

nal C

hine

se

med

icin

e dr

ugs

Swin

e flu

Aft

er n

ew h

uman

tran

smis

sibl

e H

1N1

(sw

ine

flu) v

irus

es w

ere

repo

rted

in M

exic

o an

d th

e U

nite

d St

ates

in A

pril

2009

, th

e W

orld

Hea

lth O

rgan

izat

ion

(WH

O) a

nnou

nced

the

emer

genc

e of

a n

ovel

influ

enza

A v

irus

. Mos

t gov

ernm

ents

in

the

wor

ld h

ave

been

ale

rted

and

are

mon

itori

ng th

e si

tuat

ion

clos

ely.

As

one

of th

e of

ficia

l res

pons

es to

the

H1N

1 pa

ndem

ic, t

he C

hine

se g

over

nmen

t has

rele

ased

thre

e ed

ition

s of

a d

ocum

ent e

ntitl

ed “R

ecom

men

ded

Sche

mes

for

Pand

emic

Influ

enza

A D

iagn

oses

and

Tre

atm

ents

”. T

he th

ird

editi

on re

com

men

ded

the

use

of n

ot o

nly

two

targ

eted

an

ti-flu

dru

gs, o

selta

miv

ir a

nd z

anam

ivir,

but

als

o fo

ur a

nti-

flu T

CM

(Tra

ditio

nal C

hine

se M

edic

ine)

pre

scri

ptio

ns.

Sinc

e th

en, T

CM

has

pla

yed

a si

gnifi

cant

role

in fi

ghtin

g th

e pa

ndem

ic. T

CM

dru

gs c

ompr

ise

mul

tiple

com

poun

ds

regu

latin

g m

ultip

le ta

rget

s fo

r a

give

n cl

ass

of m

edic

al in

dica

tions

, and

are

tuna

ble

to th

e sy

mpt

oms

of th

e in

divi

dual

. T

his

revi

ew s

umm

ariz

es a

nti-

influ

enza

age

nts

from

TC

M, i

nclu

ding

com

poun

ds, h

erbs

, and

TC

M p

resc

ript

ions

, and

su

gges

ts th

at, b

y fu

rthe

r in

vest

igat

ing

TC

M th

eory

and

min

ing

TC

M d

atab

ases

, a b

ette

r dr

ug d

isco

very

par

adig

m m

ay

aris

e –

one

that

can

be

bene

ficia

l to

both

TC

M a

nd m

oder

n m

edic

ine.

Ge

et a

l. 20

10

Com

bina

tion

ther

apy

The

pand

emic

pot

entia

l of i

nflue

nza

viru

ses h

as e

ngag

ed a

larg

e po

rtio

n of

the

antiv

iral

dru

g di

scov

ery

rese

arch

com

mu-

nity

in th

e de

velo

pmen

t of n

umer

ous a

ntiv

iral

age

nts,

with

the

ultim

ate

goal

to su

pple

men

t effe

ctiv

e im

mun

izat

ion

whe

n ne

w st

rain

s ari

se, e

spec

ially

aft

er a

n an

tigen

ic sh

ift. A

ntiv

iral

age

nts a

gain

st in

fluen

za A

targ

ets d

iffer

ent r

eplic

atio

n st

eps

of th

e vi

rus l

ife c

ycle

s. S

ome

of th

e ag

ents

are

ana

logu

es o

f bio

mol

ecul

es re

quir

ed d

urin

g vi

rus i

nfec

tion

and

othe

rs a

re

insp

ired

from

nat

ural

pla

nt e

xtra

cts.

In th

is re

view

, we

sum

mar

ize

thei

r mec

hani

sms o

f act

ion

duri

ng th

e in

fluen

za li

fe

cycl

e in

vitr

o an

d th

e effi

caci

es o

f com

bina

tiona

l the

rapi

es w

ith th

ese

agen

ts a

gain

st th

e in

fluen

za v

irus

infe

ctio

ns in

viv

o.

Kai

hats

u an

d Ba

rnar

d 20

12

Com

bina

tion

ther

apy

Cur

rent

ly a

vaila

ble

agen

tsTh

e em

erge

nce

of p

ande

mic

H1N

1 in

fluen

za v

irus

es in

Apr

il 20

09 a

nd th

e co

ntin

uous

evo

lutio

n of

hig

hly

path

ogen

ic

H5N

1 in

fluen

za v

irus

es u

nder

scor

e th

e ur

genc

y of

nov

el a

ppro

ache

s to

chem

othe

rapy

for h

uman

influ

enza

infe

ctio

n.

Ant

i-infl

uenz

a dr

ugs a

re c

urre

ntly

lim

ited

to th

e ne

uram

inid

ase

inhi

bito

rs (o

selta

miv

ir a

nd z

anam

ivir

) and

to M

2 io

n ch

anne

l blo

cker

s (am

anta

dine

and

rim

anta

dine

), al

thou

gh re

sist

ance

to th

e la

tter

cla

ss d

evel

ops r

apid

ly. P

oten

tial t

arge

ts

for t

he d

evel

opm

ent o

f new

ant

i-infl

uenz

a ag

ents

incl

ude

the

vira

l pol

ymer

ase

(and

end

onuc

leas

e), t

he h

emag

glut

inin

, an

d th

e no

n-st

ruct

ural

pro

tein

NS1

. The

limita

tions

of m

onot

hera

py a

nd th

e em

erge

nce

of d

rug-

resi

stan

t var

iant

s mak

e co

mbi

natio

n ch

emot

hera

py th

e lo

gica

l the

rape

utic

opt

ion.

Her

e w

e re

view

the

expe

rim

enta

l dat

a on

com

bina

tion

chem

o-th

erap

y w

ith c

urre

ntly

ava

ilabl

e ag

ents

and

the

deve

lopm

ent o

f new

age

nts a

nd th

erap

y ta

rget

s.

Gov

orko

va a

nd

Web

ster

201

0

Prot

ease

inhi

bito

rsA

mbr

oxol

Cla

rith

rom

ycin

Neu

ram

inid

ase

inhi

bito

rsC

ombi

natio

n th

erap

y

Influ

enza

A v

irus

(IAV

) is o

ne o

f the

mos

t com

mon

infe

ctio

us p

atho

gens

in h

uman

s. S

ince

IVA

gen

ome

does

not

hav

e th

e pr

oces

sing

pro

teas

e fo

r the

vir

al m

embr

ane

fusi

on g

lyco

prot

ein

prec

urso

rs, e

ntry

of’

this

vir

us in

to c

ells

is d

eter

min

ed

prim

arily

by

host

cel

lula

r, tr

ypsi

n-ty

pe, p

roce

ssin

g pr

otea

ses t

hat p

rote

olyt

ical

ly a

ctiv

ate

the

fusi

on g

lyco

prot

ein

prec

ur-

sors

of I

AV. A

t lea

st fi

ve d

iffer

ent p

roce

ssin

g pr

otea

ses h

ave

been

iden

tified

in th

e ai

rway

s of a

nim

als a

nd h

uman

s. Th

ese

prot

ease

s det

erm

ine

the

infe

ctio

us o

rgan

trop

ism

of I

AV in

fect

ion

as w

ell a

s the

effi

cien

cy o

f vir

al m

ultip

licat

ion

in th

e ai

rway

, and

som

etim

es in

the

brai

n. P

rote

ases

in th

e up

per r

espi

rato

ry tr

act a

re su

ppre

ssed

by

secr

etor

y le

ukop

rote

ase

inhi

bito

r, an

d th

ose

in th

e lo

wer

resp

irat

ory

trac

t are

supp

ress

ed b

y pu

lmon

ary

surf

acta

nt w

hich

, by

adso

rptio

n, in

hibi

ts

the

inte

ract

ion

betw

een

the

prot

ease

s and

vir

al m

embr

ane

prot

eins

. Sin

ce p

rote

ase

activ

ities

pre

dom

inat

e ov

er th

ose

of

endo

geno

us in

hibi

tory

com

poun

ds u

nder

nor

mal

air

way

con

ditio

ns, a

dmin

istr

atio

n of

pro

teas

e in

hibi

tors

in th

e ea

rly-

stag

e of

infe

ctio

n si

gnifi

cant

ly su

ppre

sses

vir

al e

ntry

and

vir

al m

ultip

licat

ion.

Sev

eral

vir

al n

eura

min

idas

e in

hibi

tors

are

us

ed c

linic

ally

as a

nti-i

nflue

nza

viru

s age

nts,

bas

ed o

n th

eir i

nhib

itory

act

ion

oil v

iral

rele

ase

from

infe

cted

cel

ls. F

ur-

ther

mor

e, p

rote

ase

inhi

bito

rs o

f vir

al e

ntry

cou

ld b

e po

tent

ially

use

ful a

gain

st in

fluen

za v

irus

as w

ell a

s neu

ram

inic

lase

in

hibi

tor-

resi

stan

t vir

uses

. We

also

foun

d th

at a

mbr

oxol

, a m

ucol

ytic

and

ant

i-oxi

dant

age

nt, u

p-re

gula

tes t

he le

vels

of

endo

geno

us p

rote

ase

inhi

bito

ry c

ompo

unds

in th

e ai

rway

flui

ds in

ear

ly-p

hase

infe

ctio

n, a

nd th

at c

lari

thro

myc

in, a

mac

-ro

tide

antib

iotic

, inc

reas

es Ig

A le

vels

and

muc

osal

imm

unity

thro

ugh

augm

enta

tion

of in

terl

euki

n-12

leve

ls in

the

airw

ay.

The

com

bina

tion

of n

eura

min

idas

e in

hibi

tors

and

pro

teas

e in

hibi

tors

, cla

rith

rom

ycin

or a

mbr

oxol

, cou

ld b

e po

tent

ially

us

ed a

s a p

oten

t ant

i-infl

uenz

a th

erap

y to

min

imiz

e th

e em

erge

nce

of d

rug-

resi

stan

t mut

ant v

irus

es.

Kid

o et

al.

2007


134

Tabl

e 3.

des

ign,

syn

thes

is a

nd e

valu

atio

n of

new

ant

i-in

flue

nza

drug

s

dru

g de

sign

and

an

tivi

ral a

ctiv

ity

eval

uati

onD

rug

data

base

of

400

0 co

mpo

unds

Doc

king

sim

ulat

ion

Viab

ility

ass

ayPl

aque

ass

ayBe

nzbr

omar

one

Dic

lazu

ril

RNA

pol

ymer

ase

We

perf

orm

ed a

doc

king

sim

ulat

ion

targ

etin

g th

e in

terf

ace

of P

A in

tera

ctin

g w

ith P

B1 u

sing

a d

rug

data

base

incl

ud-

ing

sim

ilar

to 4

000

com

poun

ds. W

e th

en c

ondu

cted

cel

l via

bilit

y as

say

and

plaq

ue a

ssay

usi

ng IA

V/W

SN/3

3. F

inal

ly

we

exam

ined

thei

r an

ti-IA

V m

echa

nism

by

surf

ace

plas

mon

reso

nanc

e an

d IA

V re

plic

on a

ssay

. Res

ults

: We

foun

d th

at

benz

brom

aron

e, d

icla

zuri

l, an

d tr

enbo

lone

ace

tate

had

str

ong

anti-

IAV

act

iviti

es. W

e co

nfir

med

that

ben

zbro

mar

one

and

dicl

azur

il bo

und

with

PA

sub

unit

, and

dec

reas

ed th

e tr

ansc

ript

iona

l act

ivity

of t

he v

iral

RN

A p

olym

eras

e. C

on-

clus

ions

: Ben

zbro

mar

one

and

dicl

azur

il ha

d st

rong

ant

i-IA

V a

ctiv

ities

with

nov

el a

ctio

n m

echa

nism

, i.e

. inh

ibiti

on o

f vi

ral R

NA

pol

ymer

ase.

Gen

eral

sig

nific

ance

: Sin

ce b

enzb

rom

aron

e an

d di

claz

uril

are

alre

ady

used

in p

ublic

as

med

i-ci

nes,

thes

e co

uld

be th

e ca

ndid

ates

for

alte

rnat

ives

in c

ase

of a

n ou

tbre

ak o

f IAV

whi

ch is

resi

stan

t to

pre-

exis

ting

anti-

IAV

dru

gs.

Fuku

oka

et a

l. 20

12

Stru

ctur

e-ba

sed

drug

de

sign

Com

puta

tiona

l m

etho

dolo

gies

Vir

tual

scre

enin

gD

ocki

ngPh

arm

acop

hore

mod

elin

g20

0 ne

w d

esig

ned

ligan

ds

The

M2

chan

nel p

rote

in o

n th

e in

fluen

za A

vir

us m

embr

ane

has

beco

me

the

mai

n ta

rget

of t

he a

nti-

flu d

rugs

am

an-

tadi

ne a

nd r

iman

tadi

ne. T

he s

truc

ture

of t

he M

2 ch

anne

l pro

tein

s of

the

H3N

2 (P

DB

code

2RL

F) a

nd 2

009-

H1N

1 (G

enba

nk a

cces

sion

num

ber

GQ

3853

83) v

irus

es m

ay h

elp

rese

arch

ers

to s

olve

the

drug

-res

ista

nt p

robl

em o

f the

se tw

o ad

aman

tane

-bas

ed d

rugs

and

dev

elop

mor

e po

wer

ful n

ew d

rugs

aga

inst

influ

enza

A v

irus

. In

the

pres

ent s

tudy

, we

sear

ched

for

new

M2

chan

nel i

nhib

itors

thro

ugh

a co

mbi

natio

n of

diff

eren

t com

puta

tiona

l met

hodo

logi

es, i

nclu

ding

vi

rtua

l scr

eeni

ng w

ith d

ocki

ng a

nd p

harm

acop

hore

mod

elin

g. V

irtu

al s

cree

ning

was

per

form

ed to

cal

cula

te th

e fr

ee

ener

gies

of b

indi

ng b

etw

een

rece

ptor

M2

chan

nel p

rote

ins

and

200

new

des

igne

d lig

ands

. Aft

er th

at, p

harm

acop

hore

an

alys

is w

as u

sed

to id

entif

y th

e im

port

ant M

2 pr

otei

n-in

hibi

tor

inte

ract

ions

and

com

mon

feat

ures

of t

op b

indi

ng

com

poun

ds w

ith M

2 ch

anne

l pro

tein

s. F

inal

ly, t

he tw

o m

ost p

oten

tial c

ompo

unds

wer

e de

term

ined

as

nove

l lea

ds to

in

hibi

t M2

chan

nel p

rote

ins

in b

oth

H3N

2 an

d 20

09-H

1N1

influ

enza

A v

irus

.

Tran

et a

l. 20

11

dru

g re

sist

ance

Ada

man

tane

s and

ne

uram

inid

ase

inhi

bito

rs

Influ

enza

vir

uses

are

maj

or h

uman

pat

hoge

ns w

ith a

glo

bal d

istr

ibut

ion,

acc

ount

ing

for

mor

e th

an 5

00 0

00 a

nnua

l de

aths

wor

ldw

ide

and

with

con

side

rabl

e im

pact

on

the

qual

ity o

f life

and

pro

duct

ivity

of t

he s

ocie

ty. D

ue to

the

limite

d ef

ficac

y of

vac

cina

tion,

ant

ivir

al d

rugs

con

stitu

te a

com

plem

enta

ry a

ppro

ach

in th

e co

ntro

l and

pre

vent

ion

of in

fluen

za

infe

ctio

ns a

nd th

us p

lay

an im

port

ant r

ole

in th

e m

anag

emen

t of i

nflu

enza

out

brea

ks a

nd p

ande

mic

s. C

urre

ntly

, ada

-m

anta

nes

and

neur

amin

idas

e in

hibi

tors

(NA

Is) a

re th

e on

ly tw

o cl

asse

s of

ant

i-in

fluen

za a

gent

s ap

prov

ed fo

r cl

inic

al

use.

How

ever

, the

wor

ldw

ide

emer

genc

e an

d hi

gh p

reva

lenc

e of

ada

man

tane

-res

ista

nt v

irus

var

iant

s ha

s di

scou

rage

d th

e us

e of

the

form

er d

rugs

. NA

Is h

ave

prov

ed to

be

very

effe

ctiv

e ag

ains

t inf

luen

za A

and

B v

irus

es. N

ever

thel

ess,

os

elta

miv

ir-r

esis

tant

str

ains

hav

e al

so b

een

repo

rted

qui

te fr

eque

ntly

, as

in th

e ca

se o

f sea

sona

l H1N

1 vi

ruse

s th

at

circ

ulat

ed b

etw

een

2007

and

200

9. In

deed

, the

em

erge

nce

of d

rug-

resi

stan

t vir

us v

aria

nts

is a

lway

s a

mat

ter

of c

once

rn

beca

use

it co

uld

sign

ifica

ntly

com

prom

ise

the

usef

ulne

ss o

f suc

h in

terv

entio

n. T

his

high

light

s th

e ne

ed fo

r co

ntin

uous

m

onito

ring

of r

esis

tanc

e m

arke

rs, a

s w

ell a

s th

e de

velo

pmen

t of n

ew a

nti-

influ

enza

dru

gs a

nd c

ombi

natio

n th

erap

ies.

Pizz

orno

et a

l. 20

11


135

Inte

ract

ion

of

adam

anta

ne w

ith

M2

prot

ein

Pore

doc

king

mod

el

Inte

ract

ion

of a

min

oada

man

tane

s w

ith th

e in

fluen

za A

vir

us M

2 pr

oton

cha

nnel

was

ana

lyze

d by

doc

king

sim

ulat

ions

of

a s

erie

s of

syn

thet

ic a

min

oada

man

tane

der

ivat

ives

, of d

iffer

ing

bind

ing

affin

ity, i

nto

the

crys

tal s

truc

ture

of t

he

tran

smem

bran

e (M

2TM

) por

e. T

he p

ore

bloc

king

mod

el te

sted

in th

e ‘g

as p

hase

’ des

crib

es q

ualit

ativ

ely

the

chan

ges

on th

e re

lativ

e bi

ndin

g af

finiti

es o

f the

com

poun

ds (a

lthou

gh a

ser

ies

of h

ighl

y hy

drop

hobi

c lig

ands

whi

ch s

eem

to

have

litt

le c

apac

ity fo

r di

ffere

nt s

peci

fic in

tera

ctio

ns w

ith th

eir

rece

ptor

). T

he d

ocki

ng c

alcu

latio

ns p

redi

cted

pos

es

in w

hich

the

adam

anta

ne r

ing

is s

urro

unde

d m

ainl

y by

the

alky

l sid

e ch

ains

of V

al27

or

Ala

30 a

nd th

e lig

and’

s am

ino

grou

p is

gen

eral

ly h

ydro

gen

bond

ed w

ith h

ydro

xyls

of S

er31

or

carb

onyl

s of

Val

27 o

r ca

rbon

yls

of A

la30

, the

form

er

(Ser

31) b

eing

the

mos

t sta

ble

and

mos

t fre

quen

tly o

bser

ved.

The

bin

ding

of t

he li

gand

is a

com

prom

ise

betw

een

hydr

ogen

bon

ding

abi

lity,

whi

ch is

ele

vate

d by

a p

rim

ary

amin

o gr

oup,

and

apo

lar

inte

ract

ions

, whi

ch a

re in

crea

sed

by

the

abili

ty o

f the

lipo

phili

c m

oiet

y to

ade

quat

ely

fill a

hyd

roph

obic

poc

ket w

ithin

the

M2T

M p

ore.

A d

elic

ate

bala

nce

of th

ese

hydr

opho

bic

cont

ribu

tions

is re

quir

ed fo

r op

timal

inte

ract

ion.

Elef

ther

atos

et a

l. 20

10

Stru

ctur

e-ba

sed

drug

de

sign

Poly

mer

ase

basi

c

prot

ein

2Po

lym

eras

e in

hibi

tors

Influ

enza

vir

uses

cau

se a

sig

nific

ant l

evel

of m

orbi

dity

and

mor

talit

y in

the

popu

latio

n ev

ery

year

. The

ir re

sist

ance

to

curr

ent a

nti-

influ

enza

dru

gs in

crea

ses

the

diff

icul

ty o

f flu

trea

tmen

t. T

hus,

dev

elop

men

t of n

ew a

nti-

influ

enza

dru

gs

is n

eces

sary

in re

gard

s of

pre

vent

the

trag

edy

of in

fluen

za p

ande

mic

. The

Pol

ymer

ase

basi

c pr

otei

n 2

(PB2

) sub

unit

of in

fluen

za v

irus

RN

A p

olym

eras

e is

one

of p

oten

tial t

arge

ts fo

r ne

w d

rugs

bec

ause

the

bind

ing

of P

B2 w

ith th

e 5’

ca

p of

the

host

pre

-mRN

As

is th

e in

itial

ste

p of

the

viru

s’ pr

otei

n sy

nthe

sis.

In th

is s

tudy

, we

com

pare

d th

e bi

ndin

g po

tenc

y of

PB2

cap

bin

ding

dom

ain

with

two

smal

l mol

ecul

es, i

.e.,

RO a

nd P

PT28

, with

that

of P

B2 w

ith c

ap a

nalo

g m

7GT

P. T

he c

alcu

late

d bi

ndin

g en

ergi

es s

how

ed th

at R

O a

nd P

PT28

had

hig

her

bind

ing

affin

ity w

ith P

B2. F

urth

er

inte

ract

ion

anal

ysis

sho

wed

that

the

impo

rtan

t par

ts fo

r bi

ndin

g w

ere

the

five-

and

six-

mem

ber

hete

rocy

clic

rin

gs (t

he

6/5-

mem

ber

ring

s) o

f sm

all m

olec

ules

, as

wel

l as

the

hydr

opho

bic

part

s of

RO

and

PPT

28 w

hich

had

goo

d in

tera

c-tio

ns w

ith th

e hy

drop

hobi

c re

sidu

es in

the

bind

ing

cavi

ty. T

hus,

RO

and

PPT

28 a

re tw

o po

tent

ial a

nti-

influ

enza

dru

gs

targ

eted

PB2

, whi

ch m

ay in

hibi

t the

gro

wth

of i

nflu

enza

vir

us b

y co

mpe

titiv

ely

bind

ing

with

the

cap

stru

ctur

e bi

ndin

g do

mai

n of

PB2

.

Lv e

t al.

2011

des

ign,

syn

thes

is, i

n vi

tro

eval

uati

onTr

ansi

tion-

stat

e-ba

sed

neur

amin

idas

e in

hibi

tors

X-r

ay c

ryst

allo

grap

hic

stru

ctur

eH

ydro

phob

ic p

ocke

t

The

des

ign,

syn

thes

is, a

nd in

vitr

o ev

alua

tion

of th

e no

vel c

arbo

cycl

es a

s tr

ansi

tion-

stat

e-ba

sed

inhi

bito

rs o

f inf

luen

za

neur

amin

idas

e (N

A) a

re d

escr

ibed

. The

dou

ble

bond

pos

ition

in th

e ca

rboc

yclic

ana

logu

es p

lays

an

impo

rtan

t rol

e in

N

A in

hibi

tion

as d

emon

stra

ted

by th

e an

tivir

al a

ctiv

ity o

f 8 (I

C50

= 6

.3 µ

M) v

s 9

(IC

50 >

200

µM

). St

ruct

ure-

activ

ity

stud

ies

of a

ser

ies

of c

arbo

cycl

ic a

nalo

gues

6a-

i ide

ntifi

ed th

e 3-

pent

ylox

y m

oiet

y as

an

appa

rent

opt

imal

gro

up a

t the

C

-3 p

ositi

on w

ith a

n IC

50 v

alue

of 1

nM

for

NA

inhi

bitio

n. T

he X

-ray

cry

stal

logr

aphi

c st

ruct

ure

of 6

h bo

und

to N

A

reve

aled

the

pres

ence

of a

larg

e hy

drop

hobi

c po

cket

in th

e re

gion

cor

resp

ondi

ng to

the

glyc

erol

sub

site

of s

ialic

aci

d.

The

hig

h an

tivir

al p

oten

cy o

bser

ved

for

6h a

ppea

rs to

be

attr

ibut

ed to

a h

ighl

y fa

vora

ble

hydr

opho

bic

inte

ract

ion

in

this

poc

ket.

The

pra

ctic

al s

ynth

esis

of 6

sta

rtin

g fr

om (–

)-qu

inic

aci

d is

als

o de

scri

bed.

Kim

et a

l. 19

97

Enan

tios

elec

tive

sy

nthe

sis

(–)-

osel

tam

ivir

free

bas

e(–

)-m

ethy

l 3-e

pi-

shik

imat

e

A n

ew e

nant

iose

lect

ive

synt

hesi

s of

the

anti-

influ

enza

age

nt (–

)-os

elta

miv

ir fr

ee b

ase

(7.1

% o

vera

ll yi

eld;

98%

ee)

and

(–

)-m

ethy

l 3-e

pi-s

hiki

mat

e (1

6% o

vera

ll yi

eld;

98%

ee)

has

bee

n de

scri

bed

from

read

ily a

vaila

ble

raw

mat

eria

ls. S

harp

-le

ss a

sym

met

ric

epox

idat

ion

and

dias

tere

osel

ectiv

e Ba

rbie

r al

lyla

tion

of a

n al

dehy

de a

re th

e ke

y re

actio

ns e

mpl

oyed

in

the

inco

rpor

atio

n of

chi

ralit

y, w

hile

the

cycl

ohex

ene

carb

oxyl

ic e

ster

cor

e w

as c

onst

ruct

ed th

roug

h a

ring

clo

sing

m

etat

hesi

s re

actio

n.

Raw

at e

t al.

2012


136

Synt

heti

c st

rate

gies

4-am

ino-

1-ph

osph

ono-

DA

NA

Phos

phon

o-za

nam

ivir

Two

phos

phon

ate

com

poun

ds la

(4-a

min

o-1-

phos

phon

o-D

AN

A) a

nd 1

b (p

hosp

hono

-zan

amiv

ir) a

re s

ynth

esiz

ed a

nd

show

n m

ore

pote

nt th

an z

anam

ivir

aga

inst

the

neur

amin

idas

es o

f avi

an a

nd h

uman

influ

enza

vir

uses

, inc

ludi

ng th

e os

elta

miv

ir-r

esis

tant

str

ains

. For

the

first

tim

e, th

e pr

actic

al s

ynth

esis

of t

hese

pho

spho

nate

com

poun

ds is

real

ized

by

conv

ersi

on o

f sia

lic a

cid

to p

erac

etyl

ated

pho

spho

no-D

AN

A d

ieth

yl e

ster

(5) a

s a

key

inte

rmed

iate

in th

ree

step

s by

a

nove

l app

roac

h. In

com

pari

son

with

zan

amiv

ir, th

e hi

gh a

ffin

ity o

f la

and

1b c

an b

e pa

rtly

att

ribu

tabl

e to

the

stro

ng

elec

tros

tatic

inte

ract

ions

of t

heir

pho

spho

nate

, gro

up’s

with

the

thre

e ar

gini

ne re

sidu

es (A

rg11

8, A

rg29

2, a

nd A

rg37

1)

in th

e ac

tive

site

of n

eura

min

idas

es. T

hese

pho

spho

nate

s ar

e no

ntox

ic to

the

hum

an 2

93T

cel

ls; t

hey

prot

ect c

ells

from

in

fluen

za: v

irus

infe

ctio

n w

ith E

C50

val

ues.

in lo

w-n

anom

olar

ran

ge; i

nclu

ding

the

wild

-typ

e W

SN (H

1N1)

, the

200

9 pa

ndem

ic (H

1N1)

, the

ose

ltam

ivir

-res

ista

nt H

274Y

(H1N

1), R

G14

(H5N

1); a

nd U

dorn

(H3N

2) in

fluen

za s

trai

ns.

Shie

et a

l. 20

11

Synt

heti

c st

rate

gies

(+)-

stac

hyfli

nA

nov

el a

nd p

oten

t hem

aggl

utin

in in

hibi

tor,

(+)-

stac

hyfli

n, w

as e

ffic

ient

ly s

ynth

esiz

ed in

an

enan

tiose

lect

ive

man

ner

star

ting

from

the

(+)-

5-m

ethy

l-Wie

land

-Mie

sche

r ke

tone

. The

syn

thet

ic m

etho

d fe

atur

es a

BF(

3) c

ente

r do

t Et(

2)O

-ind

uced

cas

cade

epo

xide

-ope

ning

/rea

rran

gem

ent/

cycl

izat

ion

reac

tion

to s

tere

osel

ectiv

ely

cons

truc

t the

requ

isite

pe

ntac

yclic

rin

g sy

stem

in o

ne s

tep.

In o

rder

to r

atio

naliz

e th

e m

echa

nism

of t

he c

asca

de re

actio

n, q

uant

um c

hem

ical

ca

lcul

atio

ns o

f the

pos

sibl

e in

term

edia

ry c

arbo

catio

ns a

nd tr

ansi

tion

stat

es in

the

mod

el s

ynth

esis

wer

e ca

rrie

d ou

t. A

n al

tern

ativ

e ap

proa

ch to

syn

thes

ize

(+)-

stac

hyfli

n by

em

ploy

ing

a si

mila

r ca

scad

e re

actio

n w

as a

lso

desc

ribe

d.

Saku

rai e

t al.

2011

nuc

leop

rote

in s

cree

ning

as

say

Tryp

toph

an fl

uore

scen

ce

quen

chin

g

Rece

nt s

tudi

es h

ave

show

n th

at N

P (n

ucle

opro

tein

), w

hich

pos

sess

es m

ultip

le fu

nctio

ns in

the

vira

l life

cyc

le, i

s a

new

po

tent

ial a

nti-

influ

enza

dru

g ta

rget

. NP

inhi

bito

rs re

liabl

y in

duce

con

form

atio

nal c

hang

es in

NPs

, and

thes

e ch

ange

s m

ay c

onfe

r in

hibi

tion

of th

e in

fluen

za v

irus

. The

six

con

serv

ed tr

ypto

phan

resi

dues

in N

P ca

n be

use

d as

an

intr

insi

c pr

obe

to m

onito

r th

e ch

ange

in fl

uore

scen

ce o

f the

tryp

toph

an re

sidu

es in

the

prot

ein

upon

bin

ding

to a

n N

P in

hibi

-to

r. In

the

pres

ent s

tudy

, we

foun

d th

at th

e flu

ores

cenc

e of

reco

mbi

nant

NP

prot

eins

was

que

nche

d fo

llow

ing

the

bind

ing

of a

vaila

ble

NP

inhi

bito

rs (s

uch

as n

ucle

ozin

) in

a co

ncen

trat

ion-

and

tim

e-de

pend

ent m

anne

r, w

hich

sug

gest

s th

at th

e in

hibi

tor

indu

ced

conf

orm

atio

nal c

hang

es in

the

NPs

. The

min

imal

fluo

resc

ence

-que

nchi

ng e

ffect

and

wea

k bi

ndin

g co

nsta

nt o

f nuc

leoz

in to

the

swin

e-or

igin

influ

enza

vir

us H

1N1p

dm09

(SO

IV) N

P re

veal

ed th

at th

e SO

IV is

re

sist

ant t

o nu

cleo

zin.

We

have

use

d th

e flu

ores

cenc

e-qu

ench

ing

prop

erty

of t

rypt

opha

ns in

NPs

that

wer

e bo

und

to

ligan

ds in

a 9

6-w

ell-

plat

e-ba

sed

drug

scr

een

to a

sses

s th

e ab

ility

of p

rom

isin

g sm

all m

olec

ules

to in

tera

ct w

ith N

Ps a

nd

have

iden

tifie

d on

e ne

w a

nti-

influ

enza

dru

g, C

SV0C

0010

18, w

ith a

hig

h SI

val

ue. T

his

conv

enie

nt m

etho

d fo

r dr

ug

scre

enin

g m

ay fa

cilit

ate

the

deve

lopm

ent o

f ant

ivir

al d

rugs

that

targ

et v

irus

es o

ther

than

the

influ

enza

vir

us, s

uch

as

HIV

and

HBV

.

Hun

g et

al.

2012

neu

ram

inid

ase

enzy

mat

ic a

ssay

Reco

mbi

nant

ne

uram

inid

ase

expr

essi

onRe

com

bina

nt b

acul

ovir

us

Con

text

: Dev

elop

men

t of i

nexp

ensi

ve a

nd s

afe

enzy

mat

ic a

ssay

s to

scr

een

for

puta

tive

neur

amin

idas

e in

hibi

tors

. O

bjec

tive:

Val

idat

e th

e us

e of

reco

mbi

nant

neu

ram

inid

ase

expr

esse

d in

bac

ulov

irus

loca

ted

on th

e vi

ral s

urfa

ce

caps

ule

to d

evel

op a

neu

ram

inid

ase

inhi

bito

r sc

reen

ing

assa

y. M

ater

ials

and

met

hods

: Rec

ombi

nant

bac

ulov

irus

pa

rtic

les

disp

layi

ng n

eura

min

idas

e N

1 an

d N

3 w

ere

used

as

enzy

me

sour

ces.

The

ass

ay s

et-u

p re

quir

ed th

e us

e of

2’

-(4-

met

hylu

mbe

llife

ryl)-

alph

a-D

-ace

tyl n

eura

min

ic a

cid

as s

ubst

rate

and

ose

ltam

ivir

car

boxy

late

as

benc

hmar

k in

hibi

tor.

Resu

lts: T

he a

ssay

was

set

up

in a

sta

ndar

d 96

-wel

l pla

te. T

he w

ithin

- and

bet

wee

n-as

say

coef

ficie

nts

of v

ari-

atio

n w

ere,

on

aver

age,

less

than

10%

. The

50%

inhi

bito

ry c

once

ntra

tion

valu

es o

f the

inhi

bito

r w

ere

in g

ood

agre

e-m

ent w

ith th

ose

dete

rmin

ed b

y in

depe

nden

t kin

etic

exp

erim

ents

. Dis

cuss

ion

and

conc

lusi

ons:

The

ass

ay s

how

ed s

atis

-fa

ctor

y w

ithin

- and

bet

wee

n-as

say

repe

atab

ility

. The

obt

aine

d re

sults

sug

gest

that

reco

mbi

nant

bac

ulov

irus

exp

ress

ing

neur

amin

idas

e lo

cate

d on

the

viru

s m

embr

ane

caps

ule

can

be u

sed

to s

et u

p af

ford

able

and

relia

ble

neur

amin

idas

e in

hibi

tors

scr

eeni

ng a

ssay

s.

Kon

gkam

nerd

et

al. 2

012


137

Fluo

resc

ence

po

lari

sati

on a

ssay

Endo

nucl

ease

inhi

bito

rs

The

end

onuc

leas

e ac

tivity

of t

he in

fluen

za v

irus

PA

(N) p

rote

in is

ess

entia

l for

vir

us re

plic

atio

n an

d is

a p

rom

isin

g ta

rget

for

nove

l ant

i-in

fluen

za d

rugs

. To

faci

litat

e th

e di

scov

ery

of e

ndon

ucle

ase

inhi

bito

rs, w

e ha

ve d

evel

oped

a h

igh-

tluou

ghpu

t flu

ores

cenc

e po

lari

zatio

n (F

P) a

ssay

, util

izin

g a

nove

l flu

ores

cein

-lab

eled

com

poun

d (K

–d =

0.3

78 m

u M

) an

d a

PA(N

) con

stru

ct, t

o id

entif

y sm

all m

olec

ules

that

bin

d to

the

PA(N

) end

onuc

leas

e ac

tive

site

. Sev

eral

kno

wn

4-su

bstit

uted

2,4

-dio

xobu

tano

ic a

cid

inhi

bito

rs w

ith h

igh

and

low

aff

initi

es h

ave

been

eva

luat

ed in

this

FP-

base

d,

com

petit

ive

bind

ing

assa

y, a

nd th

ere

was

a g

ener

al c

orre

latio

n be

twee

n bi

ndin

g an

d th

e re

port

ed in

hibi

tion

of e

ndo-

nucl

ease

act

ivity

. Add

ition

ally

, we

have

dem

onst

rate

d th

e ut

ility

of t

his

assa

y fo

r id

entif

ying

end

onuc

leas

e in

hibi

tors

in

a s

mal

l div

erse

targ

eted

frag

men

t lib

rary

. The

se fr

agm

ent h

its w

ere

used

to b

uild

a fo

llow

-up

libra

ry th

at th

at le

d to

new

act

ive

com

poun

ds th

at d

emon

stra

te F

P bi

ndin

g an

d an

ti-in

fluen

za a

ctiv

ities

in p

laqu

e in

hibi

tion

assa

ys. T

he

assa

y of

fers

sig

nific

ant a

dvan

tage

s. o

ver

prev

ious

ly re

port

ed a

ssay

s an

d is

sui

tabl

e fo

r hi

gh-t

hrou

ghpu

t and

frag

men

t-ba

sed

scre

enin

g st

udie

s. A

dditi

onal

ly th

e de

mon

stra

tion

of th

e ap

plic

abili

ty o

f a m

echa

nism

-bas

ed “t

arge

ted

frag

men

t”

libra

ry s

uppo

rts

the

gene

ral p

oten

tial o

f thi

s no

vel a

ppro

ach

for

othe

r en

zym

e ta

rget

s. T

hese

resu

lts s

erve

as

a so

und

foun

datio

n fo

r th

e de

velo

pmen

t of n

ew th

erap

eutic

lead

s ta

rget

ing

influ

enza

end

onuc

leas

e.

Baug

hman

et a

l. 20

12

Hig

h-th

roug

hput

sc

reen

ing

Cyt

opat

hic

effec

t in

hibi

tory

ass

ayC

ryst

al v

iole

t upt

ake

assa

yIn

fluen

za v

irus

AC

oxsa

ckie

vir

us B

3H

erpe

s sim

plex

vir

us-1

In o

rder

to id

entif

y ne

w p

oten

tial a

ntiv

iral

dru

gs, s

mal

l am

ount

s of

ext

ract

s or

com

poun

ds h

ave

to b

e ex

amin

ed fo

r cy

toto

xici

ty a

nd a

ntiv

iral

act

ivity

in p

rim

ary

scre

enin

g us

ing

a ra

pid,

eas

y, in

expe

nsiv

e, a

nd h

ighl

y st

anda

rdis

ed te

st

syst

em. I

n th

is s

tudy

, hig

h-th

roug

hput

cyt

opat

hic

effe

ct (C

PE) i

nhib

itory

ass

ays

wer

e es

tabl

ishe

d fo

r co

xsac

kie

viru

s B3

on

HeL

a O

hio

cells

, inf

luen

za v

irus

A o

n M

adin

-Dar

by c

anin

e ki

dney

cel

ls, a

nd h

erpe

s si

mpl

ex v

irus

type

(HSV

-1)

on

gree

n m

onke

y ki

dney

cel

ls th

at m

eet t

hese

requ

irem

ents

. The

cyt

otox

ic a

nd th

e an

tivir

al e

ffect

s w

ere

quan

ti-fie

d us

ing

a cr

ysta

l vio

let u

ptak

e as

say

allo

win

g au

tom

ated

han

dlin

g of

larg

e nu

mbe

rs o

f can

dida

te a

gent

s. T

o en

sure

co

mpa

rabl

e re

sults

with

pla

que

redu

ctio

n as

says

. the

50

and

90%

pla

que

inhi

bito

ry c

once

ntra

tions

of g

uani

dine

, am

anta

dine

, and

pho

spho

nofo

rmic

aci

d w

ere

used

to s

tand

ardi

se th

e an

ti-co

xsac

kie

viru

s B3

, ant

i-in

fluen

za v

irus

A,

and

anti-

HSV

-l te

sts,

resp

ectiv

ely.

The

str

ong

corr

elat

ion

betw

een

the

antiv

iral

act

ivity

det

erm

ined

by

CPE

-inh

ibito

ry

assa

ys a

nd p

laqu

e re

duct

ion

assa

y w

as fu

rthe

r pr

oved

for

othe

r an

tivir

als.

In s

umm

ary,

low

am

ount

s of

larg

e nu

mbe

rs

of c

ompo

unds

may

be

test

ed in

expe

nsiv

ely

and

stan

dard

ised

with

in 2

4 h

(cox

sack

ie v

irus

B3

and

influ

enza

vir

us A

) or

48 h

(her

pes

sim

plex

vir

us ty

pe 1

) pos

t-in

fect

ion

usin

g C

PE in

hibi

tory

ass

ays.

Schm

idtk

e et

al.

2001

Hig

h-th

roug

hput

sc

reen

ing

Plaq

ue in

hibi

tion

assa

y Ti

me-

of-a

dditi

on

expe

rim

ent

Trad

ition

al C

hine

se

med

icin

ePr

ocya

nidi

n

In th

is re

sear

ch, w

e ha

ve e

stab

lishe

d a

high

-thr

ough

put s

cree

ning

(HT

S) p

latf

orm

bas

ed o

n th

e in

fluen

za A

vir

us

(IAV

) vRN

A p

rom

oter

. Usi

ng th

is H

TS

plat

form

, we

sele

cted

35

med

icin

al p

lant

s ou

t of 8

3 ex

ampl

es o

f tra

ditio

nal

Chi

nese

med

icin

e an

d fo

und

that

7 e

xam

ples

had

not

bee

n re

port

ed. A

fter

exa

min

ing

man

y pr

evio

us re

port

s, w

e fo

und

that

Vac

cini

um a

ngus

tifol

ium

Ait

., V

itis

vini

fera

L, a

nd C

inna

mom

um c

assi

a Pr

esl h

ad a

com

mon

act

ive

com

poun

d,

proc

yani

din,

and

then

det

erm

ined

the

anti-

IAV

effe

ct o

f pro

cyan

idin

and

exp

lore

d its

mec

hani

sm o

f act

ion.

With

a

plaq

ue in

hibi

tion

assa

y an

d a

time-

of-a

dditi

on e

xper

imen

t, w

e fo

und

that

pro

cyan

idin

cou

ld in

hibi

t the

IAV

repl

ica-

tion

at s

ever

al s

tage

s of

the

life

cycl

e. In

the

Wes

tern

blo

t and

EG

FP-L

C3

loca

lizat

ion

assa

ys, w

e fo

und

that

pro

cyan

i-di

n co

uld

inhi

bit t

he a

ccum

ulat

ion

of L

C3I

I and

the

dot-

like

aggr

egat

ion

of E

GFP

-LC

3. In

the

RT-P

CR

and

Wes

tern

bl

ot a

ssay

s, w

e fo

und

proc

yani

din

coul

d in

hibi

t the

exp

ress

ion

of A

tg7,

Atg

5, a

nd A

tg12

. Fin

ally

, by

the

bim

olec

ular

flu

ores

cenc

e co

mpl

emen

tatio

n-flu

ores

cenc

e re

sona

nce

ener

gy tr

ansf

er a

nd c

o-im

mun

opre

cipi

tatio

n as

says

, we

foun

d th

at p

rocy

anid

in c

ould

inhi

bit t

he fo

rmat

ion

of th

e A

tg5-

Atg

12/A

tg16

het

erot

rim

er a

nd th

e di

ssoc

iatio

n of

the

becl

in1/

bcl2

het

erod

imer

. In

conc

lusi

on, w

e ha

ve e

stab

lishe

d an

HT

S pl

atfo

rm a

nd id

entif

ied

proc

yani

din

as a

nov

el

and

prom

isin

g an

ti-IA

V a

gent

.

Dai

et a

l. 20

12


138

Com

puta

tion

al/

expe

rim

enta

l sc

reen

ing

Flow

cyt

omet

ry a

naly

sis

Cas

pase

-3 a

ssay

Min

i-gen

ome

assa

yTi

me-

of-a

dditi

on

expe

rim

ent

Ribo

nucl

eopr

otei

n co

mpl

ex

In th

is s

tudy

, com

puta

tiona

l and

exp

erim

enta

l scr

eeni

ng o

f an

exte

nsiv

e co

mpo

und

libra

ry id

entif

ied

TH

C19

, whi

ch

was

abl

e to

sup

pres

s in

fluen

za v

irus

repl

icat

ion.

Thi

s co

mpo

und

had

no c

ytot

oxic

effe

cts

and

did

not d

isru

pt c

ell

cycl

e pr

ogre

ssio

n or

indu

ce a

popt

osis

in M

DC

K c

ells

as

conf

irm

ed b

y W

ST-1

ass

ays,

flow

cyt

omet

ry a

naly

sis,

and

ca

spas

e-3

assa

ys. T

ime-

of-a

dditi

on e

xper

imen

ts s

how

ed th

at T

HC

19 a

cts

at a

rela

tivel

y ea

rly

stag

e of

the

vira

l life

-cy

cle.

Sub

sequ

ent m

ini-

geno

me

assa

ys re

veal

ed th

at T

HC

19 in

hibi

ted

vira

l gen

ome

repl

icat

ion

and/

or tr

ansc

ript

ion,

su

gges

ting

that

it in

terf

eres

with

one

or

mor

e of

the

vira

l com

pone

nts

that

form

the

ribo

nucl

eopr

otei

n co

mpl

exes

, na

mel

y po

lym

eras

e ba

sic

2 (P

B2),

poly

mer

ase

basi

c 1

(PB1

), po

lym

eras

e ac

idic

(PA

), nu

cleo

prot

ein

(NP)

and

vir

al

RNA

. Fin

ally

, min

i-ge

nom

e as

says

whe

re P

B2, P

B1, P

A o

r N

P fr

om A

/WSN

/33

(H1N

1) v

irus

wer

e re

plac

ed w

ith th

ose

from

A/U

dorn

/307

/197

2 (H

3N2)

vir

us e

ffect

ivel

y de

mon

stra

ted

that

TH

C19

inhi

bite

d vi

ral m

ultip

licat

ion

in a

man

ner

depe

nden

t upo

n th

e PA

sub

unit

. Tak

en to

geth

er, t

hese

resu

lts s

ugge

st th

at in

fluen

za v

irus

PA

pro

tein

is a

pot

entia

l ta

rget

for,

and

may

aid

the

deve

lopm

ent o

f, no

vel c

ompo

unds

that

inhi

bit i

nflu

enza

A v

irus

repl

icat

ion.

Yam

ada

et a

l. 20

12

Ferr

et/m

ouse

mod

elIn

tran

asal

adm

inis

trat

ion

Stac

hyfli

n de

riva

tives

The

in-v

ivo

anti-

influ

enza

-vir

us a

ctiv

ity o

f Sta

chyf

lin d

eriv

ativ

es (I

II a

nd it

s ph

osph

ate

este

r, II

I-Ph

os),

a ne

w c

lass

of

haem

aggl

utin

in fu

sion

inhi

bito

r, an

d th

e im

prov

emen

t of t

heir

abs

orpt

ion

afte

r or

al o

r in

tran

asal

adm

inis

trat

ion

wer

e st

udie

d in

mic

e, r

ats,

and

ferr

ets.

The

abs

orpt

ion

of II

I in

PEG

400

0 an

d II

I-Ph

os a

queo

us s

olut

ion

incr

ease

d ab

out

thre

e an

d fo

ur fo

ld in

AU

C a

fter

ora

l adm

inis

trat

ion

to u

ninf

ecte

d m

ice

com

pare

d w

ith th

at o

f 0.5

% H

PMC

(hyd

roxy

-pr

opyl

-met

hylc

ellu

lose

) sus

pens

ion.

Usi

ng a

mou

se in

fluen

za v

irus

infe

ctio

n m

odel

, sig

nific

ant a

nti-

influ

enza

-vir

us

activ

ity w

as o

bser

ved

in in

fect

ed m

ice

trea

ted

oral

ly w

ith th

ese

com

poun

ds d

isso

lved

in P

EG 4

000

or d

istil

led

wat

er,

resp

ectiv

ely,

but

not

in m

ice

trea

ted

with

0.5

% H

PMC

. The

in-v

ivo

anti-

influ

enza

-vir

us a

ctiv

ity in

ferr

ets,

a g

ood

mod

el fo

r in

fluen

za v

irus

infe

ctio

n in

man

, was

als

o st

udie

d. A

lthou

gh th

e co

ncen

trat

ion

of II

I in

plas

ma

was

abo

ve

the

IC50

aga

inst

the

influ

enza

vir

us s

trai

n us

ed fo

r 6h

aft

er th

e or

al a

dmin

istr

atio

n of

III i

n PE

G 4

00 to

uni

nfec

ted

ferr

ets,

no

in-v

ivo

antii

nflu

enza

-vir

us a

ctiv

ity w

as o

bser

ved

at th

e sa

me

dosa

ge g

iven

4 ti

mes

dai

ly fo

r 3

days

. The

in

tran

asal

adm

inis

trat

ion

of II

I-Ph

os, w

hich

was

exp

ecte

d to

hav

e a

mor

e no

tabl

e in

-viv

o an

ti-in

fluen

za-v

irus

act

iv-

ity, w

as e

xam

ined

. III

-Pho

s, w

hose

intr

anas

al a

bsor

ptio

n ha

d be

en im

prov

ed b

y th

e m

odifi

catio

n of

III w

ith p

hosp

hate

es

ter

in r

ats,

inhi

bite

d vi

ral r

eplic

atio

n in

the

nasa

l cav

ity a

nd s

uppr

esse

d in

fluen

za-v

irus

-ind

uced

feve

r w

hen

adm

inis

-te

red

intr

anas

ally

to in

fect

ed fe

rret

s. T

his

stud

y de

mon

stra

tes

that

intr

anas

ally

adm

inis

tere

d co

mpo

unds

with

ant

i-in

fluen

za-v

irus

act

ivity

mus

t per

mea

te th

e na

sal m

embr

anes

to p

rodu

ce th

eir

anti-

influ

enza

-vir

us e

ffect

.

Yosh

imot

o et

al.

2000

Chi

cken

egg

mod

elC

ompa

rativ

e st

udy

Am

anta

dine

Rim

anta

dine

Ose

ltam

ivir

Z

anam

ivir

Prev

ious

stud

ies h

ave

show

n th

at e

mbr

yona

ted

egg

prov

ides

a c

onve

nien

t and

eas

y to

use

syst

em fo

r in

vivo

scre

enin

g of

an

ti-in

fluen

za v

irus

inhi

bito

rs. H

owev

er, i

t is n

ot k

now

n w

heth

er th

is m

odel

is su

itabl

e fo

r tes

ting

neur

amin

idas

e (N

A)

inhi

bito

rs, t

oo. Th

eref

ore,

the

pres

ent s

tudy

des

crib

es th

e ev

alua

tion

of th

e io

n-ch

anne

l blo

cker

s am

anta

dine

and

rim

an-

tadi

ne in

com

pari

son

with

the

NA

inhi

bito

rs o

selta

miv

ir a

nd z

anam

ivir

by

usin

g th

e in

fluen

za A

vir

us h

en’s

egg

mod

el.

The

trea

tmen

t was

star

ted

imm

edia

tely

bef

ore

or a

fter

the

chal

leng

e do

se w

as p

lace

d on

the

chor

ioal

lant

oic

mem

bran

e (C

AM

). D

iffer

ence

s bet

wee

n th

e su

rviv

al ra

te o

f tre

ated

and

unt

reat

ed c

hick

em

bryo

s inf

ecte

d w

ith in

fluen

za A

vir

us

wer

e an

alyz

ed st

atis

tical

ly. A

s res

ult,

the

surv

ival

rate

of c

hick

em

bryo

s cou

ld b

e si

gnifi

cant

ly in

crea

sed

whe

n th

e tr

eat-

men

t with

am

anta

dine

, rim

anta

dine

, ose

ltam

ivir,

or z

anam

ivir

was

star

ted

befo

re th

e C

AM

was

inoc

ulat

ed w

ith o

ne e

gg

infe

ctiv

e do

se 5

0% (E

ID50

) infl

uenz

a A

vir

us. W

hen

the

drug

s wer

e ad

min

iste

red

shor

tly a

fter

vir

al in

ocul

atio

n, si

gnifi

-ca

nt a

ntiv

iral

effi

cacy

was

show

n fo

r rim

anta

dine

, ose

ltam

ivir,

and

zan

amiv

ir. A

ntiv

iral

effi

cacy

cou

ld b

e de

mon

stra

ted

excl

usiv

ely

for b

oth

osel

tam

ivir

and

zan

amiv

ir a

fter

the

embr

yos w

ere

infe

cted

with

hig

her c

halle

nge

dose

s of 1

02 EID

50

influ

enza

A v

irus

. In

conc

lusi

on, t

he N

A in

hibi

tors

ose

ltam

ivir

and

zan

amiv

ir h

ave

a si

gnifi

cant

ly b

ette

r ant

ivir

al a

ctiv

ity

agai

nst i

nflue

nza

A v

irus

than

am

anta

dine

and

rim

anta

dine

test

ed in

em

bryo

nate

d he

n’s e

ggs.

Ther

efor

e, th

is m

odel

can

be

a v

alua

ble

alte

rnat

ive

appr

oach

for i

n vi

vo p

re-t

estin

g an

ti-in

fluen

za v

irus

act

ivity

of N

A in

hibi

tors

.

Saue

rbre

i et a

l. 20

06


139

Phar

mac

okin

etic

stu

dies

Ose

ltam

ivir

Bioa

vaila

bilit

yRe

nal c

lear

ance

Stea

dy-s

tate

pla

sma

conc

entr

atio

n

Ose

ltam

ivir

is a

n et

hyl e

ster

pro

drug

of P

o 64

-080

2, a

sel

ectiv

e in

hibi

tor

of in

fluen

za v

irus

neu

ram

inid

ase.

Ora

l ad

min

istr

atio

n of

ose

ltam

ivir

del

iver

s th

e ac

tive

antiv

iral

Po

64-0

802

to th

e bl

oods

trea

m, a

nd th

us a

ll si

tes

of in

fluen

za

infe

ctio

n (lu

ng, n

asal

muc

osa,

mid

dle

ear)

are

acc

essi

ble.

The

pha

rmac

okin

etic

pro

file

of o

selta

miv

ir is

sim

ple

and

pre-

dict

able

, and

twic

e da

ily tr

eatm

ent r

esul

ts in

effe

ctiv

e an

tivir

al p

lasm

a co

ncen

trat

ions

ove

r th

e en

tire

adm

inis

trat

ion

inte

rval

. Aft

er o

ral a

dmin

istr

atio

n, o

selta

miv

ir is

read

ily a

bsor

bed

from

the

gast

roin

test

inal

trac

t and

ext

ensi

vely

con

-ve

rted

to th

e ac

tive

met

abol

ite. T

he a

bsol

ute

bioa

vaila

bilit

y of

the

activ

e m

etab

olite

from

ora

lly a

dmin

iste

red

osel

tam

i-vi

r is

80%

. The

act

ive

met

abol

ite is

det

ecta

ble

in p

lasm

a w

ithin

30

min

utes

and

reac

hes

max

imal

con

cent

ratio

ns a

fter

3

to 4

hou

rs. A

fter

pea

k pl

asm

a co

ncen

trat

ions

are

att

aine

d, th

e co

ncen

trat

ion

of th

e ac

tive

met

abol

ite d

eclin

es w

ith

an a

ppar

ent h

alf-

life

of 6

to 1

0 ho

urs.

Ose

ltam

ivir

is e

limin

ated

pri

mar

ily b

y co

nver

sion

to a

nd re

nal e

xcre

tion

of th

e ac

tive

met

abol

ite. R

enal

cle

aran

ce o

f bot

h co

mpo

unds

exc

eeds

glo

mer

ular

filtr

atio

n ra

te, i

ndic

atin

g th

at re

nal t

ubul

ar

secr

etio

n co

ntri

bute

s to

thei

r el

imin

atio

n vi

a th

e an

ioni

c pa

thw

ay. N

eith

er c

ompo

und

inte

ract

s w

ith c

ytoc

hrom

e P4

50

mix

ed-f

unct

ion

oxid

ases

or

gluc

uron

osyl

tran

sfer

ases

. The

pha

rmac

okin

etic

pro

file

of th

e ac

tive

met

abol

ite is

line

ar

and

dose

-pro

port

iona

l, w

ith le

ss th

an 2

-fol

d ac

cum

ulat

ion

over

a d

osag

e ra

nge

of o

selta

miv

ir 5

0 to

500

mg

twic

e da

ily.

Stea

dy-s

tate

pla

sma

conc

entr

atio

ns a

re a

chie

ved

with

in 3

day

s of

twic

e da

ily a

dmin

istr

atio

n, a

nd a

t a d

osag

e of

75m

g tw

ice

daily

the

stea

dy-s

tate

pla

sma

trou

gh c

once

ntra

tions

of a

ctiv

e m

etab

olite

rem

ain

abov

e th

e m

inim

um in

hibi

tory

co

ncen

trat

ion

for

all i

nflu

enza

. str

ains

test

ed. E

xpos

ure

to th

e ac

tive

met

abol

ite a

t ste

ady

stat

e is

app

roxi

mat

ely

25%

hi

gher

in e

lder

ly c

ompa

red

with

you

ng in

divi

dual

s; h

owev

er, n

o do

sage

adj

ustm

ent i

s ne

cess

ary.

In p

atie

nts

with

rena

l im

pair

men

t, m

etab

olite

cle

aran

ce d

ecre

ases

line

arly

with

cre

atin

ine

clea

ranc

e. A

dos

age

redu

ctio

n to

75m

g on

ce d

aily

is

reco

mm

ende

d fb

r pa

tient

s w

ith c

reat

inin

e cl

eara

nce

<30

ml/

min

(1.8

l/h)

. The

pha

rmac

okin

etic

s in

pat

ient

s w

ith

influ

enza

are

qua

litat

ivel

y si

mila

r to

thos

e in

hea

lthy

youn

g ad

ults

. In

vitr

o an

d in

viv

o st

udie

s in

dica

te n

o cl

inic

ally

si

gnifi

cant

dru

g in

tera

ctio

ns. N

eith

er p

arac

etam

ol (a

ceta

min

ophe

n) n

or c

imet

idin

e al

tere

d th

e ph

arm

acok

inet

ics

of

Ro 6

4-08

02. C

oadm

inis

trat

ion

of p

robe

neci

d re

sulte

d in

a 2

.5-f

old

incr

ease

in e

xpos

ure

to R

o 64

-080

2, h

owev

er, t

his

com

petit

ion

is u

nlik

ely

to re

sult

in c

linic

ally

rele

vant

effe

cts.

The

se p

rope

rtie

s m

ake

osel

tam

ivir

a. s

uita

ble

cand

idat

e fo

r us

e in

the

prev

entio

n an

d tr

eatm

ent o

f inf

luen

za.

He

et a

l. 19

99

Mul

tice

nter

clin

ical

st

udy

Zan

amiv

irA

ntiv

iral

act

ivity

Safe

tyTo

lera

bilit

yC

hina

201

0–20

11A

dole

scen

ts/a

dults

Back

grou

nd It

is th

e fir

st m

ultic

ente

r cl

inic

al s

tudy

in C

hina

to in

vest

igat

e za

nam

ivir

use

am

ong

Chi

nese

ado

lesc

ents

an

d ad

ults

with

influ

enza

-lik

e ill

ness

(ILI

) sin

ce 2

009,

whe

n in

hale

d za

nam

ivir

(REL

ENZ

A (R

)) w

as m

arke

ted

in

Chi

na. M

etho

ds A

n un

cont

rolle

d op

en-l

abel

, mul

ticen

tre

stud

y to

eva

luat

e th

e an

tivir

al a

ctiv

ity, a

nd s

afet

y of

inha

led

zana

miv

ir (a

s Ro

tadi

sk v

ia D

iskh

aler

dev

ice)

; 10

mg

adm

inis

tere

d tw

ice

daily

for

5 da

ys in

sub

ject

s ≥

12 y

ears

old

w

ith IL

I. Pa

tient

s w

ere

enro

lled

with

in 4

8 ho

urs

of o

nset

and

follo

wed

for

eigh

t day

s. P

atie

nts

wer

e de

fined

as

bein

g in

fluen

za-p

ositi

ve if

the

real

-tim

e re

vers

e tr

ansc

ript

ase-

poly

mer

ase

chai

n re

actio

n (r

RT-P

CR)

test

had

pos

itive

resu

lts.

Resu

lts A

tota

l of 4

00 p

atie

nts

≥ 12

yea

rs o

ld w

ere

scre

ened

from

11

cent

ers

in s

even

pro

vinc

es fr

om M

arch

201

0 to

Ja

nuar

y 20

11. T

hree

hun

dred

and

nin

ety-

two

patie

nts

who

took

at l

east

one

dos

e of

zan

amiv

ir w

ere

ente

red

into

the

safe

ty a

naly

sis.

The

mea

n ag

e w

as 3

3.8

year

s an

d 50

% w

ere

mal

e. C

ardi

ovas

cula

r di

seas

es a

nd d

iabe

tes

wer

e th

e m

ost

com

mon

com

orbi

ditie

s. A

ll th

e re

port

ed a

dver

se e

vent

s, s

uch

as r

ash,

nas

al a

che,

mus

cle

ache

, nau

sea,

dia

rrhe

a, h

ead-

ache

, occ

urre

d in

less

than

1%

of s

ubje

cts.

Mild

sin

us b

rady

cadi

a or

arr

hyth

mia

occ

urre

d in

four

sub

ject

s (1

%).

Mos

t of

the

adve

rse

even

ts w

ere

mild

and

did

not

requ

ire

any

chan

ge o

f tre

atm

ent.

No

seve

re a

dver

se e

vent

s (S

AE)

or

fata

l ca

ses

wer

e re

port

ed. B

ronc

hosp

asm

was

foun

d in

a 3

8 ye

ars

old

wom

an w

hose

sym

ptom

s di

sapp

eare

d af

ter

stop

ping

za

nam

ivir

and

with

out a

dditi

onal

trea

tmen

t. A

ll th

e 61

influ

enza

vir

us is

olat

es (4

3 be

fore

enr

ollm

ent,

18 d

urin

g tr

eat-

men

t) p

rove

d to

be

sens

itive

to z

anam

ivir.

Con

clus

ions

Zan

amiv

ir is

wel

l tol

erat

ed b

y C

hine

se a

dole

scen

ts a

nd a

dults

w

ith IL

Is. T

here

is n

o ev

iden

ce fo

r th

e em

erge

nce

of d

rug-

resi

stan

t iso

late

s du

ring

trea

tmen

t with

zan

amiv

ir.

Cao

et a

l. 20

12


140

Tol

erab

ility

/ph

arm

acok

inet

ics

Neu

ram

inid

ase

inhi

bito

rsA

dult/

elde

rly

volu

ntee

rsSt

eady

-sta

te p

lasm

a co

ncen

trat

ions

The

tole

rabi

lity

and

phar

mac

okin

etic

s of

Ro

64-0

802,

a p

oten

t, se

lect

ive

inhi

bito

r of

influ

enza

neu

ram

inid

ase,

and

ifs

oral

pro

drug

ose

ltam

ivir

wer

e in

vest

igat

ed in

thre

e do

uble

-blin

d, p

lace

bo-c

ontr

olle

d st

udie

s. T

wo

stud

ies

invo

lved

he

alth

y ad

ult v

olun

teer

s (1

8–55

yea

rs) (

n =

48) w

ho re

ceiv

ed s

ingl

e (2

0–10

00 m

g) o

r bi

d do

ses

(50–

500

mg)

(n =

32)

of

ose

ltam

ivir

or

plac

ebo

for

7 da

ys. H

ealth

y el

derl

y vo

lunt

eers

(gre

ater

than

or

equa

l to

65 y

ears

) (n

= 24

) rec

eive

d os

elta

miv

ir 1

00 to

200

mg

bid

or p

lace

bo fo

r 7

days

in a

thir

d st

udy.

Mea

sura

ble

plas

ma

conc

entr

atio

ns o

f the

act

ive

met

abol

ite a

ppea

red

rapi

dly

in p

lasm

a an

d w

ere

sign

ifica

ntly

hig

her

and

long

er la

stin

g th

an th

ose

of o

selta

miv

ir.

Phar

mac

okin

etic

s of

bot

h co

mpo

unds

wer

e lin

ear.

Mul

tiple

-dos

e ex

posu

re w

as p

redi

ctab

le fr

om s

ingl

e-do

se d

ata,

and

st

eady

-sta

te p

lasm

a co

ncen

trat

ions

wer

e ac

hiev

ed w

ithin

3 d

ays

of b

id d

rug

adm

inis

trat

ion.

Ose

ltam

ivir

was

wel

l tol

er-

ated

at s

ingl

e do

ses

of u

p to

100

0 m

g an

d tw

ice-

daily

dos

es o

f up

to 5

00 m

g. A

dver

se e

vent

s w

ere

mild

in in

tens

ity.

Expo

sure

to b

oth

prod

rug

and

activ

e m

etab

olite

was

incr

ease

d in

eld

erly

pat

ient

s by

app

roxi

mat

ely

25%

. How

ever

, due

to

the

wid

e sa

fety

mar

gin

of b

oth

com

poun

ds, n

o do

se a

djus

tmen

t is

nece

ssar

y fo

r el

derl

y pa

tient

s.

Mas

sare

lla e

t al.

2000

Tabl

e 4.

1. In

hibi

tors

of h

aem

aggl

utin

in

Hae

mag

glut

inin

X-r

ay c

ryst

alog

raph

yRe

cept

or b

indi

ng

Hem

aggl

utin

in (H

A) i

s th

e re

cept

or-b

indi

ng a

nd m

embr

ane

fusi

on g

lyco

prot

ein

of in

fluen

za v

irus

and

the

targ

et fo

r in

fect

ivity

-neu

tral

izin

g an

tibod

ies.

The

str

uctu

res

of th

ree

conf

orm

atio

ns o

f the

ect

odom

ain

of th

e 19

68 H

ong

Kon

g in

fluen

za v

irus

HA

hav

e be

en d

eter

min

ed b

y X

-ray

cry

stal

logr

aphy

: the

sin

gle-

chai

n pr

ecur

sor,

HA

0; th

e m

etas

tabl

e ne

utra

l-pH

con

form

atio

n fo

und

on v

irus

, and

the

fusi

on p

H-i

nduc

ed c

onfo

rmat

ion.

The

se s

truc

ture

s pr

ovid

e a

fram

e-w

ork

for

desi

gnin

g an

d in

terp

retin

g th

e re

sults

of e

xper

imen

ts o

n th

e ac

tivity

of H

A in

rece

ptor

bin

ding

, the

gen

era-

tion

of e

mer

ging

and

reem

ergi

ng e

pide

mic

s, a

nd m

embr

ane

fusi

on d

urin

g vi

ral e

ntry

.

Skeh

el a

nd W

iley

2000

Sial

ylgl

ycan

sM

olec

ular

dyn

amic

s si

mul

atio

nH

aem

aggl

utin

in-r

ecep

tor

inte

ract

ion

Reco

gniti

on o

f cel

l-su

rfac

e si

alyl

disa

ccha

ride

s by

influ

enza

A h

emag

glut

inin

(HA

) tri

gger

s th

e in

fect

ion

proc

ess

of

influ

enza

. The

cha

nges

in g

lyco

sidi

c to

rsio

nal l

inka

ge a

nd th

e re

cept

or c

onfo

rmat

ions

may

alte

r th

e bi

ndin

g sp

ecifi

city

of

HA

s to

the

sial

ylgl

ycan

s. In

this

stu

dy, 1

0-ns

mol

ecul

ar d

ynam

ics

sim

ulat

ions

wer

e ca

rrie

d ou

t to

exam

ine

the

stru

c-tu

ral a

nd d

ynam

ic b

ehav

ior

of th

e H

As

boun

d w

ith s

ialy

ldis

acch

arid

es N

eu5A

c al

pha(

2-3)

Gal

(N23

G) a

nd N

eu5A

c al

pha(

2-6)

Gal

(N26

G).

The

ana

lysi

s of

the

glyc

osid

ic to

rsio

nal a

ngle

s an

d th

e pa

ir in

tera

ctio

n en

ergy

bet

wee

n th

e re

cept

or a

nd th

e in

tera

ctin

g re

sidu

es o

f the

bin

ding

site

reve

al th

at N

23G

has

two

bind

ing

mod

es fo

r H

1 an

d H

5 an

d a

sing

le b

indi

ng m

ode

for

H3

and

H9.

For

N26

G, H

1 an

d H

3 ha

s tw

o bi

ndin

g m

odes

, and

H5

and

H9

has

a si

ngle

bin

d-in

g m

ode.

The

dir

ect a

nd w

ater

-med

iate

d hy

drog

en b

ondi

ng in

tera

ctio

ns b

etw

een

the

rece

ptor

s an

d H

As

play

dom

i-na

nt ro

les

in th

e st

ruct

ural

sta

biliz

atio

n of

the

com

plex

es. I

t is

conc

lude

d fr

om p

air

inte

ract

ion

ener

gy a

nd M

olec

ular

M

echa

nic-

Pois

son-

Boltz

-man

n Su

rfac

e A

rea

calc

ulat

ions

that

N26

G is

a b

ette

r re

cept

or fo

r H

1 w

hen

com

pare

d w

ith

N23

G. N

23G

is a

bet

ter

rece

ptor

for

H5

whe

n co

mpa

red

with

N26

G. H

owev

er, H

3 an

d H

9 ca

n re

cogn

ize

N23

G a

nd

N26

G in

equ

al b

indi

ng s

peci

ficity

due

to th

e m

argi

nal e

nerg

y di

ffere

nce

(app

roxi

mat

e to

2.5

kca

l/m

ol).

The

ord

er o

f bi

ndin

g sp

ecifi

city

of N

23G

is H

3 >

H5

> H

9 >

H1

and

N26

G is

H1

> H

3 >

H5

> H

9, re

spec

tivel

y. T

he p

ropo

sed

con-

form

atio

nal m

odel

s w

ill b

e he

lpfu

l in

desi

gnin

g in

hibi

tors

for

influ

enza

vir

us.

Priy

adar

zini

et a

l. 20

12


141

Sial

ylla

ctos

e-co

ntai

ning

gl

ycop

olym

ers

Che

moe

nzym

atic

sy

nthe

sis

Gly

co-b

iose

nsor

Gly

coar

ray

We

repo

rt a

che

moe

nzym

atic

syn

thes

is o

f cha

in-e

nd fu

nctio

naliz

ed s

ialy

llact

ose-

cont

aini

ng g

lyco

poly

mer

s w

ith d

iffer

-en

t lin

kage

s an

d th

eir

orie

nted

imm

obili

zatio

n fo

r gl

ycoa

rray

and

SPR

-bas

ed g

lyco

-bio

sens

or a

pplic

atio

ns. S

peci

fical

ly,

O-c

yana

te c

hain

-end

func

tiona

lized

sia

lylla

ctos

e-co

ntai

ning

gly

copo

lym

ers

wer

e sy

nthe

size

d by

enz

ymat

ic a

2,3-

and

al

pha

2,6-

sial

ylat

ion

of a

lact

ose-

cont

aini

ng g

lyco

poly

mer

that

was

syn

thes

ized

by

cyan

oxyl

-med

iate

d fr

ee r

adic

al

poly

mer

izat

ion.

H-1

NM

R sh

owed

alm

ost q

uant

itativ

e al

pha

2,3-

and

alp

ha 2

,6-s

ialy

latio

n. T

he O

-cya

nate

cha

in-e

nd

func

tiona

lized

sia

lylla

ctos

e-co

ntai

ning

gly

copo

lym

ers

wer

e pr

inte

d on

to a

min

e-fu

nctio

naliz

ed g

lass

slid

es v

ia is

oure

a bo

nd fo

rmat

ion

for

glyc

oarr

ay fo

rmat

ion.

Spe

cific

pro

tein

bin

ding

act

ivity

of t

he a

rray

s w

as c

onfir

med

with

alp

ha 2

,3-

and

alph

a 2,

6-si

alyl

spe

cific

bin

ding

lect

ins

toge

ther

with

inhi

bitio

n as

says

. Fur

ther

, im

mob

ilizi

ng O

-cya

nate

cha

in-

end

func

tiona

lized

sia

lylla

ctos

e-co

ntai

ning

gly

copo

lym

ers

onto

am

ine-

mod

ified

SPR

chi

p vi

a is

oure

a bo

nd fo

rmat

ion

affo

rded

SPR

-bas

ed g

lyco

-bio

sens

or, w

hich

sho

wed

spe

cific

bin

ding

act

ivity

for

lect

ins

and

influ

enza

vir

al h

emag

glu-

tinin

s (H

A).

The

se s

ialy

lolig

o-m

acro

ligan

d de

rive

d gl

ycoa

rray

and

SPR

-bas

ed g

lyco

-bio

sens

or a

re c

lose

ly to

mim

ic 3

D

natu

re p

rese

ntat

ion

of s

ialy

lolig

osac

char

ides

and

will

pro

vide

impo

rtan

t hig

h-th

roug

hput

tool

s fo

r vi

rus

diag

nosi

s an

d po

tent

ial a

ntiv

iral

dru

g ca

ndid

ates

scr

eeni

ng a

pplic

atio

ns.

Nar

la a

nd S

un

2012

Sial

ylla

ctos

eSt

ruct

ural

ana

lysi

sC

ompu

ter s

imul

atio

n

We

desi

gned

and

syn

thes

ized

nov

el tr

ival

ent a

nti-

influ

enza

reag

ents

. Sia

lylla

ctos

e w

as lo

cate

d at

the

term

inal

of e

ach

vale

nce

whi

ch a

imed

to b

lock

eac

h re

cept

or-b

indi

ng s

ite o

f the

hem

aggl

utin

in (H

A) t

rim

er o

n th

e su

rfac

e of

the

viru

s.

Stru

ctur

al a

naly

ses

wer

e ca

rrie

d ou

t with

a m

odel

whi

ch w

as c

onst

ruct

ed w

ith a

com

pute

r si

mul

atio

n. A

pre

viou

sly

repo

rted

cyc

lic g

lyco

pept

ide

bloc

ker

boun

d to

the

HA

in th

e m

odel

. The

ana

lyse

s su

gges

t tha

t the

glu

tam

ine

resi

due

in th

e cy

clic

pep

tide

bear

ing

Neu

5A a

lpha

2,3

Gal

bet

a 1,

4Glc

tris

acch

arid

e vi

a a

linke

r in

tera

cts

with

the

Gln

189

in

HA

thro

ugh

hydr

ogen

bon

ding

. The

pre

sent

ant

i-in

fluen

za re

agen

ts li

kely

inte

ract

with

a g

luta

min

e re

sidu

e in

clud

ed

in th

e vi

cini

ty o

f Gln

189.

A p

lagu

e re

duct

ion

assa

y of

the

influ

enza

vir

us, A

/PR/

8/19

34 (H

1N1)

, was

per

form

ed in

M

DC

K c

ells

to e

valu

ate

for

the

synt

hesi

zed

com

poun

ds to

inhi

bit v

iral

repl

icat

ion.

One

of t

he c

ompo

unds

sho

wed

ap

prox

imat

ely

85%

inhi

bitio

n at

the

conc

entr

atio

n of

400

mu

M a

t 4 d

egre

es °C

.

Feng

et a

l. 20

10

Sial

ic a

cid

anal

ogue

sC

2-, C

4- a

nd

C5-

subs

titut

ions

M

olec

ular

doc

king

Aut

oDoc

k3.0

5

A m

olec

ular

doc

king

tool

of A

utoD

ock3

.05

was

eva

luat

ed fo

r its

abi

lity

to re

prod

uce

expe

rim

enta

lly d

eter

min

ed a

ffin

i-tie

s of

var

ious

sia

lic a

cid

anal

ogue

s to

war

d he

mag

glut

inin

of i

nflu

enza

A v

irus

. With

the

exce

ptio

n of

thos

e w

ith a

C

6-m

odifi

ed g

lyce

rol s

ide

chai

n, th

e ex

peri

men

tal b

indi

ng a

ffin

ities

of m

ost s

ialic

aci

d an

alog

ues

(C2,

C4

and

C5-

sub-

stitu

ted)

det

erm

ined

by

vira

l hem

adso

rptio

n in

hibi

tion

assa

y, h

emag

glut

inat

ion

inhi

bitio

n as

say

and

nucl

ear

mag

netic

re

sona

nce

corr

elat

ed w

ell w

ith th

e co

mpu

tatio

nally

est

imat

ed fr

ee e

nerg

y of

bin

ding

. Sia

lic a

cid

anal

ogue

s w

ith m

odi-

fied

glyc

erol

sid

e ch

ains

sho

wed

onl

y po

or c

orre

latio

n be

twee

n th

e ex

peri

men

tally

det

erm

ined

hem

aggl

utin

in in

hibi

-to

r af

finiti

es a

nd A

utoD

ock3

.05

scor

es, s

ugge

stin

g hi

gh m

obili

ty o

f the

glu

tam

ic a

cid

side

cha

in a

t the

gly

cero

l bin

ding

po

cket

, whi

ch is

diff

icul

t to

sim

ulat

e us

ing

a fle

xi-r

igid

mol

ecul

ar d

ocki

ng a

ppro

ach.

In c

oncl

usio

n, e

xcep

t for

som

e gl

ycer

ol-s

ubst

itute

d si

alic

aci

d an

alog

ues,

the

resu

lts s

how

ed th

e ef

fect

iven

ess

of A

utoD

ock3

.05

sear

chin

g an

d sc

orin

g fu

nctio

ns in

est

imat

ing

affin

ities

of s

ialic

aci

d an

alog

ues

tow

ard

influ

enza

A h

emag

glut

inin

, mak

ing

it a

relia

ble

tool

fo

r sc

reen

ing

a da

taba

se o

f vir

tual

ly d

esig

ned

sial

ic a

cid

anal

ogue

s fo

r he

mag

glut

inin

inhi

bito

rs.

Al-q

atta

n an

d M

ordi

201

0a


142

Sial

ic a

cid

anal

ogue

sC

2-, C

5- a

nd

C6-

subs

titut

ions

Fr

agm

ent-

base

d dr

ug

desi

gn

In th

is st

udy

frag

men

t-ba

sed

drug

des

ign

is c

ombi

ned

with

mol

ecul

ar d

ocki

ng si

mul

atio

n te

chni

que,

to d

esig

n da

taba

ses

of v

irtu

al si

alic

aci

d (S

A) a

nalo

gues

with

new

subs

titut

ions

at C

2, C

5 an

d C

6 po

sitio

ns o

f SA

scaff

old.

Usi

ng sp

aces

occ

u-pi

ed b

y C

2, C

5 an

d C

6 na

tura

l moi

etie

s of S

A w

hen

boun

d to

hem

aggl

utin

in (H

A) c

ryst

allo

grap

hic

stru

ctur

e, n

ew fr

ag-

men

ts th

at a

re c

omm

erci

ally

ava

ilabl

e w

ere

dock

ed in

depe

nden

tly in

all

the

pock

ets.

The

orie

nted

frag

men

ts w

ere

then

co

nnec

ted

to th

e SA

scaff

old

with

or w

ithou

t inc

orpo

ratio

n of

link

er m

olec

ules

. The

com

plet

ed a

nalo

gues

wer

e do

cked

to

the

who

le S

A b

indi

ng si

te to

est

imat

e th

eir b

indi

ng c

onfo

rmat

ions

and

affi

nitie

s, g

ener

atin

g th

ree

data

base

s of H

A-b

ound

SA

ana

logu

es. S

elec

ted

new

ana

logu

es sh

owed

hig

her e

stim

ated

affi

nitie

s tha

n th

e na

tura

l SA

whe

n te

sted

aga

inst

H3N

2,

H5N

1 an

d H

1N1

subt

ypes

of i

nflue

nza

A. A

n im

prov

emen

t in

the

bind

ing

ener

gies

indi

cate

s tha

t fra

gmen

t-ba

sed

drug

de

sign

whe

n co

mbi

ned

with

mol

ecul

ar d

ocki

ng si

mul

atio

n is

cap

able

to p

rodu

ce v

irtu

al a

nalo

gues

that

can

bec

ome

lead

co

mpo

und

cand

idat

es fo

r ant

i-flu

drug

dis

cove

ry p

rogr

am.

Al-q

atta

n an

d M

ordi

201

0b

Sial

ic a

cid

anal

ogue

sPo

lyac

ryla

mid

e co

njug

ate

Poly

vale

nt in

hibi

tor

An

ELIS

A a

ssay

is d

escr

ibed

for m

easu

ring

the

bind

ing

of in

fluen

za v

irus

A-X

31 to

alp

ha-s

ialo

side

gro

ups t

hat a

re li

nked

to

bio

tin-la

bele

d po

lyac

ryla

mid

es. Th

e effi

cacy

of t

hese

pol

ymer

s in

inhi

bitin

g th

e ad

hesi

on o

f infl

uenz

a vi

rus t

o er

ythr

o-cy

tes (

as m

easu

red

by a

hem

aggl

utin

atio

n as

say)

was

show

n to

be

dire

ctly

rela

ted

to th

e bi

ndin

g affi

nity

of t

he p

olym

ers

for t

he v

iral

surf

ace:

the

diffe

renc

es in

inhi

bito

ry e

ffica

cy a

mon

g th

e po

lym

eric

inhi

bito

rs a

nd m

onom

eric

alp

ha-m

ethy

l si

alos

ide,

am

ong

frac

tions

of a

pol

ymer

ic, p

olyv

alen

t inh

ibito

r with

nar

row

mol

ecul

ar w

eigh

t ran

ges,

and

am

ong

poly

-m

eric

inhi

bito

rs p

repa

red

by c

opol

ymer

izat

ion

or m

odifi

catio

n of

a p

refo

rmed

pol

ymer

cha

in, a

ll co

rrel

ated

with

diff

er-

ence

s in

the

affini

ty o

f the

inhi

bito

rs fo

r the

surf

ace

of th

e vi

rus,

The

poly

mer

ic in

hibi

tors

stud

ied

had

affini

ties f

or th

e vi

ral s

urfa

ce th

at ra

nged

bet

wee

n 10

3 and

> 1

06 gre

ater

than

alp

ha-m

ethy

l sia

losi

de, o

n th

e ba

sis o

f tot

al si

alic

aci

d gr

oups

in

solu

tion,

The

role

of s

teri

c st

abili

zatio

n in

the

mec

hani

sm b

y w

hich

thes

e po

lym

ers i

nhib

it he

mag

glut

inat

ion

was

in

vest

igat

ed, Th

e ab

ility

of t

he p

olym

eric

, pol

yval

ent i

nhib

itors

to in

hibi

t the

bin

ding

of a

pol

yclo

nal a

ntib

ody

to th

e vi

ral

surf

ace

sugg

ests

that

ster

ic st

abili

zatio

n m

ay a

lso

be a

n im

port

ant e

ffect

in th

is sy

stem

.

Siga

l et a

l. 19

96

Qui

none

mol

ecul

esBi

cycl

ic q

uino

nes

Poly

mer

ic in

hibi

tors

Influ

enza

A v

irus

Usi

ng th

e pl

aque

redu

ctio

n as

say,

rela

tivel

y si

mpl

e bi

cycl

ic q

uino

ne m

olec

ules

, as w

ell a

s mul

tiple

cop

ies t

here

of c

ova-

lent

ly a

ttac

hed

to a

long

pol

yglu

tam

ate-

base

d po

lym

eric

cha

in, w

ere

exam

ined

as n

ew in

hibi

tors

of v

ario

us n

atur

ally

oc

curr

ing

stra

ins o

f infl

uenz

a A

vir

us. Th

e po

lym

er-c

onju

gate

d in

hibi

tors

wer

e fo

und

to h

ave

a fa

r gre

ater

pot

ency

(for

so

me

as h

igh

as tw

o or

ders

of m

agni

tude

whe

n a

long

spac

er a

rm w

as e

mpl

oyed

) tha

n th

eir c

orre

spon

ding

par

ent m

ol-

ecul

es a

gain

st th

e hu

man

Wuh

an in

fluen

za st

rain

. How

ever

, suc

h po

lym

eric

inhi

bito

rs fa

iled

to e

xhib

it hi

gher

pot

ency

co

mpa

red

with

thei

r sm

all m

olec

ule

pred

eces

sors

aga

inst

the

hum

an P

uert

o Ri

co a

nd a

vian

turk

ey in

fluen

za st

rain

s. Th

ese

obse

rvat

ions

, fur

ther

exp

lore

d by

mea

ns o

f mol

ecul

ar m

odel

ing,

reve

al th

e pr

evio

usly

unr

ecog

nize

d un

pred

icta

bil-

ity o

f the

ben

efits

of m

ultiv

alen

cy, p

ossi

bly

beca

use

of p

oor a

cces

sibi

lity

of th

e vi

ral t

arge

ts to

pol

ymer

ic a

gent

s.

Lars

on e

t al.

2012

Ben

zoqu

inon

es,

hydr

oqui

none

sC

onfo

rmat

iona

l cha

nge

of

haem

aggl

utin

inV

irus

-cel

l mem

bran

e fu

sion

Past

effo

rts t

o em

ploy

a st

ruct

ure-

base

d ap

proa

ch to

des

ign

an in

hibi

tor o

f the

fusi

on-i

nduc

ing

conf

orm

atio

nal c

hang

e in

th

e in

fluen

za v

irus

hem

aggl

utin

in (H

A) y

ield

ed a

fam

ily o

f sm

all b

enzo

quin

ones

and

hyd

roqu

inon

es, Th

e m

ost p

oten

t of

thes

e, te

rt-b

utyl

hyd

roqu

inon

e (T

BHQ

), in

hibi

ts b

oth

the

conf

orm

atio

nal c

hang

e in

HA

from

stra

in X

:31

influ

enza

vir

us

and

vira

l inf

ectiv

ity in

tiss

ue c

ultu

re c

ells

with

50%

inhi

bito

ry c

once

ntra

tions

in th

e m

icro

mol

ar ra

nge.

A n

ew st

ruct

ure-

base

d in

hibi

tor d

esig

n se

arch

was

beg

un w

hich

invo

lved

the

rece

ntly

refin

ed c

ryst

al st

ruct

ure

(2.1

-Ang

stro

m re

solu

-tio

n) o

f the

HA

ect

odom

ain,

new

insi

ghts

into

the

conf

orm

atio

nal c

hang

e, a

nd im

prov

emen

ts in

the

mol

ecul

ar d

ocki

ng

prog

ram

, DO

CK

. As a

resu

lt, w

e id

entifi

ed n

ew in

hibi

tors

of H

A-m

edia

ted

mem

bran

e fu

sion

. Lik

e T

BHQ

, mos

t of t

hese

m

olec

ules

inhi

bit t

he c

onfo

rmat

iona

l cha

nge,

One

of t

he n

ew c

ompo

unds

, how

ever

, fac

ilita

tes r

athe

r tha

n in

hibi

ts th

e H

A c

onfo

rmat

iona

l cha

nge,

Non

ethe

less

, the

faci

litat

or, d

iiodo

fluor

esce

in, i

nhib

its H

A-m

edia

ted

mem

bran

e fu

sion

and

, ir

reve

rsib

ly, i

nfec

tivity

. We

furt

her c

hara

cter

ized

the

effec

ts o

f inh

ibito

rs fr

om b

oth

sear

ches

on

the

conf

orm

atio

nal

chan

ge a

nd m

embr

ane

fusi

on a

ctiv

ity o

f HA

as w

ell a

s on

vira

l inf

ectiv

ity. W

e al

so is

olat

ed a

nd c

hara

cter

ized

seve

ral

mut

ants

resi

stan

t to

each

cla

ss o

f inh

ibito

r. Th

e im

plic

atio

ns o

f our

resu

lts fo

r HA

-med

iate

d m

embr

ane

fusi

on, a

nti-

influ

enza

vir

us th

erap

y, an

d st

ruct

ure-

base

d in

hibi

tor d

esig

n ar

e di

scus

sed.

Hoff

man

et a

l. 19

97


143

new

hae

mag

glut

inin

in

hibi

tors

We

repo

rted

pre

viou

sly

that

a s

mal

l mol

ecul

e na

med

CL-

3853

19 c

ould

inhi

bit H

5N1

influ

enza

vir

us in

fect

ion

by

targ

etin

g he

mag

glut

inin

, the

env

elop

e pr

otei

n m

edia

ting

viru

s en

try.

In th

e pr

esen

t stu

dy, a

nov

el s

erie

s of

der

iva-

tives

focu

sed

on th

e st

ruct

ural

var

iatio

n of

CL-

3853

19 w

ere

synt

hesi

zed

as s

peci

fic in

hibi

tors

aga

inst

the

H5

subt

ype

of in

fluen

za A

vir

uses

. The

se s

mal

l mol

ecul

es in

hibi

ted

the

low

pH

-ind

uced

con

form

atio

nal c

hang

e of

hem

aggl

u-tin

in, t

here

by b

lock

ing

vira

l ent

ry in

to h

ost c

ells

. Com

poun

d 11

was

the

mos

t act

ive

inhi

bito

r in

this

ser

ies

with

an

IC50

of 0

.22

µM. T

he s

truc

ture

act

ivity

rela

tions

hips

ana

lysi

s of

thes

e co

mpo

unds

sho

wed

that

the

3-flu

oro-

5-(t

riflu

orom

ethy

l)ben

zam

ide

moi

ety

was

ver

y im

port

ant f

or a

ctiv

ity, a

nd th

e -F

gro

up w

as a

bet

ter

subs

titue

nt g

roup

th

an -C

F3 g

roup

in th

e ph

enyl

rin

g. T

he in

hibi

tory

act

ivity

was

sen

sitiv

e to

the

benz

amid

e be

caus

e th

e ox

ygen

and

hy

drog

en o

f the

am

ide

serv

ed a

s H

-bon

d ac

cept

or a

nd d

onor

, res

pect

ivel

y.

Zhu

et a

l. 20

12

Stac

hyfli

nC

onfo

rmat

iona

l cha

nge

of

haem

aggl

utin

in

Stac

hyfli

n is

a n

ovel

com

poun

d ha

ving

H1

and

H2

subt

ype-

spec

ific

anti-

influ

enza

A v

irus

act

ivity

. Sta

chyf

lin h

as n

o in

hibi

tion

on H

3 su

btyp

e in

fluen

za A

or

influ

enza

B v

irus

es. T

he s

usce

ptib

ility

of t

he re

asso

rtan

t vir

uses

bet

wee

n H

1 an

d H

3 su

btyp

e in

fluen

za A

vir

uses

to S

tach

yflin

indi

cate

d th

at it

s ta

rget

was

vir

us-e

ncod

ed h

emag

glut

inin

(HA

). T

he

resu

lts o

f the

tim

ing

of S

tach

yflin

add

ition

aga

inst

in v

itro

viru

s in

fect

ion

and

viru

s-m

edia

ted

hem

olys

is a

ssay

sug

-ge

sted

that

the

drug

inhi

bite

d th

e H

A-m

edia

ted

viru

s-ce

ll fu

sion

pro

cess

. Mor

e di

rect

ly, S

tach

yflin

inte

rfer

ed w

ith

HA

con

form

atio

nal c

hang

e in

duce

d by

low

pH

or

heat

trea

tmen

t. T

he e

ffect

of S

tach

yflin

cou

ld n

ot b

e el

imin

ated

by

was

hing

of t

he S

tach

yflin

-tre

ated

vir

us, w

hich

cau

sed

very

spe

cific

vir

ucid

al e

ffect

. Thi

s is

a re

mar

kabl

e pr

oper

ty

amon

g sm

all m

olec

ules

whi

ch in

hibi

t low

-pH

indu

ced

HA

con

form

atio

nal c

hang

e. F

rom

thes

e fin

ding

s, w

e co

nclu

ded

that

the

mec

hani

sm o

f Sta

chyf

lin a

ctio

n is

to in

hibi

t HA

con

form

atio

nal c

hang

e w

hich

is n

eces

sary

for

viru

s-ce

ll m

em-

bran

e fu

sion

. Sta

chyf

lin m

ay b

e us

ed a

s a

tool

for

a st

udy

of m

olec

ular

mec

hani

sm o

f low

-pH

indu

ced

HA

con

form

a-tio

nal c

hang

e, a

nd o

ffers

pot

entia

l as

a ph

arm

aceu

tical

age

nt.

Yosh

imot

o et

al.

1999

(+)-

stac

hyfli

nEn

antio

sele

ctiv

e sy

nthe

sis

A n

ovel

and

pot

ent h

emag

glut

inin

inhi

bito

r, (+

)-st

achy

flin,

was

eff

icie

ntly

syn

thes

ized

in a

n en

antio

sele

ctiv

e m

anne

r st

artin

g fr

om th

e (+

)-5-

met

hyl-W

iela

nd-M

iesc

her

keto

ne. T

he s

ynth

etic

met

hod

feat

ures

a B

F(3)

cent

er d

ot E

t(2)

O-i

nduc

ed c

asca

de e

poxi

de-o

peni

ng/r

earr

ange

men

t/cy

cliz

atio

n re

actio

n to

ste

reos

elec

tivel

y co

nstr

uct t

he re

quis

ite

pent

acyc

lic r

ing

syst

em in

one

ste

p. In

ord

er to

rat

iona

lize

the

mec

hani

sm o

f the

cas

cade

reac

tion,

qua

ntum

che

mic

al

calc

ulat

ions

of t

he p

ossi

ble

inte

rmed

iary

car

boca

tions

and

tran

sitio

n st

ates

in th

e m

odel

syn

thes

is w

ere

carr

ied

out.

An

alte

rnat

ive

appr

oach

to s

ynth

esiz

e (+

)-st

achy

flin

by e

mpl

oyin

g a

sim

ilar

casc

ade

reac

tion

was

als

o de

scri

bed.

Saku

rai e

t al.

2011

Ben

zam

id d

eriv

ativ

esV

irus

-cel

l mem

bran

e fu

sion

Sequ

ence

ana

lyse

sH

aem

aggl

utin

in g

ene

Dru

g re

sist

ance

In th

e in

itial

sta

ges

of in

fluen

za v

irus

infe

ctio

n, th

e he

mag

glut

inin

(HA

) pro

tein

of i

nflu

enza

vir

us m

edia

tes

both

ad

sorp

tion

and

pene

trat

ion

of th

e vi

rus

into

the

host

cel

l. Re

cent

ly, w

e id

entif

ied

and

char

acte

rize

d BM

Y-27

709

as

an in

hibi

tor

of th

e H

1 an

d H

2 su

btyp

es o

f inf

luen

za A

vir

us th

at s

peci

fical

ly in

hibi

ts th

e H

A fu

nctio

n ne

cess

ary

for

viru

s-ce

ll m

embr

ane

fusi

on. S

tudi

es p

rese

nted

her

ein

show

that

the

inhi

bitio

n is

med

iate

d th

roug

h sp

ecifi

c in

tera

ctio

n w

ith th

e H

A p

rote

in. T

his

bind

ing

repr

esse

s th

e lo

w-p

H-i

nduc

ed c

onfo

rmat

iona

l cha

nge

of th

e H

A p

rote

in w

hich

is

a pr

ereq

uisi

te fo

r m

embr

ane

fusi

on. I

n an

att

empt

to d

efin

e th

e bi

ndin

g po

cket

with

in th

e H

A m

olec

ule,

a n

umbe

r of

dr

ug-r

esis

tant

vir

uses

hav

e be

en is

olat

ed a

nd c

hara

cter

ized

. Seq

uenc

e an

alys

es o

f the

HA

gen

e of

thes

e dr

ug-r

esis

tant

vi

ruse

s m

appe

d am

ino

acid

cha

nges

resp

onsi

ble

for

drug

resi

stan

ce to

a re

gion

loca

ted

near

the

amin

o te

rmin

us o

f H

A2.

In a

dditi

on, w

e ha

ve id

entif

ied

inac

tive

anal

ogs

of B

RY-2

7709

whi

ch a

re a

ble

to c

ompe

te o

ut th

e in

hibi

tory

act

iv-

ity o

f BM

Y-27

709.

Thi

s fin

ding

sug

gest

s th

at in

hibi

tion

of th

e H

A-m

edia

ted

mem

bran

e fu

sion

by

this

cla

ss o

f com

-po

unds

is n

ot s

olel

y th

e re

sult

of b

indi

ng w

ithin

the

HA

mol

ecul

e bu

t req

uire

s sp

ecifi

c in

tera

ctio

ns.

Luo

et a

l. 19

97


144

Podo

carp

ic a

cid

deri

vati

ves

Sequ

ence

ana

lyse

sH

aem

aggl

utin

in g

ene

Dru

g re

sist

ance

Entr

y of

influ

enza

vir

us in

to th

e ho

st c

ell i

s de

pend

ent o

n th

e fu

sion

of t

he v

iral

env

elop

e w

ith th

e en

doso

mal

mem

-br

ane

and

is m

edia

ted

by a

low

-pH

-ind

uced

cha

nge

of th

e vi

ral h

emag

glut

inin

(HA

) to

a co

nfor

mat

ion

that

is fu

so-

geni

c. A

com

poun

d re

late

d to

pod

ocar

pic

acid

(180

299]

was

iden

tifie

d th

at in

hibi

ts m

ultic

ycle

repl

icat

ion

of in

fluen

za

A/K

awas

aki/

86 (H

1N1)

Vir

us in

cul

ture

. Tre

atm

ent o

f Mad

in-D

arby

can

ine

kidn

ey (M

DC

K) c

ells

with

180

299

at 1

h

befo

re in

fect

ion

resu

lted

in th

e in

hibi

tion

of v

iral

pro

tein

syn

thes

is. A

dditi

on o

f 20

µg o

f 180

299/

ml a

t 1 h

p.i.

had

no

effe

ct, i

ndic

atin

g th

at 1

8029

9 af

fect

s an

ear

ly s

tep

of th

e in

fluen

za v

iral

repl

icat

ion

cycl

e. G

enet

ic a

naly

sis

of re

as-

sort

ants

bet

wee

n se

nsiti

ve a

nd re

sist

ant v

irus

es d

emon

stra

ted

that

hem

aggl

utin

in (H

A) c

onfe

rred

the

1802

99-r

esis

t-an

t (18

0299

(r))

phe

noty

pe. T

wel

ve in

depe

nden

t iso

late

s of

influ

enza

A/K

awas

aki/

86 w

ere

sele

cted

for

resi

stan

ce

to 1

8029

9, a

nd s

eque

nce

anal

ysis

reve

aled

that

eac

h of

thes

e vi

ruse

s co

ntai

ned

amin

o ac

id s

ubst

itutio

ns in

the

HA

. T

hese

mut

atio

ns a

re d

ispe

rsed

thro

ugho

ut th

e H

A p

rim

ary

amin

o ac

id s

eque

nce

and

clus

ter

in o

ne o

f tw

o re

gion

s: th

e in

terf

ace

betw

een

HA

(1) a

nd H

A(2

) and

in a

regi

on n

ear

the

fusi

on d

omai

n of

HA

(2).

Whe

n co

mpa

red

with

the

pare

nt

viru

s, th

e pH

-of-

inac

tivat

ion

of th

e re

sist

ant m

utan

ts w

as in

crea

sed

by 0

.3 to

0.6

pH

uni

t, su

gges

ting

that

the

mut

ant

HA

s un

derg

o th

e co

nfor

mat

iona

l cha

nge

at a

n el

evat

ed p

H. F

usio

n of

hum

an e

ryth

rocy

tes

to M

DC

K c

ells

infe

cted

w

ith p

aren

t inf

luen

za A

/Kaw

asak

i/86

was

inhi

bite

d by

180

299

(0.1

–10

µg/m

l) in

a c

once

ntra

tion-

depe

nden

t: m

anne

r, w

here

as fu

sion

of e

ryth

rocy

tes

to M

DC

K c

ells

infe

cted

with

180

299(

r) m

utan

ts w

as n

ot: a

ffect

ed. T

hese

resu

lts s

ug-

gest

that

180

299

inte

ract

s w

ith th

e ne

utra

l pH

con

form

atio

n of

influ

enza

A H

A a

nd p

reve

nts

the

low

-pH

-ind

uced

ch

ange

of H

A to

its

fuso

geni

c co

nfor

mat

ion.

Stas

chke

et a

l. 19

98

dA

S181

Sial

idas

e fu

sion

pro

tein

Influ

enza

A a

nd B

Mou

se/f

erre

t mod

el

Influ

enza

is a

hig

hly

infe

ctio

us d

isea

se c

hara

cter

ized

by

recu

rren

t ann

ual e

pide

mic

s an

d un

pred

icta

ble

maj

or w

orld

-w

ide

pand

emic

s. R

apid

spr

ead

of th

e hi

ghly

pat

hoge

nic

avia

n H

5N1

stra

in a

nd e

scal

atin

g hu

man

infe

ctio

ns b

y th

e vi

rus

have

set

off

the

alar

m fo

r a

glob

al p

ande

mic

. To

prov

ide

an u

rgen

tly n

eede

d al

tern

ativ

e tr

eatm

ent m

odal

ity fo

r in

fluen

za, w

e ha

ve g

ener

ated

a re

com

bina

nt fu

sion

pro

tein

com

pose

d of

a s

ialid

ase

cata

lytic

dom

ain

deri

ved

from

A

ctin

omyc

es v

isco

sus

fuse

d w

ith a

cel

l sur

face

-anc

hori

ng s

eque

nce.

The

sia

lidas

e fu

sion

pro

tein

is to

be

appl

ied

topi

-ca

lly a

s an

inha

lant

to re

mov

e th

e in

fluen

za v

iral

rece

ptor

s, s

ialic

aci

ds, f

rom

the

airw

ay e

pith

eliu

m. W

e de

mon

stra

te

that

a s

ialid

ase

fusi

on c

onst

ruct

, DA

S181

, effe

ctiv

ely

clea

ves

sial

ic a

cid

rece

ptor

s us

ed b

y bo

th h

uman

and

avi

an in

flu-

enza

vir

uses

. The

trea

tmen

t pro

vide

s lo

ng-l

astin

g ef

fect

and

is n

onto

xic

to th

e ce

lls. D

AS1

81 d

emon

stra

ted

pote

nt

antiv

iral

and

cel

l pro

tect

ive

effic

acie

s ag

ains

t a p

anel

of l

abor

ator

y st

rain

s an

d cl

inic

al is

olat

es o

f IFV

A a

nd IF

V B

, w

ith v

irus

repl

icat

ion

inhi

bitio

n 50

% e

ffect

ive

conc

entr

atio

ns in

the

rang

e of

0.0

4 to

0.9

nM

. Mou

se a

nd fe

rret

stu

dies

co

nfir

med

sig

nific

ant i

n vi

vo e

ffic

acy

of th

e si

alid

ase

fusi

on in

bot

h pr

ophy

lact

ic a

nd tr

eatm

ent m

odes

.

Mal

akho

v et

al.

2006

dA

S181

Phas

e II

clin

ical

tria

lBa

ckgr

ound

. DA

S181

, a n

ovel

hos

t-di

rect

ed a

ntiv

iral

in d

evel

opm

ent f

or in

fluen

za tr

eatm

ent,

was

ass

esse

d in

this

pha

se

II c

linic

al tr

ial.

Met

hods

. This

stud

y w

as a

dou

ble-

blin

d, p

lace

bo-c

ontr

olle

d ph

ase

II c

linic

al tr

ial a

sses

sing

influ

enza

vi

ral l

oad

and

patie

nt sa

fety

in o

ther

wis

e he

alth

y in

fluen

za-in

fect

ed p

artic

ipan

ts. P

artic

ipan

ts w

ere

rand

omiz

ed to

a

sing

le-d

ose,

mul

tiple

-dos

e, o

r pla

cebo

gro

up a

nd w

ere

follo

wed

for s

afet

y an

d vi

rolo

gic

outc

omes

. Res

ults

. A to

tal o

f 177

la

bora

tory

-con

firm

ed in

fluen

za-in

fect

ed p

artic

ipan

ts w

ere

enro

lled

in th

e tr

ial,

whi

ch e

ncom

pass

ed 3

influ

enza

seas

ons

from

200

9–20

11 in

bot

h th

e N

orth

ern

and

Sout

hern

Hem

isph

eres

. Thir

ty-s

even

per

cent

of p

artic

ipan

ts h

ad c

onfir

med

in

fect

ion

with

influ

enza

B, 3

3% w

ith se

ason

al H

3N2,

29%

with

pan

dem

ic 2

009

H1N

1, a

nd 1

par

ticip

ant w

as p

ositi

ve

for b

oth

influ

enza

B a

nd p

ande

mic

200

9 H

1N1.

Sig

nific

ant e

ffect

s wer

e ob

serv

ed in

rega

rd to

dec

reas

ed c

hang

e fr

om

base

line

vira

l loa

d an

d vi

ral s

hedd

ing

in th

e m

ultip

le-d

ose

grou

p co

mpa

red

with

pla

cebo

as m

easu

red

by q

uant

itativ

e po

lym

eras

e ch

ain

reac

tion

(P <

0.0

5). N

o in

stan

ces o

f H27

4Y w

ere

obse

rved

am

ong

vira

l iso

late

s fro

m th

is tr

ial.

Ove

rall,

th

e dr

ug w

as g

ener

ally

wel

l tol

erat

ed. C

oncl

usio

ns. D

AS1

81 si

gnifi

cant

ly re

duce

d vi

ral l

oad

in p

artic

ipan

ts in

fect

ed w

ith

influ

enza

, thu

s war

rant

ing

futu

re c

linic

al d

evel

opm

ent o

f thi

s nov

el h

ost-

dire

cted

ther

apy.

Mos

s et a

l. 20

12


145

Fusi

on-i

nhib

itor

y pe

ptid

esC

hole

ster

ol-c

onju

gate

d co

mpo

unds

We

prev

ious

ly d

escr

ibed

fusi

on-in

hibi

tory

pep

tides

that

are

targ

eted

to th

e ce

ll m

embr

ane

by c

hole

ster

ol c

onju

gatio

n an

d po

tent

ly in

hibi

t env

elop

ed v

irus

es th

at fu

se a

t the

cel

l sur

face

, inc

ludi

ng H

IV, p

arai

nflue

nza,

and

hen

ipav

irus

es.

How

ever

, for

vir

uses

that

fuse

insi

de o

f int

race

llula

r com

part

men

ts, f

usio

n-in

hibi

tory

pep

tides

hav

e ex

hibi

ted

very

low

an

tivir

al a

ctiv

ity. W

e pr

opos

e th

at fo

r the

se v

irus

es, t

oo, m

embr

ane

targ

etin

g vi

a ch

oles

tero

l con

juga

tion

may

yie

ld

pote

nt c

ompo

unds

. Her

e w

e co

mpa

re th

e ac

tivity

of f

usio

n-in

hibi

tory

pep

tides

der

ived

from

the

influ

enza

hem

aggl

utin

in

(HA

) and

show

that

alth

ough

the

unco

njug

ated

pep

tides

are

inac

tive,

the

chol

este

rol-c

onju

gate

d co

mpo

unds

are

effe

ctiv

e in

hibi

tors

of i

nfec

tivity

and

mem

bran

e fu

sion

. We

hypo

thes

ize

that

the

chol

este

rol m

oiet

y, by

loca

lizin

g th

e pe

ptid

es to

th

e ta

rget

cel

l mem

bran

e, a

llow

s the

pep

tides

to fo

llow

the

viru

s to

the

intr

acel

lula

r site

of f

usio

n. Th

e ch

oles

tero

l-con

ju-

gate

d pe

ptid

es tr

ap H

A in

a tr

ansi

ent i

nter

med

iate

stat

e af

ter f

usio

n is

trig

gere

d bu

t bef

ore

com

plet

ion

of th

e re

fold

ing

step

s tha

t dri

ve th

e m

ergi

ng o

f the

vir

al a

nd c

ellu

lar m

embr

anes

. Thes

e re

sults

pro

vide

pro

of o

f con

cept

for a

n an

tivir

al

stra

tegy

that

is a

pplic

able

to in

trac

ellu

larl

y fu

sing

vir

uses

, inc

ludi

ng k

now

n an

d em

ergi

ng v

iral

pat

hoge

ns.

Lee

et a

l. 20

11

Rn

A a

ptam

ers

SELE

X te

chno

logy

Hae

mag

glut

inat

ion

inhi

bitio

n as

say

In th

is s

tudy

, we

inve

stig

ated

the

effic

acy

of u

sing

hem

aggl

utin

in (H

A) a

s a

targ

et fo

r an

tivir

al th

erap

y th

roug

h nu

clei

c ac

id a

ptam

ers.

Aft

er p

urifi

catio

n of

the

rece

ptor

bin

ding

dom

ain

(HA

1) o

f HA

pro

tein

, act

ivity

of r

ecom

bina

nt H

A1

was

con

firm

ed b

y us

ing

hem

aggl

utin

atio

n as

say.

We

sele

cted

RN

A a

ptam

er c

andi

date

s af

ter

15 ro

unds

of i

tera

tive

Syst

emat

ic E

volu

tion

of L

igan

ds b

y EX

pone

ntia

l enr

ichm

ent (

SELE

X) t

arge

ting

the

biol

ogic

ally

act

ive

HA

pro

tein

. T

he s

elec

ted

RNA

apt

amer

HA

S15-

5, w

hich

spe

cific

ally

bin

ds to

HA

1, e

xhib

ited

sign

ifica

nt a

ntiv

iral

eff

icac

y ac

cord

-in

g to

the

resu

lts o

f a h

emag

glut

inat

ion

inhi

bitio

n as

say

usin

g eg

g al

lant

oic

fluid

s ha

rbor

ing

the

viru

s. T

hus,

the

RNA

ap

tam

er H

AS1

5-5,

whi

ch a

cts

by b

lock

ing

and

inhi

bitin

g th

e re

cept

or-b

indi

ng d

omai

n of

vir

al H

A, c

an b

e de

velo

ped

as a

nov

el a

ntiv

iral

age

nt a

gain

st ty

pe H

5 av

ian

influ

enza

vir

us.

Park

et a

l. 20

11


146

Tabl

e 4.

2. M

2 io

n ch

anne

l blo

cker

s

Stru

ctur

e an

d fu

ncti

on o

f M

2 pr

otei

nC

ondu

ctan

ce o

f pro

tons

Hae

mag

glut

inin

-med

iate

d m

embr

ane

fusi

onM

2-ri

man

tadi

n co

mpl

ex

The

inte

gral

mem

bran

e pr

otei

n M

2 of

influ

enza

vir

us fo

rms

pH-g

ated

pro

ton

chan

nels

in th

e vi

ral l

ipid

env

elop

e (1

). T

he lo

w p

H o

f an

endo

som

e ac

tivat

es th

e M

2 ch

anne

l bef

ore

haem

aggl

utin

in-m

edia

ted

fusi

on. C

ondu

ctan

ce o

f pr

oton

s ac

idifi

es th

e vi

ral i

nter

ior

and

ther

eby

faci

litat

es d

isso

ciat

ion

of th

e m

atri

x pr

otei

n fr

om th

e vi

ral n

ucle

o-pr

otei

ns -

a re

quir

ed p

roce

ss fo

r un

pack

ing

of th

e vi

ral g

enom

e (2

). In

add

ition

to it

s ro

le in

rele

ase

of v

iral

nuc

leo-

prot

eins

, M2

in th

e tr

ans-

Gol

gi n

etw

ork

(TG

N) m

embr

ane

prev

ents

pre

mat

ure

conf

orm

atio

nal r

earr

ange

men

t of

new

ly s

ynth

esiz

ed h

aem

aggl

utin

in d

urin

g tr

ansp

ort t

o th

e ce

ll su

rfac

e by

equ

ilibr

atin

g th

e pH

of t

he T

GN

with

that

of

the

host

cel

l cyt

opla

sm (3

). In

hibi

ting

the

prot

on c

ondu

ctan

ce o

f M2

usin

g th

e an

ti- v

iral

dru

g am

anta

dine

or

rim

anta

dine

inhi

bits

vir

al re

plic

atio

n(4–

7). H

ere

we

pres

ent t

he s

truc

ture

of t

he te

tram

eric

M2

chan

nel i

n co

mpl

ex

with

rim

anta

dine

, det

erm

ined

by

NM

R. I

n th

e cl

osed

sta

te, f

our

tight

ly p

acke

d tr

ansm

embr

ane

helic

es d

efin

e a

narr

ow c

hann

el, i

n w

hich

a ‘t

rypt

opha

n ga

te’ i

s lo

cked

by

inte

rmol

ecul

ar in

tera

ctio

ns w

ith a

spar

tic a

cid.

A c

arbo

xy-

term

inal

, am

phip

athi

c he

lix o

rien

ted

near

ly p

erpe

ndic

ular

to th

e tr

ansm

embr

ane

helix

form

s an

inw

ard-

faci

ng

base

. Low

erin

g th

e pH

des

tabi

lizes

the

tran

smem

bran

e he

lical

pac

king

and

unl

ocks

the

gate

, adm

ittin

g w

ater

to

cond

uct p

roto

ns, w

here

as th

e C

-ter

min

al b

ase

rem

ains

inta

ct, p

reve

ntin

g di

ssoc

iatio

n of

the

tetr

amer

. Rim

anta

dine

bi

nds

at fo

ur e

quiv

alen

t site

s ne

ar th

e ga

te o

n th

e lip

id- f

acin

g si

de o

f the

cha

nnel

and

sta

biliz

es th

e cl

osed

con

for-

mat

ion

of th

e po

re. D

rug-

resi

stan

ce m

utat

ions

are

pre

dict

ed to

cou

nter

the

effe

ct o

f dru

g bi

ndin

g by

eith

er in

crea

s-in

g th

e hy

drop

hilic

ity o

f the

por

e or

wea

keni

ng h

elix

– h

elix

pac

king

, thu

s fa

cilit

atin

g ch

anne

l ope

ning

.

Schn

ell a

nd C

hou

2008

Stru

ctur

e of

M2

prot

ein

Chi

mer

ic M

2 ch

anne

lD

rug-

bind

ing

site

sD

rug

resi

stan

ce

The

M2

chan

nel o

f infl

uenz

a A

is a

targ

et o

f the

ada

man

tane

fam

ily a

ntiv

iral

dru

gs. T

wo

diffe

rent

dru

g-bi

ndin

g si

tes

have

bee

n re

port

ed: o

ne in

side

the

pore

, and

the

othe

r is a

lipi

d-fa

cing

poc

ket.

A p

revi

ous s

tudy

show

ed th

at a

chi

mer

a of

M2

vari

ants

from

influ

enza

A a

nd B

that

con

tain

s onl

y th

e po

re-b

indi

ng si

te is

sens

itive

to a

man

tadi

ne in

hibi

tion,

su

gges

ting

that

the

prim

ary

site

of i

nhib

ition

is in

side

the

pore

. To

obta

in a

tom

ic d

etai

ls o

f cha

nnel

-dru

g in

tera

ctio

n,

we

dete

rmin

ed th

e st

ruct

ures

of t

he c

him

eric

cha

nnel

with

and

with

out r

iman

tadi

ne. I

nsid

e th

e ch

anne

l and

nea

r the

N

-ter

min

al e

nd, m

ethy

l gro

ups o

f Val

27 a

nd A

la30

from

four

subu

nits

form

a h

ydro

phob

ic p

ocke

t aro

und

the

adam

an-

tane

, and

the

drug

am

ino

grou

p ap

pear

s to

be in

pol

ar c

onta

ct w

ith th

e ba

ckbo

ne o

xyge

n of

Ala

30. Th

e st

ruct

ures

als

o re

veal

diff

eren

ces b

etw

een

the

drug

-bou

nd a

nd -u

nbou

nd st

ates

of t

he c

hann

el th

at c

an e

xpla

in d

rug

resi

stan

ce.

Piel

ak e

t al.

2011

Affi

nity

stu

dyA

man

tadi

ne/r

iman

tadi

ne

bind

ing

to M

2Su

rfac

e pl

asm

on re

sona

nce

Lipo

som

es

The

influ

enza

A v

irus

con

tain

s a

prot

on-s

elec

tive

ion

chan

nel (

M2)

that

is th

e ta

rget

of t

he a

dam

anta

ne fa

mily

of

drug

inhi

bito

rs. T

wo

rece

ntly

pub

lishe

d st

udie

s re

latin

g to

ada

man

tane

bin

ding

of t

he M

2 io

n ch

anne

l usi

ng X

-ray

cr

ysta

llogr

aphy

and

sol

utio

n N

MR

have

re-i

gnite

d in

tere

st in

the

pote

ntia

l use

of a

dam

anta

nes

in c

omba

ting

the

spre

ad o

f inf

luen

za A

. How

ever

, the

se tw

o st

udie

s pr

opos

e di

ffere

nt b

indi

ng s

ites

for

the

adam

anta

ne d

rugs

with

th

e X

-ray

M2/

aman

tadi

ne s

truc

ture

favo

ring

an

ion

chan

nel p

ore-

bind

ing

mod

el a

nd th

e so

lutio

n N

MR

M2/

rim

an-

tadi

ne s

truc

ture

sug

gest

ing

the

exis

tenc

e of

a li

pid-

faci

ng b

indi

ng p

ocke

t. W

e co

nduc

ted

a se

ries

of s

urfa

ce p

lasm

on

reso

nanc

e (S

PR) e

xper

imen

ts d

esig

ned

to a

ccur

atel

y m

easu

re th

e af

finity

of a

man

tadi

ne a

nd r

iman

tadi

ne to

M2

ion

chan

nels

em

bedd

ed in

1,2

-dim

yris

toyl

-sn-

glyc

ero-

phos

phoc

holin

e (D

MPC

) lip

osom

es. W

e fin

d th

at th

is c

lass

of

drug

is c

apab

le o

f bin

ding

M2

with

two

diffe

rent

aff

initi

es in

the

orde

r of

10–4

and

10–7

M, s

ugge

stin

g th

at b

oth

pro-

pose

d bi

ndin

g si

tes

are

feas

ible

. Fur

ther

mor

e, b

y ex

amin

ing

drug

bin

ding

to M

2 m

utan

t con

stru

cts

(V27

A, S

31N

, an

d D

44A

), it

was

pos

sibl

e to

pro

be th

e lo

catio

n of

the

two

bind

ing

site

s. W

e sh

ow th

at a

hig

h-af

finity

bin

ding

site

co

rres

pond

s to

the

M2

ion

chan

nel p

ore

whe

reas

the

seco

ndar

y, lo

w-a

ffin

ity b

indi

ng s

ite c

an b

e at

trib

uted

to th

e lip

id fa

ce o

f the

por

e. T

hese

SPR

resu

lts a

re in

exc

elle

nt a

gree

men

t with

the

mos

t rec

ent s

olid

-sta

te N

MR

stud

y of

am

anta

dine

-bou

nd M

2 in

lipi

d bi

laye

rs a

nd p

rovi

de in

depe

nden

t sup

port

that

the

ion

chan

nel p

ore-

bind

ing

site

is

resp

onsi

ble

for

the

phar

mac

olog

ical

act

ivity

elic

ited

by th

e ad

aman

tane

dru

gs.

Rose

nber

g an

d C

asar

otto

201

0


147

M2

ion

chan

nel i

nhib

itio

nRi

man

tadi

ne

Mol

ecul

ar d

ynam

ics

sim

ulat

ions

In o

rder

to u

nder

stan

d ho

w r

iman

tadi

ne (R

MT

) inh

ibits

the

prot

on c

ondu

ctan

ce in

the

influ

enza

A M

2 ch

anne

l via

th

e re

cent

ly p

ropo

sed

“allo

ster

ic m

echa

nism

”, m

olec

ular

dyn

amic

s si

mul

atio

ns w

ere

appl

ied

to th

e M

2-te

tram

eric

pr

otei

n w

ith fo

ur R

MTs

bou

nd o

utsi

de th

e ch

anne

l at t

he th

ree

prot

onat

ion

stat

es: t

he 0

H-c

lose

d, 1

H-i

nter

med

iate

an

d 3H

-ope

n si

tuat

ions

. In

the

0H-c

lose

d st

ate,

a n

arro

w c

hann

el w

ith th

e R

MT-

Asp

44-T

rp41

H-b

ond

netw

ork

was

fo

rmed

, the

refo

re th

e w

ater

pen

etra

tion

thro

ugh

the

chan

nel w

as c

ompl

etel

y bl

ocke

d. T

he T

rp41

-Asp

44 in

tera

c-tio

n w

as a

bsen

t in

the

1H-i

nter

med

iate

sta

te, w

hils

t the

bin

ding

of R

MT

to A

sp44

rem

aine

d, w

hich

resu

lted

in a

w

eake

ned

helix

-hel

ix p

acki

ng, t

here

fore

the

chan

nel w

as p

artia

lly p

reve

nted

. In

the

3H-o

pen

stat

e it

was

foun

d th

at

the

elec

tros

tatic

repu

lsio

n fr

om th

e th

ree

char

ged

His

37 re

sidu

es a

llow

ed th

e Tr

p41

gate

to o

pen,

per

mitt

ing

wat

er

to p

enet

rate

thro

ugh

the

chan

nel.

Thi

s ag

reed

wel

l with

the

pote

ntia

l of t

he m

eans

forc

e w

hich

is in

the

follo

win

g or

der:

0H

> 1

H >

3H

.

Inth

arat

hep

et a

l. 20

11

dis

cove

ry o

f am

anta

dine

Ev

alua

tion

of v

irus

inhi

bitio

nI-

Ada

man

tana

min

e (a

man

tadi

ne) c

ause

s a se

lect

ive,

repr

oduc

ible

, dos

e-re

late

d in

hibi

tion

of in

fluen

za in

fect

ions

in

tissu

e cu

lture

, chi

ck e

mbr

yos,

and

mic

e. Th

e co

mpo

und

is n

ot v

iruc

idal

and

app

ears

to a

ct b

y in

terf

erin

g w

ith th

e pe

n-et

ratio

n of

the

host

cel

l by

the

viru

s. In

influ

enza

infe

ctio

ns o

f mic

e, g

reat

est e

ffica

cy o

ccur

s with

trea

tmen

t at t

he ti

me

of in

fect

ion;

how

ever

, the

re is

sign

ifica

nt a

ntiv

iral

act

ivity

with

trea

tmen

t del

ayed

up

to 7

2 ho

urs a

fter

infe

ctio

n. V

irus

in

hibi

tion

is n

ot c

ompl

ete

and

surv

ivor

s are

imm

une

to a

cha

lleng

e in

fect

ion

with

the

orig

inal

infe

ctin

g vi

rus.

Dav

ies e

t al.

1964

Ada

man

tane

der

ivat

ives

3-(2

-ada

man

tyl)p

yrro

lidin

esSy

nthe

sis a

nd e

valu

atio

n

The

3-(2

-ada

man

tyl)p

yrro

lidin

es 8

a-g.

14

wer

e sy

nthe

size

d an

d ev

alua

ted

for a

ctiv

ity a

gain

st in

fluen

za A

vir

us. Th

e pa

rent

N-H

com

poun

d 14

was

seve

ral t

imes

mor

e ac

tive

than

am

anta

dine

aga

inst

H2N

2 an

d H

3N2

influ

enza

A v

irus

. Th

e co

mbi

ned

use

of N

MR

spec

tros

copy

and

com

puta

tiona

l che

mis

try

show

ed th

at th

e co

nfor

mat

ion

arou

nd th

e py

rrol

idin

e-ad

aman

tyl c

arbo

n-ca

rbon

bon

d is

tran

s and

the

pyrr

olid

ine

hete

rocy

cle

has a

n en

velo

pe c

onfo

rmat

ion

with

C-2

out

of t

he p

lane

of t

he o

ther

ring

ato

ms.

N-D

ialk

ylam

inoe

thyl

subs

titut

ion

of c

ompo

und

14 re

sulte

d in

the

pote

nt d

iam

ine

anal

ogue

s 8e,

f,g. I

nter

estin

gly,

thei

r lac

tam

am

ine

prec

urso

rs w

ere

also

act

ive.

Com

poun

ds 8

e,f,g

are

th

e fir

st a

dam

anta

ne d

eriv

ativ

es, b

eari

ng tw

o am

ine

grou

ps, r

epor

ted

to b

e ac

tive

agai

nst i

nflue

nza

A v

irus

.

Stam

atio

u et

al.

2001

Ada

man

tane

der

ivat

ives

Pyrr

olid

ine

Aze

tidin

esA

ziri

dine

s

E2-(

1-ad

aman

tyl)-

2-m

ethy

l-py

rrol

idin

es 3

and

4, 2

-(1-

adam

atity

l)-2-

met

hyl-

azet

idin

es 5

and

6, a

nd

2-(1

-ada

man

tyl)-

2-m

ethy

l-az

irid

ines

7 a

nd 8

wer

e sy

nthe

size

d an

d te

sted

for

thei

r an

tivir

al a

ctiv

ity a

gain

st in

flu-

enza

A. P

aren

t mol

ecul

es 3

, 5, a

nd 7

con

tain

the

alph

a-m

ethy

l-1-

adim

anta

n-m

etha

nam

ine

2 ph

arm

acop

hori

c m

oiet

y (r

iman

tadi

ne).

The

rin

g si

ze e

ffect

on

anti-

influ

enza

A a

ctiv

ity w

as in

vest

igat

ed. P

yrro

lidin

e 3

was

the

mos

t po

tent

ant

i-in

fluen

za v

irus

A c

ompo

und,

9-f

old

mor

e po

tent

than

rim

anta

dine

2, 2

7-fo

ld m

ore

pote

nt th

an a

man

-ta

dine

1, a

nd 2

2-fo

ld m

ore

pote

nt th

an r

ibav

irin

. Aze

tidin

es 5

and

6 w

ere

both

mar

kedl

y ac

tive

agai

nst i

nflu

enza

A

H2N

2 vi

rus,

10-

to 2

0-fo

ld m

ore

pote

nt th

an a

man

tadi

ne. A

ziri

dine

7 w

as a

lmos

t dev

oid

of a

ny a

ctiv

ity a

gain

st

H2N

2 vi

rus

but e

xhib

ited

bord

erlin

e ac

tivity

aga

inst

H3N

2 in

fluen

za A

str

ain.

Thu

s, it

app

ears

that

cha

ngin

g th

e fiv

e-, t

o fo

ur- t

o a

thre

e-m

embe

red

ring

resu

lts in

a d

rop

of a

ctiv

ity a

gain

st in

fluen

za A

vir

us.

Zoi

dis e

t al.

2006

Ada

man

tane

der

ivat

ives

Azo

lo-a

dam

anta

nes

Rim

anta

dine

-res

ista

nt st

rain

s

Che

mot

hera

py a

nd c

hem

opro

phyl

axis

of i

nflu

enza

is o

ne o

f the

mos

t im

port

ant d

irec

tions

of h

ealth

pro

tect

ion

activ

ity. D

ue to

the

high

rat

e of

dru

g-re

sist

ant s

trai

ns o

f inf

luen

za v

irus

, the

re is

a n

eed

for

the

sear

ch a

nd fu

rthe

r de

velo

pmen

t of n

ew p

oten

t ant

ivir

als

agai

nst i

nflu

enza

with

a b

road

spe

ctru

m o

f act

ivity

. In

the

pres

ent s

tudy

, a

set o

f di-

, tri

- and

tetr

azol

e de

riva

tives

of a

dam

anta

ne w

as e

ffic

ient

ly p

repa

red

and

thei

r an

ti-in

fluen

za a

ctiv

ities

ev

alua

ted

agai

nst r

iman

tadi

ne-r

esis

tant

str

ain

A/P

uert

o Ri

co/8

/34.

In g

ener

al, d

eriv

ativ

es o

f tet

razo

le p

osse

ssed

the

high

est v

irus

-inh

ibiti

ng a

ctiv

ity. W

e de

mon

stra

ted

that

sev

eral

com

poun

ds o

f thi

s se

t exh

ibite

d m

uch

high

er a

ctiv

-ity

than

the

curr

ently

use

d an

tivir

al r

iman

tadi

ne, a

com

poun

d of

rela

ted

stru

ctur

e. M

oreo

ver,

we

show

ed th

at th

ese

azol

o- w

ere

sign

ifica

ntly

less

toxi

c. T

his

stud

y de

mon

stra

tes

that

influ

enza

vir

uses

can

be

inhi

bite

d by

ada

man

tyla

-zo

les

and

thus

hav

e po

tent

ial f

or d

evel

opin

g an

tivir

al a

gent

s w

ith a

n al

tern

ate

mec

hani

sm o

f act

ion.

Zar

ubae

v et

al.

2010


148

new

M2

ion

chan

nel

inhi

bito

rsT

he A

/M2

prot

on c

hann

el o

f inf

luen

za A

vir

us is

a ta

rget

for

the

anti-

influ

enza

dru

gs a

man

tadi

ne a

nd r

iman

ta-

dine

, who

se e

ffect

iven

ess

was

dim

inis

hed

by th

e ap

pear

ance

of n

atur

ally

occ

urri

ng p

oint

mut

ants

in th

e A

/M2

chan

nel p

ore,

am

ong

whi

ch th

e m

ost c

omm

on a

re S

31N

, V27

A, a

nd L

26F.

We

have

syn

thes

ized

and

cha

ract

eriz

ed

the

prop

ertie

s of

a s

erie

s of

com

poun

ds, o

rigi

nally

der

ived

from

the

A/M

2 in

hibi

tor

BL-1

743.

A le

ad c

ompo

und

emer

ging

from

thes

e in

vest

igat

ions

, spi

ro[5

.5]u

ndec

an-3

-am

ine,

is a

n ef

fect

ive

inhi

bito

r of

wild

-typ

e A

/M2

chan

-ne

ls a

nd L

26F

and

V27

A m

utan

t ion

cha

nnel

s in

vitr

o an

d al

so in

hibi

ts re

plic

atio

n of

reco

mbi

nant

mut

ant v

irus

es

bear

ing

thes

e m

utat

ions

in p

laqu

e re

duct

ion

assa

ys. D

iffer

ence

s in

the

inhi

bitio

n ki

netic

s be

twee

n BL

-174

3, k

now

n to

bin

d in

side

the

A/M

2 ch

anne

l por

e, a

nd a

man

tadi

ne w

ere

expl

oite

d to

dem

onst

rate

com

petit

ion

betw

een

thes

e co

mpo

unds

, con

sist

ent w

ith th

e co

nclu

sion

that

am

anta

dine

bin

ds in

side

the

chan

nel p

ore.

Inhi

bitio

n by

all

of th

ese

com

poun

ds w

as s

how

n to

be

volta

ge-i

ndep

ende

nt, s

ugge

stin

g th

at th

eir

char

ged

grou

ps a

re w

ithin

the

N-t

erm

inal

ha

lf of

the

pore

, pri

or to

the

sele

ctiv

ity fi

lter

that

def

ines

the

regi

on o

ver

whi

ch th

e tr

ansm

embr

ane

pote

ntia

l oc

curs

. The

se fi

ndin

gs n

ot o

nly

help

to d

efin

e th

e lo

catio

n an

d m

echa

nism

of b

indi

ng o

f M2

chan

nel-

bloc

king

dr

ugs

but a

lso

dem

onst

rate

the

feas

ibili

ty o

f dis

cove

ring

new

inhi

bito

rs th

at ta

rget

this

bin

ding

site

in a

num

ber

of

aman

tadi

ne-r

esis

tant

mut

ants

.

Bala

nnik

et a

l. 20

09

Synt

heti

c st

rate

gies

Mic

row

ave-

assi

sted

thre

e-co

mpo

nent

one

-pot

cy

cloc

onde

nsat

ion

met

hod

Ada

man

tyl m

oiet

y

A m

icro

wav

e-as

sist

ed th

ree-

com

pone

nt o

ne-p

ot c

yclo

cond

ensa

tion

met

hod

was

app

lied

for

the

synt

hesi

s of

nov

el

N-(

1-th

ia-4

-aza

spir

o[4.

5] d

ecan

-4-y

l)car

boxa

mid

e co

mpo

unds

car

ryin

g an

ada

man

tyl m

oiet

y. T

he s

truc

ture

s of

the

com

poun

ds w

ere

conf

irm

ed b

y sp

ectr

al a

nd e

lem

enta

l ana

lysi

s. A

ll co

mpo

unds

wer

e ev

alua

ted

for

antiv

iral

act

ivity

ag

ains

t inf

luen

za A

(H1N

1 an

d H

3N2)

and

influ

enza

B v

irus

in M

DC

K c

ell c

ultu

res.

The

com

poun

ds d

ispl

ayed

a

conf

ined

str

uctu

re-a

ctiv

ity re

latio

nshi

p. T

he N

-(2,

8-di

met

hyl-

3-ox

o-1-

thia

-4-a

zasp

iro[

4.5]

dec

-4-y

l)ada

man

tane

-1-

carb

oxam

ide

3b w

as th

e m

ost p

oten

t inh

ibito

r [an

tivir

al E

C50

: 1.4

µM

aga

inst

influ

enza

A/H

3N2

viru

s]. I

ts s

tron

g in

hibi

tory

effe

ct in

a v

irus

hem

olys

is a

ssay

sup

port

s th

at 3

b ac

ts a

s an

influ

enza

vir

us fu

sion

inhi

bito

r by

pre

vent

ing

the

conf

orm

atio

nal c

hang

e of

the

influ

enza

vir

us h

emag

glut

inin

at l

ow p

H.

Gok

tas e

t al.

2012

Enca

psul

atio

n of

ad

aman

tane

s in

lip

osom

es

The

aim

of t

he p

rese

nt s

tudy

was

to e

ncap

sula

te m

anno

syla

ted

1-am

inoa

dam

anta

ne a

nd m

anno

syla

ted

adam

an-

tyltr

ipep

tides

, nam

ely

[(2R

)-N

-(ad

aman

t-1-

yl)-

3-(a

lpha

,bet

a-D

-man

nopy

rano

sylo

xy)-

2-m

ethy

lpro

pana

mid

e an

d (2

R)-N

[3-(

alph

a-D

-man

nopy

rano

sylo

xy)-

2-m

ethy

lpro

pano

yl]-

D,L

-(ad

aman

t-2-

yl)g

lycy

l-L-

alan

yl-D

-iso

glut

amin

e]

in li

poso

mes

. The

cha

ract

eriz

atio

n of

lipo

som

es, s

ize

and

surf

ace

mor

phol

ogy

was

per

form

ed u

sing

dyn

amic

ligh

t sc

atte

ring

(DLS

) and

ato

mic

forc

e m

icro

scop

y (A

FM).

The

resu

lts h

ave

reve

aled

that

the

enca

psul

atio

n of

exa

min

ed

com

poun

ds c

hang

es th

e si

ze a

nd s

urfa

ce o

f lip

osom

es. A

fter

the

conc

anav

alin

A (C

onA

) was

add

ed to

the

lipos

ome

prep

arat

ion,

incr

ease

in li

poso

me

size

and

thei

r ag

greg

atio

n ha

s be

en o

bser

ved.

The

enl

arge

men

t of L

ipos

omes

w

as a

scri

bed

to th

e sp

ecifi

c bi

ndin

g of

the

Con

A to

the

man

nose

pre

sent

on

the

surf

ace

of th

e pr

epar

ed li

poso

mes

. T

hus,

it h

as b

een

show

n th

at th

e ad

aman

tyl m

oiet

y fr

om m

anno

syla

ted

1-am

inoa

dam

anta

ne a

nd m

anno

syla

ted

adam

anty

ltrip

eptid

es c

an b

e us

ed a

s an

anc

hor

in th

e lip

id b

ilaye

r fo

r ca

rboh

ydra

te m

oiet

y ex

pose

d on

the

lipos

ome

surf

ace.

Stim

ac e

t al.

2012


149

Effec

tive

ness

and

saf

ety

Am

anta

dine

Ri

man

tadi

neC

hild

ren/

elde

rly

Back

grou

nd T

he e

ffect

iven

ess

and

safe

ty o

f am

anta

dine

(AM

T) a

nd r

iman

tadi

ne (R

MT

) for

pre

vent

ing

and

trea

ting

influ

enza

A in

adu

lts h

as b

een

syst

emat

ical

ly re

view

ed. H

owev

er, l

ittle

is k

now

n ab

out t

hese

trea

tmen

ts in

chi

ldre

n an

d th

e el

derl

y. O

bjec

tives

To

syst

emat

ical

ly re

view

the

effe

ctiv

enes

s an

d sa

fety

of A

MT

and

RM

T in

pre

vent

ing

and

trea

ting

influ

enza

A in

chi

ldre

n an

d th

e el

derl

y. S

earc

h m

etho

ds W

e se

arch

ed th

e C

ochr

ane

Cen

tral

Reg

iste

r of

C

ontr

olle

d Tr

ials

(CEN

TR

AL)

(The

Coc

hran

e Li

brar

y 20

11, I

ssue

2) w

hich

con

tain

s th

e C

ochr

ane

Acu

te R

espi

ra-

tory

Infe

ctio

ns (A

RI) G

roup

’s Sp

ecia

lised

Reg

iste

r, M

EDLI

NE

(196

6 to

June

wee

k 3,

201

1) a

nd E

MBA

SE (1

980

to

June

201

1). S

elec

tion

crite

ria

Rand

omis

ed c

ontr

olle

d tr

ials

(RC

Ts) o

r qu

asi-

RC

Ts c

ompa

ring

AM

T a

nd/ o

r R

MT

w

ith p

lace

bo, c

ontr

ol, o

ther

ant

ivir

als

or d

iffer

ent d

oses

or

sche

dule

s of

AM

T o

r R

MT,

or

both

, or

no in

terv

en-

tion,

in c

hild

ren

and

the

elde

rly.

Dat

a co

llect

ion

and

anal

ysis

Tw

o re

view

aut

hors

inde

pend

ently

sel

ecte

d tr

ials

for

incl

usio

n an

d as

sess

ed m

etho

dolo

gica

l qua

lity.

We

reso

lved

dis

agre

emen

ts b

y co

nsen

sus.

In a

ll co

mpa

riso

ns e

xcep

t fo

r on

e, w

e se

para

tely

ana

lyse

d th

e tr

ials

in c

hild

ren

and

the

elde

rly

usin

g Re

view

Man

ager

sof

twar

e. M

ain

resu

lts A

to

tal o

f 12

stud

ies

invo

lvin

g 24

94 p

artic

ipan

ts (1

586

child

ren

and

adol

esce

nts

and

908

elde

rly)

com

pare

d A

MT

and

R

MT

with

pla

cebo

, par

acet

amol

(one

tria

l; 69

chi

ldre

n) o

r za

nam

ivir

(tw

o tr

ials

; 545

sen

iors

). A

ll st

udie

s w

ere

RCTs

bu

t mos

t wer

e st

ill s

usce

ptib

le to

bia

s. T

wo

tria

ls in

the

elde

rly

had

a hi

gh r

isk

of b

ias

beca

use

of in

com

plet

e ou

t-co

me

data

. In

one

of th

ose

tria

ls th

ere

was

als

o a

lack

of o

utco

me

asse

ssm

ent b

lindi

ng. R

isk

of b

ias

was

unc

lear

in

10 s

tudi

es d

ue to

unc

lear

ran

dom

seq

uenc

e ge

nera

tion

and

allo

catio

n co

ncea

lmen

t. O

nly

two

tria

ls in

chi

ldre

n w

ere

cons

ider

ed to

hav

e a

low

ris

k of

bia

s. A

MT

was

effe

ctiv

e in

pre

vent

ing

influ

enza

A in

chi

ldre

n. A

tota

l of 7

73 p

artic

-ip

ants

wer

e in

clud

ed in

this

out

com

e (r

isk

ratio

(RR)

0.1

1; 9

5% c

onfid

ence

inte

rval

(CI)

0.0

4 to

0.3

0). T

he a

ssum

ed

risk

of i

nflu

enza

in th

e co

ntro

l gro

up w

as 1

0 pe

r 10

0 an

d th

e co

rres

pond

ing

risk

in th

e R

MT

gro

up w

as o

ne p

er

100

(95%

CI 0

to 3

). T

he q

ualit

y of

the

evid

ence

was

con

side

red

low

. For

trea

tmen

t pur

pose

s, R

MT

was

ben

efi-

cial

for

abat

ing

feve

r on

day

thre

e of

trea

tmen

t. Fo

r th

is p

urpo

se o

ne s

tudy

was

sel

ecte

d w

ith lo

w r

isk

of b

ias

and

incl

uded

69

child

ren

(RR

0.36

; 95%

CI 0

.14

to 0

.91)

. The

ass

umed

ris

k w

as 3

8 pe

r 10

0 an

d th

e co

rres

pond

ing

risk

in

the

RM

T g

roup

was

14

per

100,

95%

CI 5

to 3

4. T

he q

ualit

y of

the

evid

ence

was

mod

erat

e. R

MT

did

not

sho

w a

pr

ophy

lact

ic e

ffect

aga

inst

influ

enza

in th

e el

derl

y, b

ut th

e qu

ality

of e

vide

nce

was

con

side

red

very

low

. The

re w

ere

103

part

icip

ants

(RR

0.45

; 95%

CI 0

.14

to 1

.41,

for

an a

ssum

ed r

isk

of 1

7 pe

r 10

0 an

d a

corr

espo

ndin

g ri

sk in

the

RM

T g

roup

of 7

per

100

, 95%

CI 2

to 2

3). W

e di

d no

t ide

ntify

any

AM

T tr

ials

in th

e el

derl

y th

at m

et o

ur in

clus

ion

crite

ria.

The

re w

as n

o ev

iden

ce o

f adv

erse

effe

cts

of A

MT

and

RM

T in

chi

ldre

n or

an

adve

rse

effe

ct o

f RM

T in

the

elde

rly.

We

did

not i

dent

ify a

ny A

MT

tria

ls in

the

elde

rly

that

met

our

incl

usio

n cr

iteri

a. A

utho

rs’ c

oncl

usio

ns A

MT

is

effe

ctiv

e in

pre

vent

ing

influ

enza

A in

chi

ldre

n bu

t the

NN

TB

is h

igh

(NN

TB:

12

(95%

CI 9

to 1

7). R

MT

pro

babl

y he

lps

the

abat

emen

t of f

ever

on

day

thre

e of

trea

tmen

t, bu

t the

qua

lity

of th

e ev

iden

ce is

poo

r. D

ue to

the

smal

l nu

mbe

r of

ava

ilabl

e st

udie

s, w

e co

uld

not r

each

a d

efin

itive

con

clus

ion

on th

e sa

fety

of A

MT

or

the

effe

ctiv

enes

s of

R

MT

in p

reve

ntin

g in

fluen

za in

chi

ldre

n an

d th

e el

derl

y.

Gal

vao

et a

l. 20

12


150

Res

ista

nce

to a

dam

anta

nes

281

influ

enza

A is

olat

esA

ustr

alia

Euro

peA

sia

The

ada

man

tane

s (a

man

tadi

ne a

nd r

iman

tadi

ne) w

ere

the

initi

al a

ntiv

iral

s lic

ense

d fo

r us

e ag

ains

t inf

luen

za A

vi

ruse

s an

d ha

ve b

een

used

in s

ome

coun

trie

s to

con

trol

sea

sona

l inf

luen

za a

nd h

ave

also

bee

n st

ockp

iled

for

pote

ntia

l pan

dem

ic u

se. W

hile

hig

h ra

tes

of re

sist

ance

hav

e be

en o

bser

ved

in re

cent

yea

rs w

ith A

(H3)

vir

uses

, the

ra

tes

of re

sist

ance

with

A(H

1) v

irus

es h

as v

arie

d w

idel

y. In

this

stu

dy w

e an

alys

ed 2

81 h

uman

influ

enza

A v

irus

es

isol

ated

in 2

007

that

wer

e re

ferr

ed to

the

WH

O C

olla

bora

ting

Cen

tre

for

Refe

renc

e an

d Re

sear

ch in

Mel

bour

ne,

mai

nly

from

Aus

tral

ia a

nd th

e su

rrou

ndin

g re

gion

s, fo

r ev

iden

ce o

f res

ista

nce

to a

dam

anta

nes

and

a su

bset

of

thes

e w

as e

xam

ined

for

resi

stan

ce to

the

neur

amin

idas

e in

hibi

tors

(NIs

). W

e fo

und

that

the

rate

s of

acd

aman

tane

re

sist

ance

in A

(H3)

vir

uses

con

tinue

d to

incr

ease

in m

ost c

ount

ries

in 2

007

but a

dis

tinct

var

iatio

n w

as s

een

with

A

(H1)

resi

stan

ce le

vels

. A(H

1) v

irus

es fr

om A

ustr

alia

, New

Zea

land

and

Eur

ope

had

low

rat

es o

f res

ista

nce

(2–9

%)

whe

reas

vir

uses

from

a n

umbe

r of

Sou

th E

ast (

SE) A

sian

cou

ntri

es h

ad h

igh

rate

s of

resi

stan

ce (3

3–10

0%).

Thi

s di

ffere

nce

can

be a

ttri

bute

d to

the

spre

ad o

f A/B

risb

ane/

59/2

007-

like

viru

ses

to m

any

part

s of

the

wor

ld w

ith th

e ex

cept

ion

of S

E A

sia

whe

re A

/Hon

g K

ong/

2652

/200

6-lik

e vi

ruse

s co

ntin

ue to

pre

dom

inat

e. W

hen

thes

e tw

o A

(H1)

su

bgro

ups

wer

e co

mpa

red

for

thei

r in

vitr

o se

nsiti

vity

to th

e ot

her

clas

s of

influ

enza

ant

ivir

al d

rugs

, the

neu

ram

ini-

dase

inhi

bito

rs, n

o di

ffere

nce

was

see

n be

twee

n th

e gr

oups

with

bot

h sh

owin

g no

rmal

leve

ls o

f sen

sitiv

ity to

thes

e dr

ugs,

The

find

ing

of re

duci

ng A

(H1)

resi

stan

ce r

ates

in A

ustr

alia

and

ris

ing

leve

ls in

SE

Asi

a in

200

7, re

vers

es th

e tr

end

seen

in 2

006

whe

n A

(H1)

resi

stan

ce le

vels

wer

e ri

sing

in A

ustr

alia

and

els

ewhe

re b

ut re

mai

ned

low

in m

ost o

f SE

Asi

a.

Barr

et a

l. 20

08

Res

ista

nce

to a

dam

anta

nes

Pyro

sequ

enci

ngC

onfir

mat

ory

sequ

ence

an

alys

isPh

enot

ypic

test

ing

Back

grou

nd a

dam

anta

nes

have

bee

n us

ed to

trea

t inf

luen

za A

vir

us in

fect

ions

for

man

y ye

ars.

Stu

dies

hav

e sh

own

a lo

w in

cide

nce

of re

sist

ance

to th

ese

drug

s am

ong

circ

ulat

ing

influ

enza

vir

uses

; how

ever

, the

ir u

se is

ris

ing

wor

ldw

ide

and

drug

resi

stan

ce h

as b

een

repo

rted

am

ong

influ

enza

A (H

5N1)

vir

uses

isol

ated

from

pou

ltry

and

hum

an b

eing

s in

Asi

a. W

e so

ught

to a

sses

s ad

aman

tane

resi

stan

ce a

mon

g in

fluen

za A

vir

uses

isol

ated

dur

ing

the

past

dec

ade

from

cou

ntri

es p

artic

ipat

ing

in W

HO

’s gl

obal

influ

enza

sur

veill

ance

net

wor

k. M

etho

ds w

e an

alys

ed

data

for

influ

enza

fiel

d is

olat

es th

at w

ere

obta

ined

wor

ldw

ide

and

subm

itted

to th

e W

HO

Col

labo

ratin

g C

ente

r fo

r In

fluen

za a

t the

US

Cen

ters

for

Dis

ease

Con

trol

and

Pre

vent

ion

betw

een

Oct

1, 1

994,

and

Mar

31,

200

5. W

e us

ed

pyro

sequ

enci

ng, c

onfir

mat

ory

sequ

ence

ana

lysi

s, a

nd p

heno

typi

c te

stin

g to

det

ect d

rug

resi

stan

ce a

mon

g ci

rcul

at-

ing

influ

enza

A H

3N2

(n =

652

4), H

1N1

(n =

589

), an

d H

1N2

(n =

83)

vir

uses

. Fin

ding

s M

ore

than

700

0 in

fluen

za

A fi

eld

isol

ates

wer

e sc

reen

ed fo

r sp

ecifi

c am

inoa

cid

subs

titut

ions

in th

e M

2 ge

ne k

now

n to

con

fer

drug

resi

stan

ce.

Dur

ing

the

deca

de o

f sur

veill

ance

a s

igni

fican

t inc

reas

e in

dru

g re

sist

ance

was

not

ed, f

rom

0.4

% in

199

4-19

95 to

12

.3%

in 2

003–

2004

. Thi

s in

crea

se in

the

prop

ortio

n of

resi

stan

t vir

uses

was

wei

ghte

d he

avily

by

thos

e ob

tain

ed

from

Asi

a w

ith 6

1% o

f res

ista

nt v

irus

es is

olat

ed s

ince

200

3 be

ing

from

peo

ple

in A

sia.

Inte

rpre

tatio

n ou

r da

ta r

aise

co

ncer

ns a

bout

the

appr

opri

ate

use

of a

dam

anta

nes

and

draw

att

entio

n to

the

impo

rtan

ce o

f tra

ckin

g th

e em

er-

genc

e an

d sp

read

of d

rug-

resi

stan

t inf

luen

za A

vir

uses

.

Brig

ht e

t al.

2005


151

Tabl

e 4.

3. In

hibi

tors

of v

iral

Rn

A p

olym

eras

e

Poly

mer

ase

basi

c

prot

ein

2C

ap-b

indi

ng d

omai

nC

ap a

nalo

gs

Influ

enza

vir

uses

cau

se a

sig

nific

ant l

evel

of m

orbi

dity

and

mor

talit

y in

the

popu

latio

n ev

ery

year

. The

ir re

sist

ance

to

curr

ent a

nti-

influ

enza

dru

gs in

crea

ses

the

diff

icul

ty o

f flu

trea

tmen

t. T

hus,

dev

elop

men

t of n

ew a

nti-

influ

enza

dru

gs

is n

eces

sary

in re

gard

s of

pre

vent

the

trag

edy

of in

fluen

za p

ande

mic

. The

Pol

ymer

ase

basi

c pr

otei

n 2

(PB2

) sub

unit

of in

fluen

za v

irus

RN

A p

olym

eras

e is

one

of p

oten

tial t

arge

ts fo

r ne

w d

rugs

bec

ause

the

bind

ing

of P

B2 w

ith th

e 5’

ca

p of

the

host

pre

-mRN

As

is th

e in

itial

ste

p of

the

viru

s’ pr

otei

n sy

nthe

sis.

In th

is s

tudy

, we

com

pare

d th

e bi

ndin

g po

tenc

y of

PB2

cap

bin

ding

dom

ain

with

two

smal

l mol

ecul

es, i

.e.,

RO a

nd P

PT28

, with

that

of P

B2 w

ith c

ap a

nalo

g m

7GT

P. T

he c

alcu

late

d bi

ndin

g en

ergi

es s

how

ed th

at R

O a

nd P

PT28

had

hig

her

bind

ing

affin

ity w

ith P

B2. F

urth

er

inte

ract

ion

anal

ysis

sho

wed

that

the

impo

rtan

t par

ts fo

r bi

ndin

g w

ere

the

five-

and

six-

mem

ber

hete

rocy

clic

rin

gs (t

he

6/5-

mem

ber

ring

s) o

f sm

all m

olec

ules

, as

wel

l as

the

hydr

opho

bic

part

s of

RO

and

PPT

28 w

hich

had

goo

d in

tera

c-tio

ns w

ith th

e hy

drop

hobi

c re

sidu

es in

the

bind

ing

cavi

ty. T

hus,

RO

and

PPT

28 a

re tw

o po

tent

ial a

nti-

influ

enza

dru

gs

targ

eted

PB2

, whi

ch m

ay in

hibi

t the

gro

wth

of i

nflu

enza

vir

us b

y co

mpe

titiv

ely

bind

ing

with

the

cap

stru

ctur

e bi

ndin

g do

mai

n of

PB2

.

Lv e

t al.

2011

Res

ista

nce

to a

dam

anta

nes

USA

S31N

subs

titut

ion

Influ

enza

A a

nd B

Thi

s re

port

des

crib

es n

ew fi

ndin

gs re

gard

ing

the

resi

stan

ce to

ada

man

tane

s of

influ

enza

A v

irus

es c

urre

ntly

ci

rcul

atin

g in

the

Uni

ted

Stat

es a

nd p

rovi

des

inte

rim

reco

mm

enda

tions

that

thes

e dr

ugs

not b

e us

ed d

urin

g th

e re

mai

nder

of t

he 2

005–

2006

influ

enza

sea

son.

Am

anta

dine

als

o is

use

d to

trea

t sym

ptom

s of

Par

kins

on d

isea

se a

nd

may

con

tinue

to b

e us

ed fo

r th

is in

dica

tion.

Res

ista

nce

of in

fluen

za A

vir

uses

to a

dam

anta

nes

can

occu

r sp

onta

ne-

ousl

y or

em

erge

rap

idly

dur

ing

trea

tmen

t. A

sin

gle

poin

t mut

atio

n in

the

codo

ns fo

r am

ino

acid

s at

pos

ition

s 26

, 27,

30

, 31,

or

34 o

f the

M2

prot

ein

can

conf

er c

ross

-res

ista

nce

to b

oth

aman

tadi

ne a

nd r

iman

tadi

ne. N

eith

er re

plic

a-tio

n, tr

ansm

issi

on, n

or v

irul

ence

of a

dam

anta

ne-r

esis

tant

influ

enza

A v

irus

es a

re im

pair

ed b

y th

e po

int m

utat

ions

co

nfer

ring

resi

stan

ce. A

rece

nt re

port

on

the

glob

al p

reva

lenc

e of

ada

man

tane

-res

ista

nt in

fluen

za A

vir

uses

indi

-ca

ted

a si

gnifi

cant

incr

ease

of d

rug

resi

stan

ce, f

rom

1.8

% d

urin

g th

e 20

01–2

002

influ

enza

sea

son

to 1

2.3%

dur

ing

the

2003

–200

4 se

ason

. In

the

Uni

ted

Stat

es, t

he fr

eque

ncy

of a

dam

anta

ne re

sist

ance

incr

ease

d fr

om 1

.9%

dur

ing

the

2003

–200

4 in

fluen

za s

easo

n to

11%

dur

ing

the

2004

-05

seas

on (C

DC

, unp

ublis

hed

data

, 200

5). I

n co

ntra

st to

ad

aman

tane

resi

stan

ce, n

eura

min

idas

e in

hibi

tor

resi

stan

ce re

mai

ns r

are

wor

ldw

ide.

Sin

ce th

e be

ginn

ing

of th

e 20

05–2

006

influ

enza

sur

veill

ance

sea

son,

WH

O a

nd N

REV

SS la

bora

tori

es h

ave

test

ed a

tota

l of 3

8,93

2 sp

ecim

ens

for

influ

enza

vir

uses

; 155

7 (4

.0%

) tes

ted

posi

tive.

Am

ong

the

1557

influ

enza

vir

uses

, 149

9 (9

6.3%

) wer

e in

fluen

za

A v

irus

es, a

nd 5

8 (3

.7%

) wer

e in

fluen

za B

vir

uses

. A to

tal o

f 765

(51.

0%) o

f the

149

9 in

fluen

za A

vir

uses

hav

e be

en

subt

yped

; 760

(99.

3%) w

ere

influ

enza

A (H

3N2)

vir

uses

, and

five

(0.7

%) w

ere

influ

enza

A (H

1N1)

vir

uses

. Dur

ing

Oct

ober

1, 2

005-

Janu

ary

14, 2

006,

a to

tal o

f 123

influ

enza

A v

irus

es c

olle

cted

from

23

stat

es w

ere

test

ed a

t CD

C

for

adam

anta

ne re

sist

ance

. Am

ong

the

120

influ

enza

A (H

3N2)

vir

uses

test

ed, 1

09 (9

1%) d

emon

stra

ted

the

S31N

su

bstit

utio

n in

the

M2

prot

ein

that

con

fers

resi

stan

ce to

am

anta

dine

and

rim

anta

dine

. Con

vent

iona

l seq

uenc

ing

on

a su

bset

of 2

0 vi

ruse

s co

nfir

med

this

sub

stitu

tion.

Am

ong

the

thre

e in

fluen

za A

(H1N

1) v

irus

es te

sted

, non

e co

n-ta

ined

any

mut

atio

ns a

ssoc

iate

d w

ith re

sist

ance

. As

of Ja

nuar

y 14

, all

U.S

. inf

luen

za v

irus

es s

cree

ned

for

antiv

iral

re

sist

ance

at C

DC

had

dem

onst

rate

d su

scep

tibili

ty to

neu

ram

inid

ase

inhi

bito

rs. P

roce

dure

s fo

r vi

rus

prop

agat

ion,

RN

A e

xtra

ctio

n, a

nd p

yros

eque

ncin

g fo

r ad

aman

tane

resi

stan

ce h

ave

been

des

crib

ed p

revi

ousl

y.

Brig

ht e

t al.

2006


152

Poly

mer

ase/

endo

nucl

ease

Cap

-bin

ding

Hos

t ada

ptat

ion

mec

hani

sms

The

het

erot

rim

eric

RN

A-d

epen

dent

RN

A p

olym

eras

e of

influ

enza

vir

uses

cat

alyz

es R

NA

repl

icat

ion

and

tran

scri

ptio

n ac

tiviti

es in

infe

cted

cel

l nuc

lei.

The

nuc

leot

ide

poly

mer

izat

ion

activ

ity is

com

mon

to b

oth

repl

icat

ion

and

tran

scri

p-tio

n pr

oces

ses,

with

an

addi

tiona

l cap

-sna

tchi

ng fu

nctio

n be

ing

empl

oyed

dur

ing

tran

scri

ptio

n to

ste

al s

hort

5’-c

appe

d RN

A p

rim

ers

from

hos

t mRN

As.

Cap

-bin

ding

, end

onuc

leas

e, a

nd p

olym

eras

e ac

tiviti

es h

ave

long

bee

n st

udie

d bi

o-ch

emic

ally

, but

str

uctu

ral s

tudi

es o

n th

e po

lym

eras

e an

d its

sub

units

hav

e be

en h

inde

red

by d

iffic

ultie

s in

pro

duci

ng

suff

icie

nt q

uant

ities

of m

ater

ial.

Rece

ntly

, bec

ause

of h

eigh

tene

d ef

fort

and

adv

ance

s in

exp

ress

ion

and

crys

talli

zatio

n te

chno

logi

es, a

ser

ies

of h

igh

reso

lutio

n st

ruct

ures

of i

ndiv

idua

l dom

ains

hav

e be

en d

eter

min

ed. T

hese

she

d lig

ht o

n in

trin

sic

activ

ities

of t

he p

olym

eras

e, in

clud

ing

cap

snat

chin

g, s

ubun

it as

soci

atio

n, a

nd n

ucle

ocyt

opla

smic

tran

spor

t, an

d op

en u

p th

e po

ssib

ility

of s

truc

ture

-gui

ded

deve

lopm

ent o

f new

pol

ymer

ase

inhi

bito

rs. F

urth

erm

ore,

the

activ

ity

of in

fluen

za p

olym

eras

e is

hig

hly

host

- and

cel

l typ

e-sp

ecifi

c, b

eing

dep

ende

nt o

n th

e id

entit

y of

a fe

w k

ey a

min

o ac

id

posi

tions

in th

e di

ffere

nt s

ubun

its, e

spec

ially

in th

e C

-ter

min

al re

gion

of P

B2. N

ew s

truc

ture

s de

mon

stra

te th

e su

rfac

e ex

posu

re o

f the

se re

sidu

es, c

onsi

sten

t with

idea

s th

at th

ey m

ight

mod

ulat

e in

tera

ctio

ns w

ith h

ost-

spe

cific

fact

ors

that

en

hanc

e or

rest

rict

act

ivity

. Rec

ent p

rote

omic

and

gen

ome-

wid

e in

tera

ctom

e an

d RN

A in

terf

eren

ce s

cree

ns h

ave

sug-

gest

ed th

e id

entit

ies

of s

ome

of th

ese

pote

ntia

l reg

ulat

ors

of p

olym

eras

e fu

nctio

n.

Boiv

in e

t al.

2010

Ada

ptiv

e ev

olut

ion

Hos

t ada

ptat

ion

mec

hani

sms

Ada

ptiv

e ev

olut

ion

is c

hara

cter

ized

by

posi

tive

and

para

llel,

or re

peat

ed s

elec

tion

of m

utat

ions

. Mou

se a

dapt

atio

n of

in

fluen

za A

vir

us (I

AV) p

rodu

ces

viru

lent

mut

ants

that

dem

onst

rate

pos

itive

and

par

alle

l evo

lutio

n of

mut

atio

ns in

th

e he

mag

glut

inin

(HA

) rec

epto

r an

d no

n-st

ruct

ural

pro

tein

1 (N

S1) i

nter

fero

n an

tago

nist

gen

es. W

e no

w p

rese

nt a

ge

nom

ic a

naly

sis

of a

ll 11

gen

es o

f 39

mou

se a

dapt

ed IA

V v

aria

nts

from

10

repl

icat

e ad

apta

tion

expe

rim

ents

. Mut

a-tio

ns w

ere

map

ped

on th

e pr

imar

y an

d st

ruct

ural

map

s of

eac

h pr

otei

n an

d sp

ecifi

c m

utat

ions

wer

e va

lidat

ed w

ith

resp

ect t

o vi

rule

nce,

repl

icat

ion,

and

RN

A p

olym

eras

e ac

tivity

. Mou

se a

dapt

ed (M

A) v

aria

nts

obta

ined

aft

er 1

2 or

20

–21

seri

al in

fect

ions

acq

uire

d on

ave

rage

5.8

and

7.9

non

syno

nym

ous

mut

atio

ns p

er g

enom

e of

11

gene

s, re

spec

-tiv

ely.

Am

ong

a to

tal o

f 115

non

syno

nym

ous

mut

atio

ns, 5

1 de

mon

stra

ted

prop

ertie

s of

nat

ural

sel

ectio

n in

clud

ing

27 p

aral

lel m

utat

ions

. The

gre

ates

t deg

ree

of p

aral

lel e

volu

tion

occu

rred

in th

e H

A re

cept

or a

nd r

ibon

ucle

ocap

sid

com

pone

nts,

pol

ymer

ase

subu

nits

(PB1

, PB2

, PA

) and

NP.

Mut

atio

ns o

ccur

red

in h

ost n

ucle

ar tr

affic

king

fact

or

bind

ing

site

s as

wel

l as

site

s of

vir

us-v

irus

pro

tein

sub

unit

inte

ract

ion

for

NP,

NS1

, HA

and

NA

pro

tein

s. A

dapt

ive

regi

ons

incl

uded

cap

bin

ding

and

end

onuc

leas

e do

mai

ns in

the

PB2

and

PA p

olym

eras

e su

buni

ts. F

our

mut

atio

ns in

N

S1 re

sulte

d in

loss

of b

indi

ng to

the

host

cle

avag

e an

d po

lyad

enyl

atio

n sp

ecifi

city

fact

or (C

PSF3

0) s

ugge

stin

g th

at a

re

duct

ion

in in

hibi

tion

of h

ost g

ene

expr

essi

on w

as b

eing

sel

ecte

d. T

he m

ost p

reva

lent

mut

atio

ns in

PB2

and

NP

wer

e sh

own

to in

crea

se v

irul

ence

but

diff

ered

in th

eir

abili

ty to

enh

ance

repl

icat

ion

and

dem

onst

rate

d ep

ista

tic e

ffect

s. S

ev-

eral

pos

itive

ly s

elec

ted

RNA

pol

ymer

ase

mut

atio

ns d

emon

stra

ted

incr

ease

d vi

rule

nce

asso

ciat

ed w

ith >

300%

enh

ance

d po

lym

eras

e ac

tivity

. Ada

ptiv

e m

utat

ions

that

con

trol

hos

t ran

ge a

nd v

irul

ence

wer

e id

entif

ied

by th

eir

repe

ated

sel

ec-

tion

to c

ompr

ise

a de

fined

mod

el fo

r st

udyi

ng IA

V e

volu

tion

to in

crea

sed

viru

lenc

e in

the

mou

se.

Ping

et a

l. 20

11


153

Poly

mer

ase

PB2

subu

nit

Tem

pera

ture

-dep

ende

nt

poly

mer

ase

activ

ity

Mos

t avi

an in

fluen

za A

vir

uses

, whi

ch p

refe

rent

ially

repl

icat

e at

the

high

tem

pera

ture

s fo

und

in th

e di

gest

ive

trac

t of

bir

ds, h

ave

a gl

utam

ic a

cid

at re

sidu

e 62

7 of

the

vira

l RN

A p

olym

eras

e PB

2 su

buni

t (G

lu-6

27),

whe

reas

the

hum

an

viru

ses,

whi

ch o

ptim

ally

repl

icat

e at

the

low

tem

pera

ture

s ob

serv

ed in

the

hum

an re

spir

ator

y tr

act,

have

a ly

sine

(L

ys-6

27).

The

mec

hani

sm o

f act

ion

for

this

mut

atio

n is

stil

l not

und

erst

ood,

alth

ough

inte

ract

ion

with

hos

t fac

tors

ha

s be

en p

ropo

sed

to p

lay

a m

ajor

role

. In

this

stu

dy, w

e ex

plor

ed a

n al

tern

ativ

e, y

et re

late

d, h

ypot

hesi

s th

at th

is P

B2

mut

atio

n m

ay a

lter

the

tem

pera

ture

-dep

ende

nt e

nzym

atic

pol

ymer

ase

activ

ity o

f the

vir

al p

olym

eras

e. F

irst

, the

avi

an

poly

mer

ase

prot

ein,

whi

ch w

as p

urifi

ed fr

om b

acul

ovir

us e

xpre

ssio

n sy

stem

, ind

eed

rem

aine

d si

gnifi

cant

ly a

ctiv

e at

hi

gher

tem

pera

ture

s (i.

e. 3

7 an

d 42

°C),

whe

reas

the

hum

an E

627K

mut

ant d

rast

ical

ly lo

st a

ctiv

ity a

t the

se h

igh

tem

-pe

ratu

res.

Sec

ond,

our

ste

ady-

stat

e ki

netic

s da

ta re

veal

ed th

at th

e hu

man

E62

7K m

utan

t pol

ymer

ase

is c

atal

ytic

ally

m

ore

activ

e th

an th

e av

ian

Glu

-627

pol

ymer

ase

at 3

4 °C

. Im

port

antly

, the

E62

7K m

utat

ion

elev

ates

app

aren

t K(c

at) a

t lo

w te

mpe

ratu

res

with

litt

le e

ffect

on

Km

, sug

gest

ing

that

the

E627

K m

utat

ion

alte

rs th

e bi

oche

mic

al s

teps

invo

lved

in

enzy

me

cata

lysi

s ra

ther

than

the

inte

ract

ion

with

the

inco

min

g N

TP.

Thi

rd, t

his

tem

pera

ture

-dep

ende

nt k

inet

ic im

pact

of

the

hum

an E

627K

mut

atio

n w

as a

lso

obse

rved

with

diff

eren

t RN

A te

mpl

ates

, with

diff

eren

t pri

mer

s an

d al

so in

the

pres

ence

of n

ucle

opro

tein

. In

conc

lusi

on, o

ur s

tudy

sug

gest

s th

at th

e am

ino

acid

seq

uenc

e va

riat

ions

at r

esid

ue 6

27 o

f PB

2 su

buni

t can

dir

ectly

alte

r th

e en

zym

e ki

netic

s of

influ

enza

pol

ymer

ase.

Agg

arw

al e

t al.

2011

Poly

mer

ase

PB2

subu

nit

Hum

an-t

o-hu

man

tr

ansm

issi

on

Back

grou

nd: T

he id

entif

icat

ion

of m

utat

ions

that

con

fer

uniq

ue p

rope

rtie

s to

a p

atho

gen,

suc

h as

hos

t ran

ge, i

s of

fun-

dam

enta

l im

port

ance

in th

e fig

ht a

gain

st d

isea

se. T

his

pape

r de

scri

bes

a no

vel m

etho

d fo

r id

entif

ying

am

ino

acid

site

s th

at d

istin

guis

h sp

ecifi

c se

ts o

f pro

tein

seq

uenc

es, b

y co

mpa

rativ

e an

alys

is o

f mat

ched

alig

nmen

ts. T

he u

se o

f mut

ual

info

rmat

ion

to id

entif

y di

stin

ctiv

e re

sidu

es re

spon

sibl

e fo

r fu

nctio

nal v

aria

nts

mak

es th

is a

ppro

ach

high

ly s

uita

ble

for

anal

yzin

g la

rge

sets

of s

eque

nces

. To

supp

ort m

utua

l inf

orm

atio

n an

alys

is, w

e de

velo

ped

the

AVA

NA

sof

twar

e, w

hich

ut

ilize

s se

quen

ce a

nnot

atio

ns to

sel

ect s

ets

for

com

pari

son,

acc

ordi

ng to

use

r-sp

ecifi

ed c

rite

ria.

The

met

hod

pres

ente

d w

as a

pplie

d to

an

anal

ysis

of i

nflu

enza

A P

B2 p

rote

in s

eque

nces

, with

the

obje

ctiv

e of

iden

tifyi

ng th

e co

mpo

nent

s of

ad

apta

tion

to h

uman

-to-

hum

an tr

ansm

issi

on, a

nd re

cons

truc

ting

the

mut

atio

n hi

stor

y of

thes

e co

mpo

nent

s. R

esul

ts:

We

com

pare

d ov

er 3

,000

PB2

pro

tein

seq

uenc

es o

f hum

an-t

rans

mis

sibl

e an

d av

ian

isol

ates

, to

prod

uce

a ca

talo

gue

of

site

s in

volv

ed in

ada

ptat

ion

to h

uman

-to-

hum

an tr

ansm

issi

on. T

his

anal

ysis

iden

tifie

d 17

cha

ract

eris

tic s

ites,

five

of

whi

ch h

ave

been

pre

sent

in h

uman

-tra

nsm

issi

ble

stra

ins

sinc

e th

e 19

18 S

pani

sh fl

u pa

ndem

ic. S

ixte

en o

f the

se s

ites

are

loca

ted

in fu

nctio

nal d

omai

ns, s

ugge

stin

g th

ey m

ay p

lay

func

tiona

l rol

es in

hos

t-ra

nge

spec

ifici

ty. T

he c

atal

ogue

of

cha

ract

eris

tic s

ites

was

use

d to

der

ive

sequ

ence

sig

natu

res

from

his

tori

cal i

sola

tes.

The

se s

igna

ture

s, a

rran

ged

in

chro

nolo

gica

l ord

er, r

evea

l an

evol

utio

nary

tim

elin

e fo

r th

e ad

apta

tion

of th

e PB

2 pr

otei

n to

hum

an h

osts

. Con

clus

ion:

By

pro

vidi

ng th

e m

ost c

ompl

ete

eluc

idat

ion

to d

ate

of th

e fu

nctio

nal c

ompo

nent

s pa

rtic

ipat

ing

in P

B2 p

rote

in a

dapt

a-tio

n to

hum

ans,

this

stu

dy d

emon

stra

tes

that

mut

ual i

nfor

mat

ion

is a

pow

erfu

l too

l for

com

para

tive

char

acte

riza

tion

of

sequ

ence

set

s. In

add

ition

to c

onfir

min

g pr

evio

usly

repo

rted

find

ings

, sev

eral

nov

el c

hara

cter

istic

site

s w

ithin

PB2

are

re

port

ed. S

eque

nce

sign

atur

es g

ener

ated

usi

ng th

e ch

arac

teri

stic

site

s ca

talo

gue

char

acte

rize

con

cise

ly th

e ad

apta

tion

char

acte

rist

ics

of in

divi

dual

isol

ates

. Evo

lutio

nary

tim

elin

es d

eriv

ed fr

om s

igna

ture

s of

ear

ly h

uman

influ

enza

isol

ates

su

gges

t tha

t cha

ract

eris

tic v

aria

nts

emer

ged

rapi

dly,

and

rem

aine

d re

mar

kabl

y st

able

thro

ugh

subs

eque

nt p

ande

mic

s.

In a

dditi

on, t

he s

igna

ture

s of

hum

an-i

nfec

ting

H5N

1 is

olat

es s

ugge

st th

at th

is a

vian

sub

type

has

low

pan

dem

ic p

oten

-tia

l at p

rese

nt, a

lthou

gh it

pre

sent

s m

ore

hum

an a

dapt

atio

n co

mpo

nent

s th

an m

ost a

vian

sub

type

s

Mio

tto

et a

l. 20

08


154

Inhi

bito

r of

pol

ymer

ase

PA(C

) sub

unit

Chl

orog

enic

aci

d

The

avi

an in

fluen

za (H

5N1)

vir

al R

NA

pol

ymer

ase

prot

ein

PA(C

) was

use

d as

a ta

rget

to s

cree

n ni

ne c

hlor

ogen

ic

acid

der

ivat

ives

for

thei

r po

lym

eras

e in

hibi

tor

activ

ity. A

mon

g th

em, s

even

com

poun

ds w

ere

PAC

liga

nds,

and

fo

ur in

hibi

ted

influ

enza

RN

A p

olym

eras

e ac

tivity

. The

se re

sults

aid

in th

e de

sign

of a

nti-

influ

enza

age

nts

base

d on

ca

ffeoy

lqui

nic

acid

.

Li e

t al.

2012

b

Inhi

bito

r of

pol

ymer

ase

PA(C

) sub

unit

Lico

rice

-der

ived

co

mpo

unds

PA(C

) sub

unit

from

avi

an in

fluen

za (H

5N1)

vir

al R

NA

pol

ymer

ase

was

use

d in

this

wor

k as

a ta

rget

in th

e sc

reen

ing

for

anti-

influ

enza

age

nts

from

lico

rice

-der

ived

com

poun

ds. A

s a

resu

lt, 1

8 be

ta-g

lycy

rrhe

tinic

aci

d w

as s

ugge

sted

to b

e PA

(C) l

igan

d by

flex

ible

doc

king

, and

was

then

con

firm

ed b

y re

laxa

tion-

edite

d N

MR

. The

resu

lt of

ApG

pri

mer

ext

en-

sion

ass

ay in

dica

ted

that

this

PA

(C) l

igan

d ca

n in

hibi

t the

pol

ymer

ase

activ

ity, a

nd th

us m

ay p

oten

tially

be

valu

able

as

anti-

influ

enza

lead

com

poun

d. T

his

wor

k va

lidat

ed th

e po

ssib

ility

of s

cree

ning

pol

ymer

ase

inhi

bito

rs b

y us

ing

PAc

as a

ta

rget

, and

pro

vide

d a

star

ting

poin

t for

the

furt

her

disc

over

y of

new

ant

i-in

fluen

za d

rugs

.

Li e

t al.

2012

a

nuc

leos

ide

anal

ogue

T-70

5A

ntiv

iral

act

ivity

ev

alua

tion

T-70

5 (6

-flu

oro-

3-hy

drox

y-2-

pyra

zine

carb

oxam

ide)

has

bee

n fo

und

to h

ave

pote

nt a

nd s

elec

tive

inhi

bito

ry a

ctiv

ity

agai

nst i

nflu

enza

vir

us. I

n an

in v

itro

plaq

ue re

duct

ion

assa

y, T

-705

sho

wed

pot

ent i

nhib

itory

act

ivity

aga

inst

influ

enza

A

, B, a

nd C

vir

uses

, with

50%

inhi

bito

ry c

once

ntra

tions

(IC

(50)

s) o

f 0.0

13 to

0.4

8 µg

/ml,

whi

le it

sho

wed

no

cyto

-to

xici

ty a

t con

cent

ratio

ns u

p to

100

0 µg

/ml i

n M

adin

-Dar

by c

anin

e ki

dney

cel

ls. T

he s

elec

tivity

inde

x fo

r in

fluen

za

viru

s w

as m

ore

than

200

0. It

was

als

o ac

tive

agai

nst a

neu

ram

inid

ase

inhi

bito

r-re

sist

ant v

irus

and

som

e am

anta

dine

-re

sist

ant v

irus

es. T

-705

sho

wed

wea

k ac

tivity

aga

inst

non

-inf

luen

za v

irus

RN

A v

irus

es, w

ith th

e IC

(50)

s be

ing

high

er

for

non-

influ

enza

vir

us R

NA

vir

uses

than

for

influ

enza

vir

us, a

nd it

had

no

activ

ity a

gain

st D

NA

vir

uses

. Ora

lly

adm

inis

tere

d T-

705

at 1

00 m

g/kg

of b

ody

wei

ght/

day

(fou

r tim

es a

day

) for

5 d

ays

sign

ifica

ntly

redu

ced

the

mea

n pu

lmon

ary

viru

s yi

elds

and

the

rate

of m

orta

lity

in m

ice

infe

cted

with

influ

enza

vir

us A

/PR/

8/34

(3 ×

102 P

FU).

The

se

resu

lts s

ugge

st th

at T

-705

may

be

a co

mpo

und

that

is u

sefu

l and

hig

hly

sele

ctiv

e ag

ains

t inf

luen

za v

irus

infe

ctio

ns a

nd

that

has

a m

ode

of a

ctio

n di

ffere

nt fr

om th

ose

of c

omm

erci

ally

ava

ilabl

e dr

ugs,

suc

h as

am

anta

dine

, rim

anta

dine

, and

ne

uram

inid

ase

inhi

bito

rs.

Furu

ta e

t al.

2002

nuc

leos

ide

anal

ogue

T-70

5D

ose-

depe

nden

t effe

ct

GT

P-co

mpe

titio

n

T-70

5, a

sub

stitu

ted

pyra

zine

com

poun

d, h

as b

een

foun

d to

exh

ibit

pote

nt a

nti-

influ

enza

vir

us a

ctiv

ity in

vitr

o an

d in

vi

vo. I

n a

time-

of-a

dditi

on s

tudy

, it w

as in

dica

ted

that

T-7

05 ta

rget

ed a

n ea

rly

to m

iddl

e st

age

of th

e vi

ral r

eplic

atio

n cy

cle

but h

ad n

o ef

fect

on

the

adso

rptio

n or

rele

ase

stag

e. T

he a

nti-

influ

enza

vir

us a

ctiv

ity o

f T-7

05 w

as a

tten

uate

d by

add

ition

of p

urin

es a

nd p

urin

e nu

cleo

side

s, in

clud

ing

aden

osin

e, g

uano

sine

, ino

sine

, and

hyp

oxan

thin

e, w

here

as

pyri

mid

ines

did

not

affe

ct it

s ac

tivity

. T-7

05-4

-rib

ofur

anos

yl-5

’-tri

phos

phat

e (T

-705

RTP)

and

T-7

05-4

-rib

ofur

anos

yl-

5’-m

onop

hosp

hate

(T-7

05R

MP)

wer

e de

tect

ed in

MD

CK

cel

ls tr

eate

d w

ith T

-705

. T-7

05RT

P in

hibi

ted

influ

enza

vir

us

RNA

pol

ymer

ase

activ

ity in

a d

ose-

depe

nden

t and

a G

TP-

com

petit

ive

man

ner.

Unl

ike

riba

viri

n, T

-705

did

not

hav

e an

influ

ence

on

cellu

lar

DN

A o

r RN

A s

ynth

esis

. Inh

ibiti

on o

f cel

lula

r IM

P de

hydr

ogen

ase

by T

-705

RM

P w

as a

bout

15

0-fo

ld w

eake

r th

an th

at b

y ri

bavi

rin

mon

opho

spha

te, i

ndic

atin

g th

e sp

ecifi

city

of t

he a

nti-

influ

enza

vir

us a

ctiv

ity

and

low

er le

vel o

f cyt

otox

icity

of T

-705

. The

se re

sults

sug

gest

that

T-7

05RT

P, w

hich

is g

ener

ated

in in

fect

ed c

ells

, may

fu

nctio

n as

a s

peci

fic in

hibi

tor

of in

fluen

za v

irus

RN

A p

olym

eras

e an

d co

ntri

bute

s to

the

sele

ctiv

e an

ti-in

fluen

za v

irus

ac

tivity

of T

-705

.

Furu

ta e

t al.

2005


155

nuc

leos

ide

anal

ogue

sT-

705

Pera

miv

irA

ntiv

iral

act

ivity

ev

alua

tion

Mou

se m

odel

Favi

pira

vir

(T-7

05),

an in

fluen

za v

irus

RN

A p

olym

eras

e in

hibi

tor,

and

pera

miv

ir, a

n in

fluen

za v

irus

neu

ram

inid

ase

inhi

bito

r, w

ere

eval

uate

d al

one

and

in c

ombi

natio

n ag

ains

t pan

dem

ic in

fluen

za A

/Cal

iforn

ia/0

4/20

09 (H

1N1)

vir

us

infe

ctio

ns in

mic

e. In

fect

ed m

ice

wer

e tr

eate

d tw

ice

daily

for

5 da

ys s

tart

ing

4 h

afte

r vi

rus

chal

leng

e. F

avip

irav

ir w

as

40%

, 70%

, and

100

% p

rote

ctiv

e at

20,

40,

and

100

mg/

kg/d

ay. P

eram

ivir

was

30%

pro

tect

ive

at 0

.5 m

g/kg

/day

, but

inef

-fe

ctiv

e at

low

er d

oses

whe

n us

ed a

s m

onot

hera

py. C

ombi

natio

ns o

f fav

ipir

avir

and

per

amiv

ir in

crea

sed

the

num

bers

of

sur

vivo

rs b

y 10

–50%

whe

n th

e 0.

025,

0.0

5, a

nd 0

.1 m

g/kg

/day

dos

es o

f per

amiv

ir w

ere

com

bine

d w

ith 2

0 m

g/kg

/day

fa

vipi

ravi

r an

d w

hen

all d

oses

of p

eram

ivir

wer

e co

mbi

ned

with

40

mg/

kg/d

ay fa

vipi

ravi

r. T

hree

-dim

ensi

onal

ana

lysi

s of

dru

g in

tera

ctio

ns u

sing

the

Mac

Syne

rgy

met

hod

indi

cate

s st

rong

syn

ergy

for

thes

e dr

ug c

ombi

natio

ns. I

n ad

ditio

n,

an in

crea

se in

life

span

for

grou

ps o

f mic

e tr

eate

d w

ith d

rug

com

bina

tions

, com

pare

d to

the

mos

t effe

ctiv

e m

ono-

ther

apy

grou

p, w

as o

bser

ved

for

the

0.02

5, 0

.05,

and

0.1

mg/

kg/d

ay d

oses

of p

eram

ivir

com

bine

d w

ith fa

vipi

ravi

r at

the

20 m

g do

se le

vel.

The

refo

re, t

he 2

0 m

g/kg

/d d

ose

of fa

vipi

ravi

r w

as s

elec

ted

for

furt

her

com

bina

tion

stud

ies.

Incr

ease

d su

rviv

al w

as e

xhib

ited

whe

n th

is d

ose

was

com

bine

d w

ith p

eram

ivir

dos

es o

f 0.1

, 0.2

5 an

d 0.

5 m

g/kg

/day

(1 m

g/kg

/day

of

per

amiv

ir a

lone

was

100

% p

rote

ctiv

e in

this

exp

erim

ent)

. Im

prov

ed b

ody

wei

ght r

elat

ive

to e

ither

com

poun

d al

one

was

evi

dent

usi

ng 0

.25,

0.5

, and

1 m

g/kg

/day

of p

eram

ivir.

Sig

nific

ant r

educ

tions

in lu

ng h

emor

rhag

e sc

ore

and

lung

w

eigh

t wer

e ev

iden

t on

day

6 po

st-i

nfec

tion.

In a

dditi

on, v

irus

tite

rs w

ere

redu

ced

sign

ifica

ntly

on

day

4 po

st-i

nfec

tion

by c

ombi

natio

n th

erap

y co

ntai

ning

favi

pira

vir

com

bine

d w

ith p

eram

ivir

at 0

.25

and

0.5

mg/

kg/d

ay. T

hese

dat

a de

mon

-st

rate

that

com

bina

tions

of f

avip

irav

ir a

nd p

eram

ivir

per

form

bet

ter

than

sub

optim

al d

oses

of e

ach

com

poun

d al

one

for

the

trea

tmen

t of i

nflu

enza

vir

us in

fect

ions

in m

ice.

Tarb

et e

t al.

2012

nuc

leos

ide

anal

ogue

sPe

ram

ivir

Phas

e I o

f clin

ical

rese

arch

Pera

miv

ir is

a n

ovel

influ

enza

neu

ram

inid

ase

inhi

bito

r. In

this

art

icle

, hyd

roph

ilic

inte

ract

ion

chro

mat

ogra

phy

coup

led

with

tand

em m

ass s

pect

rom

etry

was

dev

elop

ed to

det

erm

ine

pera

miv

ir in

hum

an p

lasm

a. Th

e po

sitiv

e io

n M

RM m

ode

was

per

form

ed a

nd th

e pr

ecur

sor t

o th

e pr

oduc

t ion

tran

sitio

ns o

f m/z

329

≥ 1

00 a

nd 2

85 ≥

138

wer

e us

ed to

mea

sure

pe

ram

ivir

and

Ro

64-0

802

(I.S

.). C

hrom

atog

raph

ic se

para

tion

was

per

form

ed o

n an

Am

ide-

80 c

olum

n w

ith a

ceto

nitr

ile-

wat

er-f

orm

ic a

cid

(70:

30:0

.1, v

/v/v

, 0.5

ml/m

in).

The

met

hod

was

line

ar o

ver a

con

cent

ratio

n ra

nge

of 1

0–10

000

ng/

ml.

The

aver

age

inte

r-da

y/in

tra-

day

prec

isio

n va

lues

wer

e 3.

7 ±

1.8%

and

4.3

± 1

.8%

, res

pect

ivel

y, w

hile

the

accu

racy

val

ues

wer

e 97

.0 ±

4.8

%. T

his

met

hod

has

been

Suc

cess

fully

app

lied

to P

hase

I of

clin

ical

rese

arch

of p

eram

ivir.

Li e

t al.

2009

nuc

leos

ide

anal

ogue

sV

iram

idin

eRi

bavi

rin

Ant

ivir

al a

ctiv

ity

eval

uatio

nM

ouse

mod

el

Vir

amid

ine,

the

3-ca

rbox

amid

ine

deri

vativ

e of

rib

avir

in, w

as e

ffect

ive

agai

nst a

spe

ctru

m o

f inf

luen

za A

(H1N

1, H

3N2

and

H5N

1) a

nd B

vir

uses

in v

itro,

with

the

50%

effe

ctiv

e co

ncen

trat

ion

(EC

50) r

angi

ng fr

om 2

to 3

2 µg

/ml.

The

mea

n 50

% c

ytot

oxic

con

cent

ratio

n (C

C50

) in

the

MD

CK

cel

ls u

sed

in th

ese

expe

rim

ents

was

760

µg/

ml.

Riba

viri

n, r

un in

pa

ralle

l, ha

d a

sim

ilar

antiv

iral

spe

ctru

m, w

ith E

C50

val

ues

rang

ing

from

0.6

to 5

.5 µ

g/m

l; th

e m

ean

CC

50 fo

r ri

bavi

-ri

n w

as 5

60 µ

g/m

l. O

ral g

avag

e ad

min

istr

atio

ns o

f vir

amid

ine

or r

ibav

irin

to m

ice

infe

cted

with

influ

enza

A/N

WS/

33

(H1N

1), A

/Vic

tori

a/3/

75 (H

3N2)

, B[H

ong

Kon

g/5/

72 o

r B/

Sich

uan/

379/

99 v

irus

es w

ere

high

ly e

ffect

ive

in p

reve

ntin

g de

ath,

less

enin

g de

clin

e in

art

eria

l oxy

gen

satu

ratio

n, in

hibi

tion

Of l

ung

cons

olid

atio

n an

d re

duci

ng lu

ng v

irus

tite

rs.

The

min

imum

effe

ctiv

e do

se o

f vir

amid

ine

in th

ese

stud

ies

rang

ed fr

om 1

5 to

31

mg/

kg/d

ay, d

epen

ding

upo

n th

e vi

rus

infe

ctio

n, w

hen

adm

inis

tere

d tw

ice

daily

for

5 da

ys b

egin

ning

4 h

pre

-vir

us e

xpos

ure.

The

LD

50 o

f the

com

poun

d w

as

610

mg/

kg/d

ay. R

ibav

irin

’s m

inim

um e

ffect

ive

dose

var

ied

betw

een

18 a

nd 3

7.5

mg/

kg/d

ay w

ith th

e LD

50 d

eter

min

ed

to b

e 22

0 m

g/kg

/day

. Vir

amid

ine’s

eff

icac

y w

as a

lso

seen

aga

inst

an

influ

enza

A/N

WS/

33 (H

IN I)

vir

us in

fect

ion

in

mic

e, w

hen

the

com

poun

d w

as a

dmin

iste

red

in th

e dr

inki

ng w

ater

, the

min

imum

effe

ctiv

e do

se b

eing

50

dose

of e

ach,

w

as p

rote

ctiv

e 10

0 m

g/kg

/day

. Del

ay o

f the

initi

atio

n of

eith

er v

iram

idin

e or

rib

avir

in th

erap

y, u

sing

the

appr

oxim

ate

1/3

LD50

dos

e of

eac

h, w

as p

rote

ctiv

e as

late

as

48 h

aft

er e

xpos

ure

to th

e A

/NW

S/33

vir

us. W

hile

bot

h co

mpo

unds

ap

pear

to h

ave

sim

ilar

effic

acy

agai

nst i

nflu

enza

vir

us in

fect

ions

, whe

n on

e co

nsid

ers

the

less

er to

xici

ty, v

iram

idin

e m

ay w

an-a

nt fu

rthe

r ev

alua

tion

as a

pos

sibl

e th

erap

y fo

r in

fluen

za.

Sidw

ell e

t al.

2005


156

nuc

leos

ide

anal

ogue

2’-d

eoxy

-2’-

fluor

ogua

nosi

neA

ntiv

iral

act

ivity

ev

alua

tion

Chi

cken

em

bryo

mod

el

The

nuc

leos

ide

anal

og 2

’-deo

xy-2

’-flu

orog

uano

sine

(2’-f

luor

odG

uo) i

s ph

osph

oryl

ated

by

cellu

lar

enzy

mes

and

reve

rs-

ibly

inhi

bits

influ

enza

vir

us re

plic

atio

n in

chi

ck e

mbr

yo c

ells

with

in th

e fir

st 4

h o

f inf

ectio

n, R

NA

hyb

ridi

zatio

n st

ud-

ies

reve

aled

that

pri

mar

y an

d se

cond

ary

tran

scri

ptio

n of

influ

enza

vir

us R

NA

wer

e bl

ocke

d at

a c

ompo

und

conc

entr

a-tio

n of

10

µM, b

ut n

o in

hibi

tion

of c

ell p

rote

in s

ynth

esis

was

see

n ev

en a

t hig

h co

mpo

und

conc

entr

atio

ns (2

00 µ

M).

In v

itro,

the

trip

hosp

hate

of 2

’-flu

orod

Guo

is a

com

petit

ive

inhi

bito

r of

influ

enza

vir

us tr

ansc

ript

ase

activ

ity fr

om

disr

upte

d vi

rus,

with

a K

-i o

f 1.0

µM

. The

cel

lula

r po

lym

eras

es D

NA

pol

ymer

ase

alph

a an

d RN

A p

olym

eras

e II

wer

e on

ly w

eakl

y in

hibi

ted

or w

ere

insu

scep

tible

to 2

’-flu

orod

GT

P. In

kin

etic

stu

dies

with

the

influ

enza

vir

us tr

ansc

ript

ase,

2’

-flu

orod

GT

P, in

the

abse

nce

of G

TP,

blo

cked

elo

ngat

ion

of th

e vi

rus

RNA

cha

in. S

imila

rly,

by

usin

g pu

rifie

d ri

bonu

-cl

eopr

otei

n co

mpl

exes

it w

as fo

und

that

the

addi

tion

of a

sin

gle

nucl

eotid

e of

2’-f

luor

odG

TP

to th

e vi

rus

RNA

cau

sed

chai

n te

rmin

atio

n, w

hich

resu

lted

in th

e bl

ocka

ge o

f fur

ther

vir

us tr

ansc

ript

ion,

Fur

ther

mor

e, th

e sp

ecifi

city

for

influ

-en

za v

irus

tran

scri

ptas

e w

as c

onfir

med

whe

n th

e tr

ansc

ript

ase

from

par

tially

resi

stan

t vir

us w

as fo

und

to b

e 10

-fol

d le

ss s

usce

ptib

le to

2’-f

luor

odG

TP

(K-i

= 1

3.1

µM).

Tisd

ale

et a

l. 19

95

Endo

nucl

ease

inhi

bito

rs33

diff

eren

t typ

es o

f ph

ytoc

hem

ical

sM

arch

antin

sPl

agio

chin

APe

rrot

tetin

FD

ocki

ng si

mul

atio

n

Influ

enza

A p

osse

sses

an

endo

nucl

ease

with

in it

s RN

A p

olym

eras

e w

hich

com

pris

es P

A, P

B1 a

nd P

B2 s

ubun

its. T

o id

entif

y po

tent

ial n

ew a

nti-

influ

enza

com

poun

ds in

our

cur

rent

stu

dy, w

e sc

reen

ed 3

3 di

ffere

nt ty

pes

of p

hyto

chem

i-ca

ls u

sing

a P

A e

ndon

ucle

ase

inhi

bitio

n as

say

in v

itro

and

an a

nti-

influ

enza

A v

irus

ass

ay. T

he m

arch

antin

s ar

e m

ac-

rocy

clic

bis

bibe

nzyl

s fo

und

in li

verw

orts

, and

pla

gioc

hin

A a

nd p

erro

ttet

in F

are

mar

chan

tin-r

elat

ed p

hyto

chem

ical

s.

We

foun

d fr

om o

ur s

cree

n th

at m

arch

antin

A, B

, E, p

lagi

ochi

n A

and

per

rott

etin

F in

hibi

t inf

luen

za P

A e

ndon

ucle

ase

activ

ity in

vitr

o. T

hese

com

poun

ds h

ave

a 3,

4-di

hydr

oxyp

hene

thyl

gro

up in

com

mon

, ind

icat

ing

the

impo

rtan

ce o

f thi

s m

oiet

y fo

r th

e in

hibi

tion

of P

A e

ndon

ucle

ase.

Doc

king

sim

ulat

ions

of m

arch

antin

E w

ith P

A e

ndon

ucle

ase

sugg

est a

pu

tativ

e “f

ittin

g an

d ch

elat

ing

mod

el” a

s th

e m

echa

nism

und

erly

ing

PA e

ndon

ucle

ase

inhi

bitio

n. T

he d

ocki

ng a

min

o ac

ids

are

wel

l con

serv

ed b

etw

een

influ

enza

A a

nd B

. In

a cu

lture

d ce

ll sy

stem

, mar

chan

tin E

was

furt

her

foun

d to

in

hibi

t the

gro

wth

of b

oth

H3N

2 an

d H

1N1

influ

enza

A v

irus

es, a

nd m

arch

antin

A, E

and

per

rote

in F

sho

wed

inhi

bi-

tory

pro

pert

ies

tow

ards

the

grow

th o

f inf

luen

za B

. The

se m

arch

antin

s al

so d

ecre

ased

the

vira

l inf

ectiv

ity ti

ter,

with

m

arch

antin

E s

how

ing

the

stro

nges

t act

ivity

in th

is a

ssay

. We

addi

tiona

lly id

entif

ied

a ch

emic

al g

roup

that

is c

onse

rved

am

ong

diffe

rent

ant

i-in

fluen

za c

hem

ical

s in

clud

ing

mar

chan

tins,

gre

en te

a ca

tech

ins

and

dihy

drox

y ph

enet

hylp

heny

l-ph

thal

imid

es. O

ur p

rese

nt re

sults

indi

cate

that

mar

chan

tins

are

cand

idat

e an

ti-in

fluen

za d

rugs

and

dem

onst

rate

the

utili

ty o

f the

PA

end

onuc

leas

e as

say

in th

e sc

reen

ing

of p

hyto

chem

ical

s fo

r an

ti-in

fluen

za c

hara

cter

istic

s.

Iwai

et a

l. 20

11

Endo

nucl

ease

and

cap

-bi

ndin

g in

hibi

tors

Co-

crys

tal s

truc

ture

sC

ap-s

natc

hing

mec

hani

smD

iket

o co

mpo

unds

Gre

en te

a ca

tech

in

The

vir

al p

olym

eras

e, w

hich

per

form

s tr

ansc

ript

ion

and

repl

icat

ion

of th

e RN

A g

enom

e, is

an

attr

activ

e ta

rget

for

antiv

iral

dru

gs s

ince

pot

ent p

olym

eras

e in

hibi

tors

cou

ld d

irec

tly s

top

vira

l rep

licat

ion

at a

n ea

rly

stag

e. R

ecen

t str

uc-

tura

l stu

dies

on

func

tiona

l dom

ains

of t

he h

eter

otri

mer

ic p

olym

eras

e, w

hich

com

pris

es s

ubun

its P

A, P

B1 a

nd P

B2,

open

the

way

to a

str

uctu

re b

ased

app

roac

h to

opt

imis

e in

hibi

tors

of v

iral

repl

icat

ion.

In p

artic

ular

, the

uni

que

cap-

snat

chin

g m

echa

nism

of v

iral

tran

scri

ptio

n ca

n be

inhi

bite

d by

targ

etin

g ei

ther

the

PB2

cap-

bind

ing

or P

A e

ndon

ucle

-as

e do

mai

ns. H

ere

we

desc

ribe

hig

h re

solu

tion

X-r

ay c

o-cr

ysta

l str

uctu

res

of th

e 20

09 p

ande

mic

H1N

1 (p

H1N

1) P

A

endo

nucl

ease

dom

ain

with

a s

erie

s of

spe

cific

inhi

bito

rs, i

nclu

ding

four

dik

eto

com

poun

ds a

nd a

gre

en te

a ca

tech

in, a

ll of

whi

ch c

hela

te th

e tw

o cr

itica

l man

gane

se io

ns in

the

activ

e si

te o

f the

enz

yme.

Com

pari

son

of th

e bi

ndin

g m

ode

of

the

diffe

rent

com

poun

ds a

nd th

at o

f a m

onon

ucle

otid

e ph

osph

ate

high

light

s, fi

rstly

, how

diff

eren

t sub

stitu

ent g

roup

s on

the

basi

c m

etal

bin

ding

sca

ffold

can

be

orie

ntat

ed to

bin

d in

dis

tinct

sub

-poc

kets

with

in th

e ac

tive

site

cav

ity, a

nd

seco

ndly

, the

pla

stic

ity o

f cer

tain

str

uctu

ral e

lem

ents

of t

he a

ctiv

e si

te c

avity

, whi

ch re

sult

in in

duce

d fit

bin

ding

. The

se

resu

lts w

ill b

e im

port

ant i

n op

timis

ing

the

desi

gn o

f mor

e po

tent

inhi

bito

rs ta

rget

ing

the

cap-

snat

chin

g en

donu

clea

se

activ

ity o

f inf

luen

za v

irus

pol

ymer

ase.

Kow

alin

ski e

t al.

2012


157

Tran

scri

ptas

e an

d en

donu

clea

se

inhi

bito

rsFl

utim

ide

3-su

bstit

uted

2,

4-di

oxob

utan

oic

acid

s

A n

ovel

ant

i-in

fluen

za v

irus

com

poun

d, fl

utim

ide,

was

iden

tifie

d in

ext

ract

s of

a re

cent

ly id

entif

ied

fung

al s

peci

es,

Del

itsch

ia c

onfe

rtas

pora

. The

com

poun

d, a

sub

stitu

ted

2,6-

dike

topi

pera

zine

, sel

ectiv

ely

inhi

bite

d th

e ca

p-de

pend

ent

tran

scri

ptas

e of

influ

enza

A a

nd B

vir

uses

and

had

no

effe

ct o

n th

e ac

tiviti

es o

f oth

er p

olym

eras

es. S

imila

r to

the

3-su

bstit

uted

2,4

-dio

xobu

tano

ic a

cids

, a s

erie

s of

tran

scri

ptas

e in

hibi

tors

whi

ch w

e de

scri

bed

prev

ious

ly, t

his

inhi

bito

r, w

hich

is a

nat

ural

pro

duct

, affe

cted

nei

ther

the

initi

atio

n no

r th

e el

onga

tion

of in

fluen

za v

irus

mRN

A s

ynth

esis

, but

it

spec

ifica

lly ta

rget

ed th

e ca

p-de

pend

ent e

ndon

ucle

ase

of th

e tr

ansc

ript

ase.

Add

ition

ally

, the

com

poun

d w

as in

hibi

-to

ry to

the

repl

icat

ion

of in

fluen

za A

and

B v

irus

es in

cel

l cul

ture

. The

sel

ectiv

e an

tivir

al p

rope

rtie

s of

this

com

poun

d fu

rthe

r de

mon

stra

te th

e ut

ility

of i

nflu

enza

vir

us e

ndon

ucle

ase

as a

targ

et o

f ant

ivir

al a

gent

s.

Tom

assi

ni e

t al.

1996

Phos

phor

othi

oate

ol

igon

ucle

otid

esIn

a p

revi

ous

stud

y a

15-m

er p

hosp

horo

thio

ate

olig

onuc

leot

ide

(S-O

N) d

eriv

ed fr

om th

e pa

ckag

ing

sign

al in

the

5’ e

nd

of s

egm

ent 1

(PB2

) of i

nflu

enza

A v

irus

(des

igna

ted

5-15

b) p

rove

d m

arke

dly

inhi

bito

ry to

vir

us re

plic

atio

n. H

ere

we

inve

stig

ated

whe

ther

ana

logo

us in

hibi

tory

S-O

Ns

targ

etin

g th

e 5’

end

of s

egm

ents

2 (P

B1) a

nd 3

(PA

) cou

ld b

e id

enti-

fied

and

whe

ther

vir

al re

sist

ance

to S

-ON

s ca

n be

dev

elop

ed. S

imila

r to

our

ear

lier

resu

lt, 2

0-m

er S

-ON

s re

prod

ucin

g th

e 5’

end

s of

seg

men

ts 2

or

3 (c

ompl

emen

tary

to th

e 3’

-cod

ing

regi

ons

of P

B1 a

nd P

A, r

espe

ctiv

ely)

exe

rted

a p

ower

ful

antiv

iral

act

ivity

aga

inst

a v

arie

ty o

f inf

luen

za A

vir

us s

ubty

pes

in M

OC

K c

ells

. Ser

ial p

assa

ge o

f the

A/T

aiw

an/1

/86

H1N

1 st

rain

in th

e pr

esen

ce o

f S-O

N 5

-15b

or

its a

ntis

ense

as5

-15b

ana

logu

e sh

owed

that

mut

ant v

irus

es w

ith re

duce

d su

scep

tibili

ty to

the

S-O

N c

ould

inde

ed b

e ge

nera

ted,

alth

ough

the

resi

stan

t vir

uses

dis

play

ed re

duce

d re

plic

ativ

e fit

ness

. Seq

uenc

ing

the

resi

stan

t vir

uses

iden

tifie

d m

utat

ions

in th

e PB

1, P

B2, P

A a

nd M

1 ge

nes.

Intr

oduc

tion

of

thes

e ch

ange

s in

to th

e A

/PR/

8/34

H1N

1 st

rain

by

reve

rse

gene

tics,

sug

gest

ed th

at a

ltera

tions

to R

NA

func

tion

in th

e pa

ckag

ing

regi

ons

of s

egm

ents

2 a

nd 3

wer

e im

port

ant i

n de

velo

ping

resi

stan

ce to

S-O

N in

hibi

tion.

How

ever

, man

y of

th

e ot

her

sequ

ence

cha

nges

indu

ced

by S

-ON

trea

tmen

t wer

e m

arke

dly

dele

teri

ous

to v

irus

fitn

ess.

We

conc

lude

that

pa

ckag

ing

sign

als

in th

e in

fluen

za A

vir

us p

olym

eras

e se

gmen

ts p

rovi

de fe

asib

le ta

rget

s fo

r nu

clei

c ac

id-b

ased

ant

ivi-

rals

that

may

be

diff

icul

t for

the

viru

s to

eva

de th

roug

h re

sist

ance

mut

atio

ns.

Gia

nnec

chin

i et

al. 2

011

5’-c

appe

d sh

ort

phos

phor

othi

oate

R

nA

frag

men

tsC

ap d

ecoy

Lipo

som

e en

caps

ulat

ion

We

have

sho

wn

prev

ious

ly th

at th

e 5’

-cap

ped

shor

t pho

spho

dies

ter

RNA

frag

men

ts, C

ap d

ecoy

, (G

m 1

2 nt

) are

pot

ent

inhi

bito

rs o

f inf

luen

za v

irus

RN

A p

olym

eras

e ge

ne e

xpre

ssio

n. H

ere

we

inve

stig

ate

the

mod

ified

cap

ped

RNA

der

iva-

tive

cont

aini

ng p

hosp

horo

thio

ate

olig

onuc

leot

ides

(Cap

dec

oy) a

s a

pote

ntia

l inf

luen

za v

irus

RN

A p

olym

eras

e in

hibi

-to

r. T

he m

odifi

ed 5

’-cap

ped

shor

t pho

spho

roth

ioat

e RN

A fr

agm

ents

(Gm

s 12

–15

nt) w

ith th

e 5’

-cap

ped

stru

ctur

e (m

7Gpp

pGm

) wer

e sy

nthe

size

d by

T7

RNA

pol

ymer

ase.

The

5’-c

appe

d sh

ort R

NA

frag

men

ts (G

ms

12–1

5 nt

) wer

e en

caps

ulat

ed in

lipo

som

e pa

rtic

ulat

es a

nd te

sted

for

thei

r in

hibi

tory

effe

cts

on in

fluen

za v

irus

RN

A p

olym

eras

e ge

ne

expr

essi

on in

the

clon

e 76

cel

ls. T

he 1

2–15

nt l

ong

Gm

s RN

A fr

agm

ents

sho

wed

hig

hly

inhi

bito

ry e

ffect

s. B

y co

ntra

st,

the

inhi

bito

ry e

ffect

s of

the

13 n

t lon

g sh

ort R

NA

frag

men

ts (G

m 1

3 nt

) wer

e co

nsid

erab

ly le

ss in

com

pari

son

with

the

5’-c

appe

d sh

ort p

hosp

horo

thio

ate

RNA

frag

men

ts (G

ms

12–1

5 nt

). In

par

ticul

ar, t

he v

ario

us G

ms

RNA

cha

in le

ngth

s sh

owed

no

sign

ifica

nt d

iffer

ence

s in

the

inhi

bitio

n of

influ

enza

vir

us R

NA

pol

ymer

ase

gene

exp

ress

ion.

Fur

ther

mor

e,

the

capp

ed R

NA

with

a p

hosp

horo

thio

ate

back

bone

was

resi

stan

t to

nucl

ease

act

ivity

. The

se p

hosp

horo

thio

ate

RNA

fr

agm

ents

exh

ibite

d hi

gher

inhi

bito

ry a

ctiv

ity th

an th

e 5’

-cap

ped

shor

t RN

A fr

agm

ents

(Gm

12

nt).

The

se d

ecoy

s m

ay

prov

e to

be

usef

ul in

ant

i-in

fluen

za v

irus

ther

apeu

tics.

Tado

et a

l. 20

01


158

Shor

t cap

ped

olig

onuc

leot

ides

The

RN

A-d

epen

dent

RN

A p

olym

eras

e of

influ

enza

vir

us tr

ansc

ribe

s m

esse

nger

RN

A th

roug

h a

uniq

ue c

ap s

cave

ngin

g m

echa

nism

. Vir

al e

nzym

e bi

nds

to th

e ca

p st

ruct

ure

of h

ost m

RNA

, cle

aves

the

mol

ecul

e 9-

15 b

ases

dow

nstr

eam

of

the

cap,

and

use

s th

e sh

ort c

appe

d ol

igon

ucle

otid

e as

a p

rim

er fo

r m

RNA

syn

thes

is. P

revi

ousl

y, w

e ha

ve s

how

n th

at th

e vi

ral p

olym

eras

e ca

n ef

ficie

ntly

bin

d ca

pped

RN

As

shor

ter

than

9 n

ucle

otid

es in

leng

th, b

ut th

e vi

ral e

nzym

e ca

nnot

ut

ilize

thes

e RN

As

as p

rim

ers.

For

this

reas

on, t

hese

sho

rt c

appe

d ol

igon

ucle

otid

es a

re p

oten

t inh

ibito

rs o

f inf

luen

za

viru

s tr

ansc

ript

ion.

In th

ese

stud

ies,

it is

now

sho

wn

that

sho

rt c

appe

d ol

igom

ers

inhi

bit c

appe

d-RN

A d

epen

dent

tr

ansc

ript

ion

at th

e in

itial

ste

p of

cap

bin

ding

. In

cont

rast

, low

con

cent

ratio

ns o

f the

se s

hort

cap

ped

RNA

s ca

n ac

tu-

ally

stim

ulat

e vi

ral t

rans

crip

tion

prim

ed w

ith h

igh

conc

entr

atio

ns o

f the

din

ucle

otid

e A

pG. A

noth

er c

appe

d RN

A

deri

vativ

e co

ntai

ning

pho

spho

roth

ioat

e ol

igon

ucle

otid

es w

as a

lso

inve

stig

ated

as

a po

tent

ial p

olym

eras

e in

hibi

tor.

Thi

s lo

nger

cap

ped

RNA

was

abl

e to

bin

d to

the

poly

mer

ase,

but

cou

ld n

ot b

e cl

eave

d to

pri

mer

leng

th b

y th

e en

zym

e as

soci

ated

end

onuc

leas

e. T

hus,

the

capp

ed p

hosp

horo

thio

ate

RNA

inhi

bite

d ca

p-pr

imed

tran

scri

ptio

n at

the

step

of

cap

bind

ing.

How

ever

, in

cont

rast

to th

e sh

ort c

appe

d ol

igon

ucle

otid

e, it

als

o in

hibi

ted

ApG

pri

med

vir

al tr

ansc

ript

ion.

Cia

nci e

t al.

1997

Endo

nucl

ease

inhi

bito

rs4-

subs

titut

ed

2,4-

diox

obut

anoi

c ac

ids

We

prev

ious

ly id

entifi

ed a

seri

es o

f com

poun

ds w

hich

spec

ifica

lly in

hibi

ted

the

tran

scri

ptio

n of

influ

enza

A a

nd B

vir

uses

. Th

e co

mpo

unds

, 4-s

ubst

itute

d 2,

4-di

oxob

utan

oic

acid

s, se

lect

ivel

y ta

rget

ed th

e ca

p-de

pend

ent e

ndon

ucle

ase

activ

ity o

f th

e tr

ansc

ript

ase

com

plex

. Add

ition

ally

, sev

eral

of t

hese

com

poun

ds e

ffect

ivel

y in

hibi

ted

the

repl

icat

ion

of in

fluen

za v

irus

bu

t not

oth

er v

irus

es in

cel

l cul

ture

ass

ays.

Her

e, w

e re

port

on

the

anti-

influ

enza

vir

us a

ctiv

ities

of o

ther

pot

ent d

eriv

a-tiv

es o

f the

seri

es e

valu

ated

in b

oth

in v

itro

and

in v

ivo

infe

ctiv

ity a

ssay

s. Th

ese

com

poun

ds in

hibi

ted

the

repl

icat

ion

of

influ

enza

vir

us in

yie

ld re

duct

ion

assa

ys, w

ith 5

0% in

hibi

tory

con

cent

ratio

ns ra

ngin

g fr

om 0

.18

to 0

.71

µM. Th

ese

50%

in

hibi

tory

con

cent

ratio

ns w

ere

sim

ilar t

o th

ose

obse

rved

for i

nhib

ition

of i

n vi

tro

tran

scri

ptio

n (0

.32

to 0

.54

µM).

One

se

lect

ed c

ompo

und

also

elic

ited

a do

se-d

epen

dent

inhi

bitio

n of

influ

enza

vir

us re

plic

atio

n in

mic

e fo

llow

ing

an u

pper

re

spir

ator

y tr

act c

halle

nge.

Thes

e st

udie

s dem

onst

rate

the

antiv

iral

effi

cacy

of t

his i

nhib

itor c

lass

and

ther

eby

esta

blis

h th

e ut

ility

of i

nflue

nza

viru

s end

onuc

leas

e as

a c

hem

othe

rape

utic

targ

et.

Has

tings

et a

l. 19

96

Rep

licat

ion/

tr

ansc

ript

ion

inhi

bito

rsC

ompu

tatio

nal a

nd

expe

rim

enta

l scr

eeni

ngC

ompo

und

libra

ry

In th

is st

udy,

com

puta

tiona

l and

exp

erim

enta

l scr

eeni

ng o

f an

exte

nsiv

e co

mpo

und

libra

ry id

entifi

ed T

HC

19, w

hich

w

as a

ble

to su

ppre

ss in

fluen

za v

irus

repl

icat

ion.

This

com

poun

d ha

d no

cyt

otox

ic e

ffect

s and

did

not

dis

rupt

cel

l cyc

le

prog

ress

ion

or in

duce

apo

ptos

is in

MD

CK

cel

ls a

s con

firm

ed b

y W

ST-1

ass

ays,

flow

cyt

omet

ry a

naly

sis,

and

cas

pase

-3

assa

ys. T

ime-

of-a

dditi

on e

xper

imen

ts sh

owed

that

TH

C19

act

s at a

rela

tivel

y ea

rly

stag

e of

the

vira

l life

cycl

e. S

ubse

-qu

ent m

ini-

geno

me

assa

ys re

veal

ed th

at T

HC

19 in

hibi

ted

vira

l gen

ome

repl

icat

ion

and/

or tr

ansc

ript

ion,

sugg

estin

g th

at

it in

terf

eres

with

one

or m

ore

of th

e vi

ral c

ompo

nent

s tha

t for

m th

e ri

bonu

cleo

prot

ein

com

plex

es, n

amel

y po

lym

eras

e ba

sic

2 (P

B2),

poly

mer

ase

basi

c 1

(PB1

), po

lym

eras

e ac

idic

(PA

), nu

cleo

prot

ein

(NP)

and

vir

al R

NA

. Fin

ally

, min

i-ge

nom

e as

says

whe

re P

B2, P

B1, P

A o

r NP

from

A/W

SN/3

3 (H

1N1)

vir

us w

ere

repl

aced

with

thos

e fr

om A

/Udo

rn/3

07/1

972

(H3N

2) v

irus

effe

ctiv

ely

dem

onst

rate

d th

at T

HC

19 in

hibi

ted

vira

l mul

tiplic

atio

n in

a m

anne

r dep

ende

nt u

pon

the

PA

subu

nit.

Take

n to

geth

er, t

hese

resu

lts su

gges

t tha

t infl

uenz

a vi

rus P

A p

rote

in is

a p

oten

tial t

arge

t for

, and

may

aid

the

deve

lopm

ent o

f, no

vel c

ompo

unds

that

inhi

bit i

nflue

nza

A v

irus

repl

icat

ion.

Yam

ada

et a

l. 20

12


159

Shor

t int

erfe

ring

Rn

ARN

A p

olym

eras

e of

influ

enza

vir

us is

a sp

ecifi

c en

zym

e ne

cess

ary

for t

he v

iral

repl

icat

ion.

A si

RNA

aga

inst

the

RNA

po

lym

eras

e an

d th

e RN

A p

olym

eras

e in

hibi

tor L

-742

,001

redu

ced

accu

mul

atio

n of

vir

al R

NA

s in

the

infe

cted

cel

ls.

L-74

2,00

1 st

rong

ly in

hibi

ted

viru

s re-

grow

th a

fter

rem

oval

of t

he a

gent

from

the

cultu

re, w

here

as th

e ne

uram

inid

ase

inhi

bito

r zan

amiv

ir d

id n

ot. L

-742

,001

-res

ista

nt m

utan

ts sh

owed

a Th

r-20

to A

la su

bstit

utio

n in

the

PA su

buni

t of R

NA

po

lym

eras

e. Th

e dr

ug-r

esis

tant

vir

us sh

owed

a sl

ight

redu

ctio

n in

the

susc

eptib

ility

to L

-742

,001

in b

oth

the

plaq

ue a

ssay

(t

hree

fold

redu

ctio

n) a

nd e

nzym

e as

say

(tw

o- to

thre

e-fo

ld re

duct

ion)

. The

resi

stan

ce le

vels

wer

e lo

wer

than

thos

e of

za

nam

ivir-

resi

stan

t mut

ants

in th

e pl

aque

ass

ay. A

gain

st z

anam

ivir-

resi

stan

t mut

ants

, L-7

42,0

01 re

tain

ed th

e sa

me

anti-

vira

l act

ivity

as a

gain

st th

e w

ild-t

ype

stra

in. Th

ese

resu

lts in

dica

te th

at L

-742

,001

is m

ost l

ikel

y to

act

at t

he P

A su

buni

t, an

d po

sses

ses a

uni

que

profi

le. I

t is s

ugge

sted

that

PA

subu

nit o

f RN

A p

olym

eras

e is

a p

rom

isin

g ta

rget

for a

nti-

influ

enza

vi

rus a

gent

s.

Nak

azaw

a et

al.

2008

Tabl

e 4.

4. In

hibi

tors

of n

eura

min

idas

e

neu

ram

inid

ase

Rele

ase

and

spre

ad o

f pr

ogen

y vi

rion

sVi

rus e

ntry

into

cel

l

Influ

enza

vir

us n

eura

min

idas

e (N

A) p

lays

an

esse

ntia

l rol

e in

rele

ase

and

spre

ad o

f pro

geny

vir

ions

, fol

low

ing

the

intr

acel

lula

r vi

ral r

eplic

atio

n cy

cle.

To

test

whe

ther

NA

cou

ld a

lso

faci

litat

e vi

rus

entr

y in

to c

ell,

we

infe

cted

cul

ture

s of

hum

an a

irw

ay e

pith

eliu

m w

ith h

uman

and

avi

an in

fluen

za v

irus

es in

the

pres

ence

of t

he N

A in

hibi

tor

osel

tam

ivir

ca

rbox

ylat

e. T

wen

ty- t

o 50

0-fo

ld le

ss c

ells

bec

ame

infe

cted

in d

rug-

trea

ted

vers

us n

ontr

eate

d cu

lture

s (P

< 0

.000

1) 7

h

afte

r vi

rus

appl

icat

ion,

indi

catin

g th

at th

e dr

ug s

uppr

esse

d th

e in

itiat

ion

of in

fect

ion.

The

se d

ata

dem

onst

rate

that

vi

ral N

A p

lays

a ro

le e

arly

in in

fect

ion,

and

they

pro

vide

furt

her

ratio

nale

for

the

prop

hyla

ctic

use

of N

A in

hibi

tors

.

Mat

roso

vich

et

al. 2

004

neu

ram

inid

ase

Enzy

mat

ic a

ctiv

ityEn

docy

tic p

athw

ays

Vir

us re

plic

atio

n

N2

neur

amin

idas

e (N

A) g

enes

of t

he 1

957

and

1968

pan

dem

ic in

fluen

za v

irus

str

ains

pos

sess

ed a

vian

-lik

e lo

w-p

H

stab

ility

of s

ialid

ase

activ

ity, u

nlik

e m

ost e

pide

mic

str

ains

. We

gene

rate

d fo

ur re

vers

e-ge

netic

s vi

ruse

s fr

om a

gen

etic

ba

ckgr

ound

of A

/WSN

/33

(H1N

1) th

at in

clud

ed p

aren

tal N

2 N

As

of 1

968

pand

emic

(H3N

2) a

nd e

pide

mic

(H2N

2)

stra

ins

or th

eir

coun

terp

art N

2 N

As

in w

hich

the

low

-pH

sta

bilit

y of

the

sial

idas

e ac

tivity

was

cha

nged

by

subs

titu-

tions

of o

ne o

r tw

o am

ino

acid

resi

dues

. We

foun

d th

at th

e tr

ansf

ecta

nt v

irus

es b

eari

ng lo

w-p

H-s

tabl

e si

alid

ase

(WSN

/Sta

ble-

NA

s) s

how

ed 2

5- to

80-

times

-gre

ater

abi

lity

to re

plic

ate

in M

adin

-Dar

by c

anin

e ki

dney

(MD

CK

) cel

ls

than

did

the

tran

sfec

tant

vir

uses

bea

ring

low

-pH

-uns

tabl

e si

alid

ase

(WSN

/ Uns

tabl

e-N

As)

. Enz

ymat

ic a

ctiv

ities

of

WSN

/Sta

ble-

NA

s w

ere

dete

cted

in e

ndos

omes

of M

DC

K c

ells

aft

er 9

0 m

in o

f vir

us in

tern

aliz

atio

n by

in s

itu fl

uore

s-ce

nt d

etec

tion

with

5-b

rom

o-4-

chlo

ro-i

ndol

e-3-

yl-c

t-N

-ace

tyin

eura

min

ic a

cid

and

Fast

Red

Vio

let L

B. In

hibi

tion

of

sial

idas

e ac

tivity

of W

SN/S

tabl

e-N

As

on th

e en

docy

tic p

athw

ay b

y pr

etre

atm

ent w

ith 4

-gua

nidi

no-2

,4-d

ideo

xy-N

-ac

etyl

neur

amin

ic a

cid

(zan

amiv

ir) r

esul

ted

in a

sig

nific

ant d

ecre

ase

in p

roge

ny v

irus

es. I

n co

ntra

st, t

he e

nzym

atic

ac

tiviti

es o

f WSN

/Uns

tabl

e-N

As,

the

repl

icat

ion

of w

hich

had

no

effe

ct o

n pr

etre

atm

ent w

ith z

anam

ivir,

wer

e un

de-

tect

able

in c

ells

und

er th

e sa

me

cond

ition

s. H

emad

sorp

tion

assa

ys o

f tra

nsfe

ctan

t-vi

rus-

infe

cted

cel

ls re

veal

ed th

at

the

low

-pH

sta

bilit

y of

the

sial

idas

e ha

d no

effe

ct o

n th

e pr

oces

s of

rem

oval

of s

ialic

aci

d fr

om h

emag

glut

inin

in th

e G

olgi

regi

ons.

Mor

eove

r, hi

gh ti

ters

of v

irus

es w

ere

reco

vere

d fr

om th

e lu

ngs

of m

ice

infe

cted

with

WSN

/Sta

ble-

NA

s on

day

3 a

fter

intr

anas

al in

ocul

atio

n, b

ut W

SN/U

nsta

ble-

NA

s w

ere

clea

red

from

the

lung

s of

the

mic

e. T

hese

resu

lts

indi

cate

that

sia

lidas

e ac

tivity

in la

te e

ndos

ome/

lyso

som

e tr

affic

enh

ance

s in

fluen

za A

vir

us re

plic

atio

n.

Suzu

ki e

t al.

2005


160

FAn

AA

ntiv

iral

act

ivity

ev

alua

tion

Susc

eptib

ility

to th

e ne

uram

inid

ase-

inhi

bitin

g eff

ects

of a

synt

hetic

ana

log

of n

eura

min

ic a

cid,

2-d

eoxy

-2,3

-deh

ydro

-N-

trifl

uoro

acet

ylne

uram

inic

aci

d (F

AN

A) w

as fo

und

to v

ary

amon

g di

ffere

nt st

rain

s of i

nflue

nza

A v

irus

acc

ordi

ng to

the

neur

amin

idas

e th

ey c

onta

in. I

n pa

rtic

ular

, NW

S (H

0N1)

vir

us a

nd re

com

bina

nt st

rain

s whi

ch d

eriv

e th

eir n

eura

min

idas

e fr

om N

WS

are

espe

cial

ly su

scep

tible

to F

AN

A a

s mea

sure

d by

the

conc

entr

atio

ns o

f inh

ibito

r whi

ch re

duce

rate

s of e

lu-

tion

from

red

cells

, in

vitr

o ne

uram

inid

ase

activ

ity u

sing

fetu

in a

s a su

bstr

ate,

and

vir

us re

plic

atio

n in

cel

l cul

ture

und

er

agar

. Gro

wth

in ti

ssue

cul

ture

of X

-7 (H

0N2)

vir

us a

nd a

ll re

com

bina

nt v

irus

es c

onta

inin

g N

2 ne

uram

inid

ase

is a

ppro

xi-

mat

ely

50–2

00 ti

mes

less

susc

eptib

le to

inhi

bitio

n by

FA

NA

.Thes

e re

sults

pro

vide

furt

her e

vide

nce

that

the

inhi

bito

ry

effec

ts o

f FA

NA

on

the

repl

icat

ion

of in

fluen

za v

irus

es a

re m

edia

ted

by it

s spe

cific

neu

ram

inid

ase-

inhi

bito

ry a

ctiv

ity

and

conf

irm

that

neu

ram

inid

ase

activ

ity is

nec

essa

ry fo

r th

e re

plic

atio

n of

influ

enza

vir

uses

.

Schu

lman

and

Pa

lese

197

5

Zan

amiv

ird

An

A4-

amin

o-d

An

A a

ctiv

ity

Neu

ram

inid

ase

aciti

vity

Rece

ptor

bin

ding

Cel

l fus

ion

4-G

U-D

AN

A (z

anam

ivir)

(as w

ell a

s DA

NA

and

4-A

M-D

AN

A) w

as fo

und

to in

hibi

t the

neu

ram

inid

ase

activ

ity o

f hum

an

para

influ

enza

vir

us ty

pe 3

(HPF

3). Th

e vi

ral n

eura

min

idas

e ac

tivity

is a

ttri

buta

ble

to h

emag

glut

inin

-neu

ram

inid

ase

(HN

), an

env

elop

e pr

otei

n es

sent

ial f

or v

iral a

ttac

hmen

t and

for f

usio

n m

edia

ted

by th

e ot

her e

nvel

ope

prot

ein,

F. W

hile

ther

e is

no

evid

ence

that

HN

’s ne

uram

inid

ase

activ

ity is

ess

entia

l for

rece

ptor

bin

ding

and

sync

ytiu

m fo

rmat

ion,

we

foun

d th

at

4-G

U-D

AN

A p

reve

nted

hem

adso

rptio

n an

d fu

sion

of p

ersi

sten

tly in

fect

ed c

ells

with

uni

nfec

ted

cells

. In

plaq

ue a

ssay

s, 4-

GU

-DA

NA

redu

ced

the

num

ber (

but n

ot th

e ar

ea) o

f pla

ques

if p

rese

nt o

nly

duri

ng th

e ad

sorp

tion

peri

od a

nd re

duce

d pl

aque

are

a (b

ut n

ot n

umbe

r) if

add

ed o

nly

afte

r the

90-

min

ads

orpt

ion

peri

od. 4

-GU

-DA

NA

als

o re

duce

d th

e ar

ea o

f pl

aque

s for

med

by

a ne

uram

inid

ase-

defic

ient

var

iant

, con

firm

ing

that

its i

nter

fere

nce

with

cel

l-cel

l fus

ion

is u

nrel

ated

to

inhi

bitio

n of

neu

ram

inid

ase

activ

ity. Th

e or

der-

of-m

agni

tude

low

er 5

0% in

hibi

tory

con

cent

ratio

ns o

f 4-G

U-D

AN

A (a

nd

also

DA

NA

and

4-A

M-D

AN

A) f

or p

laqu

e ar

ea re

duct

ion

and

for i

nhib

ition

in th

e fu

sion

ass

ay th

an fo

r red

ucin

g pl

aque

nu

mbe

r or b

lock

ing

hem

adso

rptio

n in

dica

te th

e pa

rtic

ular

effi

cacy

of t

hese

sial

ic a

cid

anal

ogs i

n in

terf

erin

g w

ith c

ell-c

ell

fusi

on. I

n ce

ll lin

es e

xpre

ssin

g in

fluen

za v

irus

hem

aggl

utin

in (R

A) a

s the

onl

y vi

ral p

rote

in, w

e fo

und

that

4-G

U-D

AN

A

had

no e

ffect

on

hem

adso

rptio

n bu

t did

inhi

bit H

A2b

-red

blo

od c

ell f

usio

n, a

s jud

ged

by b

oth

lipid

mix

ing

and

cont

ent

mix

ing.

Thus

, 4-G

U-D

AN

A c

an in

terf

ere

with

bot

h in

fluen

za v

irus

- and

HPF

3-m

edia

ted

fusi

on. Th

e re

sults

indi

cate

that

(i)

in H

PF3,

4-G

U-D

AN

A a

nd it

s ana

logs

hav

e an

affi

nity

not

onl

y fo

r the

neu

ram

inid

ase

activ

e si

te o

f HN

but

als

o fo

r site

s im

port

ant f

or re

cept

or b

indi

ng a

nd c

ell f

usio

n an

d (ii

) sia

lic a

cid-

base

d in

hibi

tors

of i

nflue

nza

viru

s neu

ram

inid

ase

can

also

exe

rt a

dir

ect,

nega

tive

effe

ct o

n th

e fu

soge

nic

func

tion

of th

e ot

her

enve

lope

pro

tein

, HA

.

Gre

enga

rd e

t al.

2000

Zan

amiv

irM

ultic

ente

r clin

ical

stud

yA

ntiv

iral

act

ivity

Safe

tyTo

lera

bilit

yC

hina

2010

–201

1A

dole

scen

ts/a

dults

Back

grou

nd It

is th

e fir

st m

ultic

ente

r cl

inic

al s

tudy

in C

hina

to in

vest

igat

e za

nam

ivir

use

am

ong

Chi

nese

ado

lesc

ents

an

d ad

ults

with

influ

enza

-lik

e ill

ness

(ILI

) sin

ce 2

009,

whe

n in

hale

d za

nam

ivir

(REL

ENZ

A) w

as m

arke

ted

in C

hina

. M

etho

ds A

n un

cont

rolle

d op

en-l

abel

, mul

ticen

tre

stud

y to

eva

luat

e th

e an

tivir

al a

ctiv

ity, a

nd s

afet

y of

inha

led

zan-

amiv

ir (a

s Ro

tadi

sk v

ia D

iskh

aler

dev

ice)

; 10

mg

adm

inis

tere

d tw

ice

daily

for

5 da

ys in

sub

ject

s ≥

12 y

ears

old

with

ILI.

Patie

nts

wer

e en

rolle

d w

ithin

48

hour

s of

ons

et a

nd fo

llow

ed fo

r ei

ght d

ays.

Pat

ient

s w

ere

defin

ed a

s be

ing

influ

enza

-po

sitiv

e if

the

real

-tim

e re

vers

e tr

ansc

ript

ase-

poly

mer

ase

chai

n re

actio

n (r

RT-P

CR)

test

had

pos

itive

resu

lts. R

esul

ts

A to

tal o

f 400

pat

ient

s ≥

12 y

ears

old

wer

e sc

reen

ed fr

om 1

1 ce

nter

s in

sev

en p

rovi

nces

from

Mar

ch 2

010

to Ja

nuar

y 20

11. T

hree

hun

dred

and

nin

ety-

two

patie

nts

who

took

at l

east

one

dos

e of

zan

amiv

ir w

ere

ente

red

into

the

safe

ty

anal

ysis

. The

mea

n ag

e w

as 3

3.8

year

s an

d 50

% w

ere

mal

e. C

ardi

ovas

cula

r di

seas

es a

nd d

iabe

tes

wer

e th

e m

ost

com

mon

com

orbi

ditie

s. A

ll th

e re

port

ed a

dver

se e

vent

s, s

uch

as r

ash,

nas

al a

che,

mus

cle

ache

, nau

sea,

dia

rrhe

a, h

ead-

ache

, occ

urre

d in

less

than

1%

of s

ubje

cts.

Mild

sin

us b

rady

cadi

a or

arr

hyth

mia

occ

urre

d in

four

sub

ject

s (1

%).

Mos

t of

the

adve

rse

even

ts w

ere

mild

and

did

not

requ

ire

any

chan

ge o

f tre

atm

ent.

No

seve

re a

dver

se e

vent

s (S

AE)

or

fata

l ca

ses

wer

e re

port

ed. B

ronc

hosp

asm

was

foun

d in

a 3

8 ye

ars

old

wom

an w

hose

sym

ptom

s di

sapp

eare

d af

ter

stop

ping

za

nam

ivir

and

with

out a

dditi

onal

trea

tmen

t. A

ll th

e 61

influ

enza

vir

us is

olat

es (4

3 be

fore

enr

ollm

ent,

18 d

urin

g tr

eat-

men

t) p

rove

d to

be

sens

itive

to z

anam

ivir.

Con

clus

ions

Zan

amiv

ir is

wel

l tol

erat

ed b

y C

hine

se a

dole

scen

ts a

nd a

dults

w

ith IL

Is. T

here

is n

o ev

iden

ce fo

r th

e em

erge

nce

of d

rug-

resi

stan

t iso

late

s du

ring

trea

tmen

t with

zan

amiv

ir.

Cao

et a

l. 20

12


161

ose

ltam

ivir

Avi

an in

fluen

zaN

6 su

btyp

eSu

scep

tibili

ty te

stin

g

Avi

an in

fluen

za v

irus

es a

re a

sou

rce

of g

enet

ic m

ater

ial t

hat c

an b

e tr

ansm

itted

to h

uman

s th

roug

h di

rect

intr

oduc

-tio

n or

rea

ssor

tmen

t. A

lthou

gh th

ere

is a

wea

lth o

f inf

orm

atio

n co

ncer

ning

glo

bal m

onito

ring

for

antiv

iral

res

ista

nce

amon

g hu

man

vir

uses

of t

he N

1 an

d N

2 ne

uram

inid

ase

(NA

) sub

type

s, in

form

atio

n co

ncer

ning

avi

an v

irus

es o

f the

se

and

othe

r N

A s

ubty

pes

is li

mite

d. W

e un

dert

ook

a su

rvei

llanc

e st

udy

to in

vest

igat

e th

e an

tivir

al s

usce

ptib

ility

of

avia

n in

fluen

za N

6 N

A v

irus

es, t

he p

redo

min

ant s

ubty

pe a

mon

g w

ild w

ater

fow

l. W

e ev

alua

ted

73 v

irus

es fr

om N

orth

A

mer

ican

duc

ks a

nd s

hore

bird

s fo

r su

scep

tibili

ty to

the

NA

inhi

bito

r os

elta

miv

ir in

a fl

uore

scen

ce-b

ased

NA

enz

yme

inhi

bitio

n as

say.

Mos

t (90

%) h

ad m

ean

IC50

val

ues

rang

ing

from

< 0

.01

to 5

.0 n

M; 1

0% w

ere

from

5.1

to 5

0.0

nM;

and

none

wer

e >

50.0

nM

. Sus

cept

ibili

ty to

ose

ltam

ivir

rem

aine

d st

able

am

ong

all i

sola

tes

colle

cted

ove

r ap

prox

i-m

atel

y th

ree

deca

des

(P ≤

0.7

4). T

wo

isol

ates

with

122

2V N

A s

ubst

itutio

n ha

d m

oder

atel

y re

duce

d su

scep

tibili

ty to

os

elta

miv

ir in

vitr

o (I

C50

, 30.

0 an

d 40

.0 n

M).

One

fiel

d sa

mpl

e w

as a

mix

ed p

opul

atio

n co

ntai

ning

an

avia

n pa

ra-

myx

ovir

us (A

PMV

) and

H4N

6 in

fluen

za v

irus

, as

reve

aled

by

elec

tron

mic

rosc

opy

and

hem

aggl

utin

atio

n in

hibi

tion

assa

ys w

ith a

pan

el o

f ant

i-A

PMV

ant

iser

a. T

his

high

light

s th

e im

port

ance

of a

war

enes

s an

d ca

refu

l exa

min

atio

n of

no

n-in

fluen

za p

atho

gens

in fi

eld

sam

ples

from

avi

an s

ourc

es. T

his

stud

y sh

owed

that

ose

ltam

ivir

-res

ista

nt N

6 N

A

avia

n in

fluen

za v

irus

es a

re r

are,

and

mus

t be

test

ed b

oth

phen

otyp

ical

ly a

nd g

enot

ypic

ally

to c

onfir

m r

esis

tanc

e.

Ston

er e

t al.

2012

ose

ltam

ivir

car

boxy

late

Zan

amiv

irPr

oduc

tion

of v

iral

pa

rtic

les

Prot

ein

synt

hesi

sLo

ng tu

bula

r vir

us-li

ke

stru

ctur

es

Back

grou

nd: N

eura

min

idas

e (N

A) i

nhib

itors

use

d fo

r in

fluen

za th

erap

y ar

e be

lieve

d to

pre

vent

the

rele

ase

of

prog

eny

viru

s fr

om th

e su

rfac

e of

an

infe

cted

cel

l. In

this

stu

dy, w

e fo

und

that

NA

inhi

bito

rs h

ave

a no

vel a

ntiv

iral

fu

nctio

n ag

ains

t an

avia

n in

fluen

za v

irus

. Res

ults

: Mad

in-D

arby

can

ine

kidn

ey c

ells

, com

mon

ly u

sed

for

the

isol

a-tio

n an

d pr

opag

atio

n of

the

influ

enza

vir

us, w

ere

infe

cted

with

an

avia

n in

fluen

za v

iral

str

ain

A/c

hick

en/G

erm

an/

N/4

9(H

10N

7) (H

10/c

hick

en) o

r a

hum

an in

fluen

za v

iral

str

ain

A/O

saka

/981

/98(

H3N

2) (H

3/O

saka

) vir

us. C

ells

wer

e in

cuba

ted

in a

med

ium

with

out o

r w

ith a

NA

inhi

bito

r, os

elta

miv

ir c

arbo

xyla

te (G

S407

1), f

rom

1 to

13

h po

st in

fec-

tion

(p.i.

). In

fect

ed c

ells

wer

e w

ashe

d 12

h p

.i. to

rem

ove

GS4

071,

incu

bate

d fo

r 1

h w

ithou

t GS4

071,

and

ass

ayed

fo

r vi

rus

prod

uctio

n. In

cuba

tion

with

GS4

071

decr

ease

d th

e pr

oduc

tion

of in

fect

ious

vir

uses

. Whe

n H

10/c

hick

en

viru

s-in

fect

ed c

ells

wer

e in

cuba

ted

with

GS4

071

from

12

to 1

3 h

p.i.

(i.e.

, 1 h

bef

ore

the

viru

s pr

oduc

tion

assa

y),

the

inhi

bito

ry e

ffect

was

cle

arly

obs

erve

d, h

owev

er, t

he s

ame

was

not

evi

dent

for

H3/

Osa

ka v

irus

-inf

ecte

d ce

lls.

Furt

herm

ore,

vir

al p

rote

in s

ynth

esis

in in

fect

ed c

ells

was

not

affe

cted

by

GS4

071.

Usi

ng a

sca

nnin

g el

ectr

on m

icro

-sc

ope,

man

y si

ngle

sph

eric

al b

uds

wer

e ob

serv

ed o

n th

e su

rfac

e of

H3/

Osa

ka v

irus

-inf

ecte

d ce

lls in

cuba

ted

with

out

GS4

071,

whe

reas

man

y ag

greg

ated

par

ticle

s w

ere

obse

rved

on

the

surf

ace

of c

ells

incu

bate

d w

ith G

S407

1. H

owev

er,

man

y lo

ng tu

bula

r vi

rus-

like

stru

ctur

es, w

ith n

o ag

greg

ated

par

ticle

s, w

ere

obse

rved

on

the

surf

ace

of H

10/c

hick

en

viru

s-in

fect

ed c

ells

incu

bate

d w

ith G

S407

1. T

he s

ame

resu

lts w

ere

obta

ined

whe

n an

othe

r N

A in

hibi

tor,

zana

miv

ir,

was

use

d. C

oncl

usio

ns: T

hese

res

ults

indi

cate

that

NA

inhi

bito

rs in

terf

ered

with

vir

us p

artic

le fo

rmat

ion

in th

e H

10/

chic

ken

viru

s-in

fect

ed c

ells

, in

whi

ch th

e in

hibi

tor

caus

ed th

e fo

rmat

ion

of lo

ng tu

bula

r vi

rus-

like

stru

ctur

es in

stea

d of

sph

eric

al v

irus

par

ticle

s.

Ush

irog

awa

and

Ohu

chi 2

011


162

Pera

miv

irEv

alua

tion

of a

ntiv

iral

ac

tivity

Mou

se m

odel

New

and

em

ergi

ng in

fluen

za v

irus

stra

ins,

such

as t

he p

ande

mic

influ

enza

A (H

1N1)

vir

us re

quire

con

stan

t vig

ilanc

e fo

r ant

ivira

l dru

g se

nsiti

vity

and

resi

stan

ce. E

ffica

cy o

f int

ram

uscu

larly

(IM

) adm

inis

tere

d ne

uram

inid

ase

(NA

) inh

ibi-

tor,

pera

miv

ir, w

as e

valu

ated

in m

ice

infe

cted

with

rece

ntly

isol

ated

pan

dem

ic A

/Cal

iforn

ia/0

4/20

09 (H

1N1,

swin

e or

igin

, m

ouse

ada

pted

) infl

uenz

a vi

rus.

A si

ngle

IM in

ject

ion

of p

eram

ivir

(four

dos

e gr

oups

), gi

ven

1 h

prio

r to

inoc

ulat

ion,

si

gnifi

cant

ly re

duce

d w

eigh

t los

s (P

< 0.

001)

and

mor

talit

y (P

< 0

.05)

in m

ice

infe

cted

with

LD

(90)

dos

e of

pan

dem

ic A

/C

alifo

rnia

/04/

2009

(H1N

1) in

fluen

za v

irus

com

pare

d to

veh

icle

gro

up. Th

ere

was

20%

surv

ival

in th

e ve

hicl

e-tr

eate

d gr

oup,

w

here

as in

the

pera

miv

ir-tr

eate

d gr

oups

, sur

viva

l inc

reas

ed in

a d

ose-

depe

nden

t man

ner w

ith 6

0, 6

0, 9

0 an

d 10

0% su

rviv

ors

for t

he 1

, 3, 1

0, a

nd 3

0 m

g/kg

dos

es, r

espe

ctiv

ely.

Wei

ght l

oss o

n da

y 4

in th

e ve

hicl

e-tr

eate

d gr

oup

was

3.4

gm

, and

in th

e pe

ram

ivir-

trea

ted

grou

ps w

as 2

.1, 1

.5, 1

.8 a

nd 1

.8 g

for t

he 1

,3, 1

0 an

d 30

mg/

kg d

ose

grou

ps, r

espe

ctiv

ely.

In th

e tr

eatm

ent

mod

el, p

eram

ivir

give

n 24

h a

fter i

nfec

tion

as a

sing

le IM

inje

ctio

n at

50

mg/

kg d

ose,

show

ed si

gnifi

cant

pro

tect

ion

agai

nst

leth

ality

and

wei

ght l

oss.

Ther

e w

as 1

3% su

rviv

al in

the

vehi

cle-

trea

ted

grou

p w

hile

in th

e pe

ram

ivir-

trea

ted

grou

p at

24,

48,

an

d 72

h p

ost i

nfec

tion,

surv

ival

was

100

, 40,

and

50%

, res

pect

ivel

y. Su

rviv

al in

the

osel

tam

ivir

grou

ps (1

0 m

g/kg

/day

twic

e a

day,

oral

ly fo

r 5 d

ays)

was

90,

30

and

20%

at 2

4, 4

8 an

d 72

h, re

spec

tivel

y. Th

ese

data

dem

onst

rate

effi

cacy

of p

aren

tera

lly

adm

inis

tere

d pe

ram

ivir

agai

nst t

he re

cent

ly is

olat

ed p

ande

mic

influ

enza

vir

us in

mur

ine

infe

ctio

n m

odel

s.

Bant

ia e

t al.

2010

Pera

miv

iro

selt

amiv

irZ

anam

ivir

Effica

cy o

f a si

ngle

in

tram

uscu

lar i

njec

tion

Mou

se m

odel

In th

e ev

ent o

f an

influ

enza

out

brea

k, a

ntiv

iral

s inc

ludi

ng th

e ne

uram

inic

lase

(NA

) inh

ibito

rs, p

eram

ivir,

ose

ltam

ivir,

and

za

nam

ivir

may

pro

vide

val

uabl

e be

nefit

whe

n va

ccin

e pr

oduc

tion

is d

elay

ed, l

imite

d, o

r can

not b

e us

ed. H

ere

we

dem

-on

stra

te th

e effi

cacy

of a

sing

le in

tram

uscu

lar i

njec

tion

of p

eram

ivir

in th

e m

ouse

influ

enza

mod

el. P

eram

ivir

pot

ently

in

hibi

ts th

e ne

uram

inid

ase

enzy

me

N9

from

H1N

9 vi

rus i

n vi

tro

with

a 5

0% in

hibi

tory

con

cent

ratio

n (I

C50

) of 1

.3 ±

0.4

nM

. On-

site

dis

soci

atio

n st

udie

s ind

icat

e th

at p

eram

ivir

rem

ains

tigh

tly b

ound

to N

9 N

A (t

1/2 >

24

h), w

here

as, z

an-

amiv

ir a

nd o

selta

miv

ir c

arbo

xyla

te d

isso

ciat

ed ra

pidl

y fr

om th

e en

zym

e (t

1/2 =

1.2

5 h)

. A si

ngle

intr

amus

cula

r inj

ectio

n of

per

amiv

ir (1

0mg/

kg) s

igni

fican

tly re

duce

s wei

ght l

oss a

nd m

orta

lity

in m

ice

infe

cted

with

influ

enza

A/H

1N1,

whi

le

osel

tam

ivir

dem

onst

rate

s no

effica

cy b

y th

e sa

me

trea

tmen

t reg

imen

. This

may

be

due

to ti

ght b

indi

ng o

f per

amiv

ir to

th

e N

I NA

enz

ymes

sim

ilar t

o th

at o

bser

ved

for N

9 en

zym

e. A

dditi

onal

effi

cacy

stud

ies i

ndic

ate

that

a si

ngle

inje

ctio

n of

pe

ram

ivir

(2–2

0 m

g/kg

) was

com

para

ble

to a

q.d

. × 5

day

cou

rse

of o

rally

adm

inis

tere

d os

elta

miv

ir (2

–20m

g/kg

/day

) in

prev

entin

g le

thal

ity in

H3N

2 an

d H

1N1

influ

enza

mod

els.

A si

ngle

intr

amus

cula

r inj

ectio

n of

per

amiv

ir m

ay su

cces

sful

ly

trea

t infl

uenz

a in

fect

ions

and

pro

vide

an

alte

rnat

e op

tion

to o

selta

miv

ir d

urin

g an

influ

enza

out

brea

k.

Bant

ia e

t al.

2006

Lani

nam

ivir

Eval

uatio

n of

ant

ivir

al

activ

ityM

ouse

/fer

ret m

odel

We

have

repo

rted

on

lani

nam

ivir

(cod

e na

me,

R-1

2548

9), a

nov

el n

eura

min

idas

e in

hibi

tor,

and

have

dis

cove

red

that

th

e la

nina

miv

ir p

rodr

ug C

S-89

58 w

orke

d as

a lo

ng-a

ctin

g ne

uram

inid

ase

inhi

bito

r in

a m

ouse

influ

enza

vir

us in

fect

ion

mod

el w

hen

it is

intr

anas

ally

adm

inis

tere

d. In

this

stud

y, C

S-89

58 w

as a

dmin

iste

red

just

onc

e 7

days

bef

ore

infe

ctio

n an

d sh

owed

sign

ifica

nt e

ffica

cy in

viv

o. Th

e effi

cacy

of a

sing

le a

dmin

istr

atio

n of

CS-

8958

aft

er v

iral

infe

ctio

n w

as th

en

com

pare

d w

ith th

at o

f rep

eate

d ad

min

istr

atio

ns o

f ose

ltam

ivir

pho

spha

te o

r zan

amiv

ir in

mic

e an

d fe

rret

s. C

S-89

58

show

ed e

ffica

cy su

peri

or o

r sim

ilar t

o th

e effi

caci

es o

f the

two

licen

sed

NA

inhi

bito

rs. C

S-89

58 a

lso

sign

ifica

ntly

re

duce

d th

e tit

ers o

f an

osel

tam

ivir-

resi

stan

t H1N

1 vi

rus w

ith a

neu

ram

inid

ase

H27

4Y su

bstit

utio

n in

a m

ouse

infe

c-tio

n m

odel

. Thes

e re

sults

sugg

est t

hat s

ince

CS-

8958

is c

hara

cter

istic

ally

long

last

ing

in th

e lu

ngs,

it m

ay b

e id

eal f

or

the

prop

hyla

xis a

nd tr

eatm

ent o

f infl

uenz

a.

Kub

o et

al.

2010

Zan

amiv

irM

ultim

eric

con

juga

tes

A se

t of t

rim

eric

and

tetr

amer

ic d

eriv

ativ

es 6

-11

of th

e in

fluen

za v

irus

neu

ram

inid

ase

inhi

bito

r zan

amiv

ir 1

have

bee

n sy

nthe

size

d by

cou

plin

g a

com

mon

mon

omer

ic z

anam

ivir

deri

vativ

e 3

onto

var

ious

mul

timer

ic c

arbo

xylic

aci

d co

re g

roup

s. Th

ese

disc

rete

mul

timer

ic c

ompo

unds

are

all

sign

ifica

ntly

mor

e an

tivira

l tha

n za

nam

ivir

and

also

show

out

stan

ding

long

-la

stin

g pr

otec

tive

activ

ity w

hen

test

ed in

mou

se in

fluen

za in

fect

ivity

exp

erim

ents

. (C

) 200

4 El

sevi

er L

td. A

ll ri

ghts

rese

rved

.

Wat

son

et a

l. 20

04


163

Poly

vale

nt z

anam

ivir

co

njug

ates

Synt

hesi

sA

ntiv

iral

effe

ct

Poly

vale

nt s

ialid

ase

inhi

bito

rs b

eari

ng 4

-gua

nidi

no-N

eu5A

c2en

der

ivat

ives

on

a po

ly-L

-glu

tam

ine

back

bone

are

de

scri

bed.

Aim

ing

for

a lo

nger

rete

ntio

n tim

e of

4-g

uani

dino

-Neu

5Ac2

en (z

anam

ivir

) in

bron

chi a

nd lu

ngs,

we

focu

sed

on s

uper

mol

ecul

es b

eari

ng 4

-gua

nidi

no-N

eu5A

c2en

der

ivat

ives

bou

nd a

t the

ir C

-7 p

ositi

on th

roug

h no

n-cl

eava

ble

alky

l eth

er li

nkag

es. W

e fir

st fo

und

that

alk

ylat

ion

of th

e 7-

hydr

oxyl

gro

up o

f sia

lic a

cid

deri

vativ

e 8

pro-

ceed

ed s

moo

thly

, and

pro

duce

d 7-

O-a

lkyl

-4-g

uani

dino

-Neu

5Ac2

en d

eriv

ativ

es 1

3, w

hich

exh

ibite

d eq

uipo

tent

inhi

bi-

tory

act

ivity

aga

inst

not

onl

y in

fluen

za A

vir

us s

ialid

ase

but a

lso

influ

enza

A v

irus

in th

e ce

ll cu

lture

. Nex

t, w

e sy

n-th

esiz

ed p

oly-

L-gl

utam

ine

bear

ing

7-O

-alk

yl-4

-gua

nidi

no-N

eu5A

c2en

der

ivat

ives

link

ed b

y am

ide

bond

s, 2

6, w

hich

sh

owed

enh

ance

d an

tivir

al a

ctiv

ity a

gain

st in

fluen

za A

vir

us a

nd m

ore

pote

nt e

ffic

acy

in v

ivo

rela

tive

to a

mon

omer

ic

sial

idas

e in

hibi

tor.

Mas

uda

et a

l. 20

03

dua

l-ta

rget

ed

bifu

ncti

onal

ant

i-in

fluen

za d

rugs

Caff

eic

acid

-zan

amiv

ir

conj

ugat

eSu

ppre

ssio

n of

pro

-in

flam

ator

y cy

toki

nes

Influ

enza

ther

apy

with

a s

ingl

e ta

rget

ed c

ompo

und

is o

ften

lim

ited

in e

ffic

acy

due

to th

e ra

pidl

y de

velo

ped

drug

re

sist

ance

. Mor

eove

r, th

e un

cont

rolle

d vi

rus-

indu

ced

cyto

kine

s co

uld

caus

e th

e hi

gh m

orta

lity

of h

uman

infe

cted

by

H5N

1 av

ian

influ

enza

vir

us. I

n th

is s

tudy

, we

expl

ored

the

nove

l dua

l-ta

rget

ed b

ifunc

tiona

l ant

i-in

fluen

za d

rugs

fo

rmed

by

conj

ugat

ion

with

ant

i-in

flam

mat

ory

agen

ts. I

n pa

rtic

ular

, the

caf

feic

aci

d (C

A)-

bear

ing

zana

miv

ir (Z

A)

conj

ugat

es Z

A-7

-CA

(1) a

nd Z

A-7

-CA

-am

ide

(7) s

how

ed s

imul

tane

ous

inhi

bitio

n of

influ

enza

vir

us n

eura

min

idas

e an

d su

ppre

ssio

n of

pro

-inf

lam

mat

ory

cyto

kine

s. T

hese

ZA

con

juga

tes

prov

ided

rem

arka

ble

prot

ectio

n of

cel

ls a

nd

mic

e ag

ains

t inf

luen

za in

fect

ions

. Int

rana

sal a

dmin

istr

atio

n of

low

dos

age

(< 1

.2 µ

mol

/kg/

day)

of Z

A c

onju

gate

s ex

hibi

ted

muc

h gr

eate

r ef

fect

than

the

com

bina

tion

ther

apy

with

ZA

and

the

anti-

infla

mm

ator

y ag

ents

in p

rote

ctio

n of

the

leth

ally

infe

cted

mic

e by

H1N

1 or

H5N

1 in

fluen

za v

irus

es.

Liu

et a

l. 20

12

Thea

flavi

nsN

eura

min

idas

e ac

tivity

as

say

Hae

mag

glut

inat

ion

inhi

bitio

n as

say

Real

-tim

e qu

antit

ativ

e PC

R A

nti-i

nflam

mat

ory

prop

ertie

s

The

thea

flavi

ns fr

actio

n (T

F80%

, with

a p

urity

of 8

0%) a

nd th

ree

thea

flavi

n (T

F) d

eriv

ativ

es fr

om b

lack

tea

have

bee

n fo

und

to e

xhib

it po

tent

inhi

bito

ry e

ffect

s ag

ains

t inf

luen

za v

irus

in v

itro.

The

y w

ere

eval

uate

d w

ith a

neu

ram

inid

ase

(NA

) act

ivity

ass

ay, a

hem

aggl

utin

atio

n (H

A) i

nhib

ition

ass

ay, a

real

-tim

e qu

antit

ativ

e PC

R (q

PCR)

ass

ay fo

r ge

ne

expr

essi

on o

f hem

aggl

utin

in (H

A) a

nd a

cyt

opat

hic

effe

ct (C

PE) r

educ

tion

assa

y. T

he e

xper

imen

tal r

esul

ts s

how

ed

that

they

all

exer

ted

sign

ifica

nt in

hibi

tory

effe

cts

on th

e N

A o

f thr

ee d

iffer

ent s

ubty

pes

of in

fluen

za v

irus

str

ains

[A/

PR/8

/34(

H1N

1), A

/Syd

ney/

5/97

( H3N

2) a

nd 8

/Jia

ngsu

/10/

2003

] with

50%

inhi

bito

ry c

once

ntra

tion

(IC

50) v

alue

s ra

ngin

g fr

om 9

.27

to 3

6.55

µg/

ml,

and

they

als

o di

spla

yed

an in

hibi

tory

effe

ct o

n H

A; t

hese

inhi

bito

ry e

ffect

s m

ight

co

nstit

ute

two

maj

or m

echa

nism

s of

thei

r an

tivir

al a

ctiv

ity. T

ime-

of-a

dditi

on s

tudi

es d

emon

stra

ted

that

TF

deri

vativ

es

mig

ht h

ave

a di

rect

effe

ct o

n vi

ral p

artic

le in

fect

ivity

, whi

ch w

as c

onsi

sten

t with

the

inhi

bito

ry e

ffect

on

HA

. Sub

se-

quen

tly, t

he in

hibi

tory

effe

ct o

f TF

deri

vativ

es o

n th

e re

plic

atio

n of

the

vira

l HA

gen

e as

ass

ayed

by

qPC

R an

d on

the

nucl

ear

loca

lizat

ion

of th

e in

fluen

za v

irus

vRN

P fu

rthe

r de

mon

stra

ted

that

they

may

pri

mar

ily a

ct d

urin

g th

e ea

rly

stag

e of

infe

ctio

n. In

tere

stin

gly,

bes

ides

the

activ

ity a

gain

st fu

nctio

nal v

iral

pro

tein

s, T

F de

riva

tives

als

o de

crea

sed

the

expr

essi

on le

vel o

f the

infla

mm

ator

y cy

toki

ne IL

-6 d

urin

g vi

ral i

nfec

tion,

exp

ress

ion

of w

hich

may

resu

lt in

ser

ious

tis

sue

inju

ry a

nd a

popt

osis

. Our

resu

lts in

dica

ted

that

TF

deri

vativ

es a

re p

oten

tial c

ompo

unds

with

ant

i-in

fluen

za

vira

l rep

licat

ion

and

anti-

infla

mm

ator

y pr

oper

ties.

The

se fi

ndin

gs w

ill p

rovi

de im

port

ant i

nfor

mat

ion

for

new

dru

g de

sign

and

dev

elop

men

t for

the

trea

tmen

t of i

nflu

enza

vir

us in

fect

ion.

Zu e

t al.

2012


164

Trad

itio

nal C

hine

se

med

icin

esW

ater

ext

ract

s fro

m

plan

tsM

ouse

mod

el

Etno

phar

mac

olog

ical

rele

vanc

e: N

eura

min

idas

e (N

A) i

nhib

itors

are

cur

rent

ly th

e m

ost e

ffect

ive

drug

s to

trea

t inf

lu-

enza

A v

irus

es in

fect

ion.

Man

y tr

aditi

onal

Chi

nese

med

icin

es (T

CM

s) h

ave

been

use

d in

the

clin

ics

to tr

eat i

nflu

-en

za. T

he a

nti-

vira

l mec

hani

sms

of th

ese

TC

Ms

and

thei

r in

hibi

tory

effe

cts

tow

ards

NA

nee

d to

be

syst

emat

ical

ly

test

ed. A

im o

f the

stu

dy: T

o ev

alua

te th

e an

ti-N

A a

ctiv

ity o

f the

TC

Ms

and

the

anti-

influ

enza

A v

irus

effe

cts

of th

e N

A in

hibi

tory

TC

Ms

in v

itro

and

in v

ivo.

Mat

eria

l and

met

hods

: We

test

ed th

e in

hibi

tory

act

ivity

of w

ater

ext

ract

s fr

om 4

39 T

CM

s to

war

ds N

A. T

he in

vitr

o an

ti-in

fluen

za v

irus

act

iviti

es o

f the

5 T

CM

s w

ere

eval

uate

d us

ing

the

stra

in A

/Cal

iforn

ia/7

/200

9 (H

1N1)

NYM

C X

-179

A o

f inf

luen

za A

vir

us. A

ran

dom

ly s

elec

ted

TC

M w

ith N

A in

hibi

-to

ry a

ctiv

ity. M

elia

toos

enda

n ex

trac

t, w

as fu

rthe

r ev

alua

ted

usin

g a

mou

se m

odel

infe

cted

with

influ

enza

A v

irus

. Re

sults

: Fiv

e T

CM

s, D

uche

snea

indi

ca, F

ocke

(Fra

gari

a in

dica

), Li

quid

amba

r fo

rmos

ana,

Lith

ospe

rmum

ery

thro

rhi-

zon,

Mel

ia to

osen

dan,

and

Pru

nella

vul

gari

s, e

xert

ed p

oten

t inh

ibito

ry a

ctiv

ity to

war

ds N

A. T

hese

TC

Ms

in th

e ra

nge

of 2

5–25

0 µg

/ml h

ad th

e ab

ility

to re

duce

vir

us-i

nduc

ed c

ytop

athi

c ef

fect

(CPE

) and

the

viru

s yi

eld

in M

DC

K c

ells

. M

elia

toos

enda

n si

gnifi

cant

ly re

duce

d de

ath

rate

and

pro

long

ed m

ean

day

to d

eath

(MD

D) o

f the

vir

al in

fect

ed m

ice.

C

oncl

usio

ns: T

his

stud

y de

scri

bes

five

TC

Ms

exer

ted

stro

ng in

hibi

tory

act

iviti

es to

war

ds N

A, a

nd e

xhib

ited

antiv

i-ra

l effe

ct a

gain

st in

fluen

za A

vir

us b

y re

duci

ng v

iral

repr

oduc

tion

and

redu

ced

CPE

of t

he v

iral

infe

cted

cel

ls. M

elia

to

osen

dan,

sig

nific

antly

redu

ced

deat

h ra

te a

nd p

rolo

nged

sur

viva

l of t

he H

1N1

vira

l inf

ecte

d m

ice.

Tian

et a

l. 20

11

Plan

t flav

onol

sG

ossy

petin

Kae

mpf

erol

Eval

uatio

n of

ant

ivir

al

activ

ity

Five

flav

onol

s (3

, 5, a

nd 9

–11)

wer

e is

olat

ed fr

om R

hodi

ola

rose

a, a

nd c

ompa

red

with

com

mer

cial

ly a

vaila

ble

fla-

vono

ids

(1, 2

, 4, 6

-8, a

nd 1

2–14

) to

faci

litat

e an

alys

is o

f the

ir s

truc

ture

-act

ivity

rela

tions

hip

(SA

R). A

ll co

mpo

unds

(1

–14)

sho

wed

neu

ram

inid

ase

inhi

bito

ry a

ctiv

ities

with

IC50

val

ues

rang

ing

from

0.8

to 5

6.9

µM. T

he in

vitr

o an

ti-in

fluen

za v

irus

act

iviti

es o

f fla

vono

ids

1-6,

8–1

2, a

nd 1

4 w

ere

eval

uate

d us

ing

two

influ

enza

vir

al s

trai

ns, H

1N1

(A/

PR/8

/34)

and

H9N

2 (A

/Chi

cken

/Kor

ea/M

S96/

96),

test

ing

thei

r ab

ility

to re

duce

vir

us-i

nduc

ed c

ytop

athi

c ef

fect

(C

PE) i

n M

DC

K c

ells

. We

foun

d th

at th

e ac

tivity

of t

hese

com

poun

ds r

ange

d fr

om 3

0.2

to 9

9.1

µM a

gain

st H

1N1-

and

18

.5 to

133

.6 µ

M a

gain

st H

9N2-

indu

ced

CPE

. Of c

ompo

unds

1–1

4, g

ossy

petin

(6) e

xhib

ited

the

mos

t pot

ent i

nhib

i-to

ry a

ctiv

ity, w

ith IC

50 v

alue

s of

0.8

and

2.6

µM

on

neur

amin

idas

es fr

om C

lost

ridi

um p

erfr

inge

ns a

nd re

com

bina

nt

influ

enza

vir

us A

(rvH

1N1)

, res

pect

ivel

y. In

con

tras

t, ka

empf

erol

(3) e

xhib

ited

the

high

est a

ctiv

ity a

gain

st tw

o in

flu-

enza

vir

uses

, H1N

1 an

d H

9N2

with

EC

50 v

alue

s of

30.

2 an

d 18

.5 µ

M, r

espe

ctiv

ely.

Act

ivity

dep

ende

d on

the

posi

tion

and

num

ber

of h

ydro

xy g

roup

s on

the

flavo

noid

s ba

ckbo

ne. I

n ki

netic

stu

dies

, all

isol

ated

com

poun

ds b

ehav

ed a

s no

ncom

petit

ive

inhi

bito

rs.

Jeon

g et

al.

2009

1,3-

diar

ylpr

op-2

-en-

1-on

e de

riva

tive

sSy

nthe

sis

Eval

uatio

n

A s

erie

s of

1,3

-dia

rylp

rop-

2-en

-1-o

ne d

eriv

ativ

es 3

a-v

have

bee

n sy

nthe

size

d an

d ev

alua

ted

for

thei

r ab

ility

to in

hibi

t ne

uram

inid

ase

(NA

). A

mon

g th

e pr

epar

ed c

ompo

unds

, the

less

lipo

phili

c de

riva

tive

3k s

how

ed th

e m

ost p

oten

t in

vitr

o in

hibi

tory

act

ivity

aga

inst

NA

with

an

IC50

val

ue o

f 1.5

µ M

.

Kin

ger e

t al.

2012


165

Cyc

lope

ntan

e de

riva

tive

sA

nov

el se

ries

of c

yclo

pent

ane

deri

vativ

es h

ave

been

foun

d to

exh

ibit

pote

nt a

nd se

lect

ive

inhi

bito

ry e

ffect

s on

influ

enza

vi

rus n

eura

min

idas

e, Th

ese

com

poun

ds, d

esig

nate

d RW

J-27

0201

, BC

X-1

827,

BC

X-1

898,

and

BC

X-1

923,

wer

e te

sted

in

par

alle

l with

zan

amiv

ir a

nd o

selta

miv

ir c

arbo

xyla

te a

gain

st a

spec

trum

of i

nflue

nza

A (H

1N1,

H3N

2, a

nd H

5N1)

an

d in

fluen

za B

vir

uses

in M

DC

K c

ells

, inh

ibiti

on o

f vir

al c

ytop

athi

c eff

ect a

scer

tain

ed v

isua

lly a

nd b

y ne

utra

l red

dye

up

take

was

use

d, w

ith 5

0% e

ffect

ive

(vir

us-in

hibi

tory

) con

cent

ratio

ns (E

C50

) det

erm

ined

. Aga

inst

the

H1N

1 vi

ruse

s A/

Baye

rn/0

7/95

, A/B

eijin

g/26

2/95

, A/P

R/8/

34, a

nd A

/Tex

as/3

6/91

, EC

(50)

s (de

term

ined

by

neut

ral r

ed a

ssay

) of t

he n

ovel

co

mpo

unds

wer

e le

ss th

an o

r equ

al to

1.5

µM

. Tw

elve

stra

ins o

f H3N

2 an

d tw

o st

rain

s of a

vian

H5N

1 vi

ruse

s wer

e in

hib-

ited

at <

0.3

µM

Influ

enza

B/B

eijin

g/18

4/93

and

B/H

arbi

n/07

/94

viru

ses w

ere

inhi

bite

d at

< 0

.2 µ

M, w

ith th

ree

othe

r B

viru

s str

ains

inhi

bite

d at

0.8

to 8

µM

, The

nove

l inh

ibito

rs w

ere

com

para

ble

in p

oten

cy to

(or s

light

ly m

ore

pote

nt th

an)

zana

miv

ir a

nd o

selta

miv

ir c

arbo

xyla

te. N

o cy

toto

xici

ty w

as se

en w

ith th

e co

mpo

unds

at c

once

ntra

tions

of l

ess t

han

or

equa

l to

1 m

M in

cel

l pro

lifer

atio

n as

says

. The

antiv

iral

act

ivity

of R

WJ-

2702

01, c

hose

n fo

r clin

ical

dev

elop

men

t, w

as

stud

ied

in g

reat

er d

etai

l. It

s pot

ency

and

that

of o

selta

miv

ir c

arbo

xyla

te d

ecre

ased

with

incr

easi

ng m

ultip

licity

of v

irus

in

fect

ion.

Tim

e-of

-add

ition

stud

ies i

ndic

ated

that

trea

tmen

t with

eith

er c

ompo

und

need

ed to

beg

in 0

to 1

2 h

afte

r vir

us

expo

sure

for o

ptim

al a

ctiv

ity. E

xpos

ure

of c

ells

to R

WJ-

2702

01 c

ause

d m

ost o

f the

vir

us to

rem

ain

cell

asso

ciat

ed, w

ith

extr

acel

lula

r vir

us d

ecre

asin

g in

a c

once

ntra

tion-

depe

nden

t man

ner.

This

is c

onsi

sten

t with

its e

ffect

as a

neu

ram

inid

ase

inhi

bito

r. RW

J-27

0201

show

s pro

mis

e in

the

trea

tmen

t of h

uman

influ

enza

vir

us in

fect

ions

.

Smee

et a

l. 20

01

Res

ista

nce

to

neur

amin

idas

e in

hibi

tors

Con

serv

ed re

sidu

esV

iral

fitn

ess

Neu

ram

inid

ase

inhi

bito

rs (N

AIs

) are

ant

ivir

als d

esig

ned

to ta

rget

con

serv

ed re

sidu

es a

t the

neu

ram

inid

ase

(NA

) enz

yme

activ

e si

te in

influ

enza

A a

nd B

vir

uses

. The

cons

erve

d re

sidu

es th

at in

tera

ct w

ith N

AIs

are

und

er se

lect

ive

pres

sure

, but

on

ly a

few

hav

e be

en li

nked

to re

sist

ance

. In

the

A/W

uhan

/359

/95

(H3N

2) re

com

bina

nt v

irus

bac

kgro

und,

we

char

ac-

teri

zed

seve

n ch

arge

d, c

onse

rved

NA

resi

dues

(111

18, R

371,

E22

7, R

152,

R22

4, E

276,

and

D15

1) th

at d

irec

tly in

tera

ct

with

the

NA

ls b

ut h

ave

not b

een

repo

rted

to c

onfe

r res

ista

nce

to N

AIs

. Thes

e N

A re

sidu

es w

ere

repl

aced

with

am

ino

acid

s tha

t pos

sess

side

cha

ins h

avin

g si

mila

r pro

pert

ies t

o m

aint

ain

thei

r ori

gina

l cha

rge.

The

NA

mut

atio

ns w

e in

tro-

duce

d si

gnifi

cant

ly d

ecre

ased

NA

act

ivity

com

pare

d to

that

of t

he A

/Wuh

an/3

59/9

5 re

com

bina

nt w

ild-t

ype

and

R292

K

(an

NA

mut

atio

n fr

eque

ntly

repo

rted

to c

onfe

r res

ista

nce)

vir

uses

, whi

ch w

ere

anal

yzed

for c

ompa

riso

n. H

owev

er, t

he

reco

mbi

nant

vir

uses

diff

ered

in re

plic

atio

n effi

cien

cy w

hen

we

seri

ally

pas

sage

d th

em in

vitr

o; th

e gr

owth

of t

he R

118K

an

d E2

27D

vir

uses

was

mos

t im

pair

ed. Th

e R2

24K

E27

6D, a

nd R

371K

mut

atio

ns c

onfe

rred

resi

stan

ce to

bot

h za

nam

ivir

an

d os

elta

miv

ir, w

hile

the

D15

1E m

utat

ion

redu

ced

susc

eptib

ility

to o

selta

miv

ir o

nly

(sim

ilar t

o 10

-fol

d) a

nd th

e R1

52K

m

utat

ion

did

not a

lter s

usce

ptib

ility

to e

ither

dru

g. B

ecau

se th

e R2

24K

mut

atio

n w

as g

enet

ical

ly u

nsta

ble

and

the

emer

-ge

nce

of th

e R3

71K

mut

atio

n in

the

N2

subt

ype

is st

atis

tical

ly u

nlik

ely,

our r

esul

ts su

gges

t tha

t onl

y th

e E2

76D

mut

atio

n is

like

ly to

em

erge

und

er se

lect

ive

pres

sure

. The

resu

lts o

f our

stud

y m

ay h

elp

to o

ptim

ize

the

desi

gn o

f NA

Is.

Yen

et a

l. 20

06

Res

ista

nce

to

adam

anta

nes

Cen

tral

/Sou

th A

mer

ica

Back

grou

nd R

ecen

t inf

luen

za a

ntiv

iral

resi

stan

ce s

tudi

es re

veal

an

alar

min

g in

crea

se in

bot

h ad

aman

tane

s an

d ne

uram

inid

ase

inhi

bito

rs (N

AIs

) res

ista

nt v

iral

str

ains

wor

ldw

ide,

par

ticul

arly

in A

sia,

Eur

ope

and

the

Uni

ted

Stat

es.

Obj

ectiv

es In

this

stu

dy, w

e ha

ve e

valu

ated

influ

enza

vir

us re

sist

ance

in C

entr

al a

nd S

outh

Am

eric

a. M

etho

ds In

flu-

enza

vir

uses

, iso

late

d fr

om s

ympt

omat

ic p

atie

nts

thro

ugho

ut C

entr

al a

nd S

outh

Am

eric

a in

200

5-20

08 w

ere

anal

yzed

fo

r in

hibi

tor

resi

stan

ce. T

he M

2 an

d N

A g

enes

of i

nflu

enza

vir

uses

wer

e se

quen

ced

and

resi

stan

ce w

as in

ferr

ed b

y co

mpa

riso

n w

ith p

ublis

hed

sequ

ence

s an

d kn

own

resi

stan

t mut

atio

ns. R

esul

ts O

ur re

sults

indi

cate

that

: (i)

resi

stan

ce

to a

dam

anta

nes

was

see

n in

the

maj

ority

(95.

5%) o

f the

influ

enza

A/H

3N2

isol

ates

but

onl

y in

one

isol

ate

of th

e in

flu-

enza

A/H

1N1

viru

ses;

(ii)

resi

stan

ce to

NA

Is b

egan

to b

e de

tect

ed in

A/H

1N1

isol

ates

from

Cen

tral

Am

eric

a in

200

8;

and

(iii)

none

of t

he in

fluen

za B

vir

uses

ana

lyze

d w

ere

resi

stan

t to

NA

Is. C

oncl

usio

ns T

hese

find

ings

sug

gest

a li

mite

d ef

fect

iven

ess

of in

fluen

za in

hibi

tors

due

to th

e de

tect

ion

of re

sist

ance

am

ong

A/H

1 an

d A

/H3

viru

ses.

Gar

cia

et a

l. 20

09


166

Phar

mac

okin

etic

stu

dies

Ose

ltam

ivir

Bioa

vaila

bilit

yRe

nal c

lear

ance

Stea

dy-s

tate

pla

sma

conc

entr

atio

n

Ose

ltam

ivir

is a

n et

hyl e

ster

pro

drug

of P

o 64

-080

2, a

sele

ctiv

e in

hibi

tor o

f infl

uenz

a vi

rus n

eura

min

idas

e. O

ral a

dmin

is-tr

atio

n of

ose

ltam

ivir

deliv

ers t

he a

ctiv

e an

tivira

l Po

64-0

802

to th

e bl

oods

trea

m, a

nd th

us a

ll si

tes o

f infl

uenz

a in

fect

ion

(lung

, nas

al m

ucos

a, m

iddl

e ea

r) a

re a

cces

sibl

e. Th

e ph

arm

acok

inet

ic p

rofil

e of

ose

ltam

ivir

is si

mpl

e an

d pr

edic

tabl

e, a

nd

twic

e da

ily tr

eatm

ent r

esul

ts in

effe

ctiv

e an

tivira

l pla

sma

conc

entr

atio

ns o

ver t

he e

ntire

adm

inis

trat

ion

inte

rval

. Afte

r or

al a

dmin

istr

atio

n, o

selta

miv

ir is

read

ily a

bsor

bed

from

the

gast

roin

test

inal

trac

t and

ext

ensi

vely

con

vert

ed to

the

activ

e m

etab

olite

. The

abso

lute

bio

avai

labi

lity

of th

e ac

tive

met

abol

ite fr

om o

rally

adm

inis

tere

d os

elta

miv

ir is

80%

. The

activ

e m

etab

olite

is d

etec

tabl

e in

pla

sma

with

in 3

0 m

inut

es a

nd re

ache

s max

imal

con

cent

ratio

ns a

fter 3

to 4

hou

rs. A

fter p

eak

plas

ma

conc

entr

atio

ns a

re a

ttai

ned,

the

conc

entr

atio

n of

the

activ

e m

etab

olite

dec

lines

with

an

appa

rent

hal

f-lif

e of

6 to

10

hou

rs. O

selta

miv

ir is

elim

inat

ed p

rim

arily

by

conv

ersi

on to

and

rena

l exc

retio

n of

the

activ

e m

etab

olite

. Ren

al c

lear

ance

of

bot

h co

mpo

unds

exc

eeds

glo

mer

ular

filtr

atio

n ra

te, i

ndic

atin

g th

at re

nal t

ubul

ar se

cret

ion

cont

ribu

tes t

o th

eir e

limin

a-tio

n vi

a th

e an

ioni

c pa

thw

ay. N

eith

er c

ompo

und

inte

ract

s with

cyt

ochr

ome

P450

mix

ed-f

unct

ion

oxid

ases

or g

lucu

rono

-sy

ltran

sfer

ases

. The

phar

mac

okin

etic

pro

file

of th

e ac

tive

met

abol

ite is

line

ar a

nd d

ose-

prop

ortio

nal,

with

less

than

2-f

old

accu

mul

atio

n ov

er a

dos

age

rang

e of

ose

ltam

ivir

50 to

500

mg

twic

e da

ily. S

tead

y-st

ate

plas

ma

conc

entr

atio

ns a

re a

chie

ved

with

in 3

day

s of t

wic

e da

ily a

dmin

istr

atio

n, a

nd a

t a d

osag

e of

75

mg

twic

e da

ily th

e st

eady

-sta

te p

lasm

a tr

ough

con

cent

ra-

tions

of a

ctiv

e m

etab

olite

rem

ain

abov

e th

e m

inim

um in

hibi

tory

con

cent

ratio

n fo

r all

influ

enza

stra

ins t

este

d. E

xpos

ure

to

the

activ

e m

etab

olite

at s

tead

y st

ate

is a

ppro

xim

atel

y 25

% h

ighe

r in

elde

rly c

ompa

red

with

you

ng in

divi

dual

s; ho

wev

er, n

o do

sage

adj

ustm

ent i

s nec

essa

ry. I

n pa

tient

s with

rena

l im

pair

men

t, m

etab

olite

cle

aran

ce d

ecre

ases

line

arly

with

cre

atin

ine

clea

ranc

e. A

dos

age

redu

ctio

n to

75

mg

once

dai

ly is

reco

mm

ende

d fb

r pat

ient

s with

cre

atin

ine

clea

ranc

e <

30 m

l/min

(1

.8 l/

h). Th

e ph

arm

acok

inet

ics i

n pa

tient

s with

influ

enza

are

qua

litat

ivel

y si

mila

r to

thos

e in

hea

lthy

youn

g ad

ults

. In

vitr

o an

d in

viv

o st

udie

s ind

icat

e no

clin

ical

ly si

gnifi

cant

dru

g in

tera

ctio

ns. N

eith

er p

arac

etam

ol (a

ceta

min

ophe

n) n

or c

imet

idin

e al

tere

d th

e ph

arm

acok

inet

ics o

f Ro

64-0

802.

Coa

dmin

istr

atio

n of

pro

bene

cid

resu

lted

in a

2.5

-fol

d in

crea

se in

exp

osur

e to

Ro

64-

0802

, how

ever

, thi

s com

petit

ion

is u

nlik

ely

to re

sult

in c

linic

ally

rele

vant

effe

cts.

Thes

e pr

oper

ties m

ake

osel

tam

ivir

a su

itabl

e ca

ndid

ate

for u

se in

the

prev

entio

n an

d tr

eatm

ent o

f infl

uenz

a.

He

et a

l. 19

99

Tabl

e 4.

5. H

ost c

ell f

acto

r ta

rget

ing

Sign

allin

g ca

scad

e bl

ocke

rsC

-Jun

kin

ase

Bloc

king

of

tran

scri

ptio

n an

d RN

A sy

nthe

sis

C-J

un N

-ter

min

al k

inas

es (

JNK

) are

act

ivat

ed in

cou

rse

of m

any

vira

l inf

ectio

ns. H

ere

we

anal

yzed

the

activ

ity o

f JN

K

inhi

bito

rs o

n in

fluen

za A

vir

us (I

AV) a

mpl

ifica

tion.

Hum

an lu

ng e

pith

elia

l cel

ls w

ere

infe

cted

with

eith

er th

e hi

ghly

pa

thog

enic

avi

an v

irus

str

ain

A/F

PV/B

ratis

lava

/79

(H7N

7) o

r th

e pa

ndem

ic s

win

e-or

igin

influ

enza

vir

us A

/Ham

-bu

rg/4

/09

(H1N

1v).

The

app

licat

ion

of th

e JN

K in

hibi

tors

SP6

0012

5 an

d A

S601

245

redu

ced

IAV

am

plifi

catio

n by

sup

-pr

essi

ng v

iral

pro

tein

and

RN

A s

ynth

esis

. Alth

ough

AS6

0124

5 ap

pear

ed to

gen

eral

ly b

lock

the

tran

scri

ptio

n of

new

ly

intr

oduc

ed g

enes

, SP6

0012

5 sp

ecifi

cally

affe

cted

vir

al R

NA

syn

thes

is. O

vere

xpre

ssio

n of

a d

omin

ant n

egat

ive

mut

ant

of S

EK/M

KK

4 an

d si

RNA

-med

iate

d su

ppre

ssio

n of

JNK

2 ex

pres

sion

con

firm

ed th

at s

peci

fic m

anip

ulat

ion

of th

e JN

K

path

way

att

enua

tes

viru

s pr

opag

atio

n. A

n IA

V m

inig

enom

e re

plic

atio

n as

say

reve

aled

that

SP6

0012

5 di

d no

t dir

ectly

af

fect

the

activ

ity o

f the

vir

al R

NA

pol

ymer

ase

com

plex

but

see

ms

to s

uppr

ess

an a

nti-

influ

enza

non

stru

ctur

al p

rote

in

1-m

edia

ted

viru

s su

ppor

tive

func

tion.

Fin

ally

, whe

n H

7N7-

or H

1N1v

-inf

ecte

d m

ice

wer

e tr

eate

d w

ith S

P600

125,

the

vira

l loa

d is

redu

ced

in lu

ngs

of tr

eate

d co

mpa

red

with

unt

reat

ed m

ice.

Our

dat

a su

gges

t tha

t thi

s cl

ass

of A

TP

com

peti-

tive

inhi

bito

rs o

nce

optim

ized

for

antiv

iral

act

ion

pote

ntia

lly re

pres

ent n

ovel

dru

gs fo

r an

tivir

al in

terv

entio

n.

Nac

ken

et a

l. 20

12


167

Sign

allin

g ca

scad

e bl

ocke

rsRa

f/M

EK/E

RK

sign

allin

g pa

thw

ayO

selta

miv

ir re

sist

ant

stra

ins

Mou

se m

odel

The

em

erge

nce

of th

e 20

09 H

1N1

pand

emic

sw

ine

influ

enza

A v

irus

is a

goo

d ex

ampl

e of

how

this

vir

al in

fect

ion

can

impa

ct h

ealth

sys

tem

s ar

ound

the

wor

ld in

a v

ery

shor

t tim

e. T

he c

ontin

uous

zoo

notic

cir

cula

tion

and

reas

sort

men

t po

tent

ial o

f inf

luen

za A

vir

uses

(IAV

) in

natu

re re

pres

ents

an

enor

mou

s pu

blic

hea

lth th

reat

to h

uman

s. B

esid

e va

ccin

a-tio

n an

tivir

als

are

need

ed to

eff

icie

ntly

con

trol

spr

eadi

ng o

f the

dis

ease

. In

the

pres

ent w

ork

we

inve

stig

ated

whe

ther

the

MEK

inhi

bito

r U

0126

, tar

getin

g th

e in

trac

ellu

lar

Raf/

MEK

/ERK

sig

nalin

g pa

thw

ay, i

s ab

le to

sup

pres

s pr

opag

atio

n of

th

e 20

09 p

ande

mic

IV H

1N1v

(v =

var

iant

) as

wel

l as

high

ly p

atho

geni

c av

ian

influ

enza

vir

uses

(HPA

IV) i

n ce

ll cu

lture

an

d al

so in

viv

o in

the

mou

se lu

ng. U

0126

sho

wed

ant

ivir

al a

ctiv

ity in

cel

l cul

ture

aga

inst

all

test

ed IA

V s

trai

ns in

clud

-in

g Ra

f/M

EK/E

RK s

igna

ling

path

way

. Fur

ther

mor

e, w

e w

ere

able

to d

emon

stra

te th

at tr

eatm

ent o

f mic

e w

ith U

0126

via

th

e ae

roso

l rou

te le

d to

inhi

bitio

n of

MEK

act

ivat

ion

in th

e lu

ng re

duct

ion

of p

roge

ny IA

V ti

ters

com

pare

d to

unt

reat

ed

cont

rols

pro

tect

ion

of IA

V in

fect

ed m

ice

agai

nst a

100

× le

thal

vir

al c

halle

nge.

Mor

eove

r, no

adv

erse

effe

cts

of U

0126

w

ere

foun

d in

cel

l cul

ture

or

in th

e m

ouse

. Thu

s, w

e co

nclu

de th

at U

0126

, by

inhi

bitin

g th

e ce

llula

r ta

rget

MEK

, has

an

antiv

iral

pot

entia

l not

onl

y in

vitr

o in

cel

l cul

ture

, but

als

o in

viv

o in

the

mou

se m

odel

.

Dro

ebne

r et a

l. 20

11

Cat

helic

idin

pep

tide

sD

ecre

ase

of p

ro-

infla

mat

ory

cyto

kine

sPr

otec

tion

agai

nst

influ

enza

The

ext

ensi

ve w

orld

-wid

e m

orbi

dity

and

mor

talit

y ca

used

by

influ

enza

A v

irus

es h

ighl

ight

s th

e ne

ed fo

r ne

w in

sigh

ts

into

the

host

imm

une

resp

onse

and

nov

el tr

eatm

ent a

ppro

ache

s. C

atio

nic

Hos

t Def

ense

Pep

tides

(CH

DP,

als

o kn

own

as a

ntim

icro

bial

pep

tides

), w

hich

incl

ude

cath

elic

idin

s an

d de

fens

ins,

are

key

com

pone

nts

of th

e in

nate

imm

une

syst

em

that

are

upr

egul

ated

dur

ing

infe

ctio

n an

d in

flam

mat

ion.

Cat

helic

idin

s ha

ve im

mun

omod

ulat

ory

and

anti-

vira

l effe

cts,

bu

t the

ir im

pact

on

influ

enza

vir

us in

fect

ion

has

not b

een

prev

ious

ly a

sses

sed.

We

ther

efor

e ev

alua

ted

the

effe

ct o

f ca

thel

icid

in p

eptid

es o

n di

seas

e ca

used

by

influ

enza

A v

irus

in m

ice.

The

hum

an c

athe

licid

in, L

L-37

, and

the

mur

ine

cath

elic

idin

, mC

RA

MP,

dem

onst

rate

d si

gnifi

cant

ant

i-vi

ral a

ctiv

ity in

viv

o, re

duci

ng d

isea

se s

ever

ity a

nd v

iral

repl

ica-

tion

in in

fect

ed m

ice

to a

sim

ilar

exte

nt a

s th

e w

ell-

char

acte

rize

d in

fluen

za v

irus

-spe

cific

ant

ivir

al d

rug

zana

miv

ir. In

vi

tro

and

in v

ivo

expe

rim

ents

sug

gest

ed th

at th

e pe

ptid

es m

ay a

ct d

irec

tly o

n th

e in

fluen

za v

irio

n ra

ther

than

via

rece

p-to

r-ba

sed

mec

hani

sms.

Influ

enza

vir

us-i

nfec

ted

mic

e tr

eate

d w

ith L

L-37

had

low

er c

once

ntra

tions

of p

ro-i

nfla

mm

ator

y cy

toki

nes

in th

e lu

ng th

an d

id in

fect

ed a

nim

als

that

had

not

bee

n tr

eate

d w

ith c

athe

licid

in p

eptid

es. T

hese

dat

a su

gges

t th

at tr

eatm

ent o

f inf

luen

za-i

nfec

ted

indi

vidu

als

with

cat

helic

idin

-der

ived

ther

apeu

tics,

or

mod

ulat

ion

of e

ndog

enou

s ca

thel

icid

in p

rodu

ctio

n m

ay p

rovi

de s

igni

fican

t pro

tect

ion

agai

nst d

isea

se.

Barl

ow e

t al.

2011

Vacu

olar

pro

ton-

AT

Pase

inhi

bito

rC

onca

nam

ycin

A

The

sel

ectiv

e in

hibi

tor

of th

e va

cuol

ar p

roto

n-A

TPa

se, c

onca

nam

ycin

A, p

ower

fully

blo

cks

influ

enza

vir

us e

ntry

into

ce

lls, i

f pre

sent

dur

ing

the

initi

al ti

mes

of v

irus

infe

ctio

n. A

ttac

hmen

t of v

irus

par

ticle

s to

cel

ls is

not

pre

vent

ed b

y co

n-ca

nam

ycin

A, r

athe

r th

e ex

it of

influ

enza

vir

us fr

om e

ndos

omes

is th

e st

ep b

lock

ed b

y th

is m

acro

lide

antib

iotic

. Inh

ibi-

tion

of in

fluen

za v

irus

ent

ry in

to c

ells

by

conc

anam

ycin

A o

r by

nig

eric

in ta

kes

plac

e un

der

acid

ic c

ondi

tions

. Mor

eove

r, if

the

pH g

radi

ent i

s ab

olis

hed

by p

re-i

ncub

atio

n of

cel

ls in

aci

dic

pH, i

nflu

enza

vir

us e

ntry

doe

s no

t occ

ur e

ven

in th

e ab

senc

e of

any

inhi

bito

rs. T

hese

resu

lts in

dica

te th

at a

cidi

c co

nditi

ons

per

se a

re n

ot s

uffic

ient

to p

rom

ote

viru

s en

try

into

cel

ls; r

athe

r th

is s

tep

of v

irus

infe

ctio

n re

quir

es a

pH

gra

dien

t.

Gui

nea

and

Car

-ra

sco

1994


168

Inhi

bito

r of

vac

uola

r-ty

pe p

roto

n pu

mp

Bafil

omyc

in A

lEn

doso

me

acid

ifica

tion

We

stud

ied

the

effe

ct o

f baf

ilom

ycin

Al (

Baf-

A1)

, a n

ovel

and

hig

hly

spec

ific

inhi

bito

r fo

r va

cuol

ar-t

ype

prot

on (V

-H+)

pu

mp,

on

the

grow

th o

f inf

luen

za A

and

B v

irus

es in

Mad

in-D

arby

can

ine

kidn

ey c

ells

. Vita

l flu

ores

cenc

e m

icro

scop

ic

stud

y sh

owed

that

Baf

-A1

indu

ced

the

com

plet

e di

sapp

eara

nce

of a

cidi

fied

com

part

men

ts s

uch

as e

ndos

omes

and

ly

soso

mes

bot

h in

infe

cted

and

uni

nfec

ted

cells

by

the

trea

tmen

t with

0.1

mu

M in

hibi

tor

for

1 h

at 3

7 °C

. In

addi

tion,

vi

rus

grow

th w

as in

hibi

ted

whe

n Ba

f-A

1 w

as p

rese

nt fr

om 1

h b

efor

e in

fect

ion

to th

e en

d of

incu

batio

n, o

r ad

ded

with

in

as e

arly

as

5–10

min

aft

er in

fect

ion.

Con

vers

ely,

the

viru

s gr

owth

was

reco

vere

d in

cor

rela

tion

with

the

reap

pear

ance

of

aci

difie

d co

mpa

rtm

ents

aft

er re

mov

al o

f Baf

-A1.

The

se d

ata

sugg

est t

hat B

af-A

1-se

nsiti

ve V

-H+

pum

ps a

re s

olel

y re

spon

sibl

e fo

r th

e ac

idifi

catio

n of

end

osom

es a

nd ly

soso

mes

, and

thus

Baf

-A1

inhi

bits

the

grow

th o

f inf

luen

za A

and

B

viru

ses

by a

ffect

ing

the

acid

ified

com

part

men

ts in

whi

ch lo

w p

H is

ess

entia

l for

the

unco

atin

g pr

oces

s of

influ

enza

vir

us

grow

th a

t an

earl

y st

age

of in

fect

ion.

Och

iai e

t al.

1995

Prot

easo

me

inhi

bito

rRe

cent

ly it

has

bee

n sh

own

that

the

proi

nfla

mm

ator

y N

F-ka

ppa

B pa

thw

ay p

rom

otes

eff

icie

nt in

fluen

za v

irus

pro

paga

-tio

n. B

ased

on

thes

e fin

ding

s, it

was

sug

gest

ed th

at N

F-ka

ppa

B bl

ocka

de m

ay b

e a

prom

isin

g ap

proa

ch fo

r an

tivir

al

inte

rven

tion.

The

cla

ssic

al v

irus

-ind

uced

act

ivat

ion

of th

e N

F-ka

ppa

B pa

thw

ay re

quir

es p

rote

asom

al d

egra

datio

n of

the

inhi

bito

r of

NF-

kapp

a B,

I ka

ppa

B. T

here

fore

, we

hypo

thes

ized

that

inhi

bitio

n of

pro

teas

omal

I ka

ppa

B de

grad

atio

n sh

ould

impa

ir in

fluen

za A

vir

us (I

AV) r

eplic

atio

n. W

e ch

ose

the

spec

ific

prot

easo

me

inhi

bito

r PS

-341

, whi

ch is

a c

lini-

cally

app

rove

d an

tican

cer

drug

als

o kn

own

as B

orte

zom

ib o

r Ve

lcad

e. A

s ex

pect

ed, P

S-34

1 tr

eatm

ent o

f inf

ecte

d A

549

cells

in a

con

cent

ratio

n ra

nge

that

was

not

toxi

c re

sulte

d in

a s

igni

fican

t red

uctio

n of

pro

geny

vir

us ti

ters

. How

ever

, we

coul

d no

t obs

erve

the

prop

osed

sup

pres

sion

of N

F-ka

ppa

B-si

gnal

ing

in v

itro.

Rat

her,

PS-3

41 tr

eatm

ent r

esul

ted

in a

n in

duct

ion

of I

kapp

a B

degr

adat

ion

and

activ

atio

n of

NF-

kapp

a B

as w

ell a

s th

e JN

K/A

P-1

path

way

. Thi

s co

inci

des

with

en

hanc

ed e

xpre

ssio

n of

ant

ivir

al g

enes

, suc

h as

inte

rleu

kin-

6 an

d, m

ost i

mpo

rtan

tly, M

xA, w

hich

is a

str

ong

inte

rfer

on

(IFN

)-in

duce

d su

ppre

ssor

of i

nflu

enza

vir

us re

plic

atio

n. T

his

sugg

ests

that

PS-

341

may

act

as

an a

ntiv

iral

age

nt v

ia

indu

ctio

n of

the

type

I IF

N re

spon

se. A

ccor

ding

ly, P

S-34

1 di

d no

t affe

ct v

irus

tite

rs in

Ver

o ce

lls, w

hich

lack

type

I IF

N

gene

s, b

ut s

tron

gly

inhi

bite

d re

plic

atio

n of

ves

icul

ar s

tom

atiti

s vi

rus

(VSV

), a

high

ly IF

N-s

ensi

tive

path

ogen

. Thu

s,

we

conc

lude

that

PS-

341

bloc

ks IA

V a

nd V

SV re

plic

atio

n by

indu

cing

an

antiv

iral

sta

te m

edia

ted

by th

e N

F-ka

ppa

B-de

pend

ent e

xpre

ssio

n of

ant

ivir

us-a

ctin

g ge

ne p

rodu

cts.

Dud

ek e

t al.

2010

Prot

easo

me

inhi

bito

rRe

duct

ion

of c

ytok

ine

rele

ase

In th

e pr

esen

t stu

dy w

e sh

ow th

at th

e pr

otea

som

e in

hibi

tor

VL-

01 le

ads

to re

duct

ion

of in

fluen

za v

irus

repl

icat

ion

in

hum

an lu

ng a

deno

carc

inom

a ep

ithel

ial c

ells

(A54

9) a

s de

mon

stra

ted

with

thre

e di

ffere

nt in

fluen

za v

irus

str

ains

, A

/Pue

rto

Rico

/8/3

4 (H

1N1)

(EC

50 v

alue

of 1

.7 µ

M),

A/R

egen

sbur

g/D

6/09

(H1N

1v) (

EC50

val

ue o

f 2.4

µM

) and

A/M

al-

lard

/Bav

aria

/1/2

006

(H5N

1) (E

C50

val

ue o

f 0.8

µM

). In

in v

ivo

expe

rim

ents

we

coul

d de

mon

stra

te th

at V

L-01

-aer

osol

-tr

eatm

ent o

f BA

LB/c

mic

e w

ith 1

4.1

mg/

kg re

sults

in n

o to

xic

side

effe

cts,

redu

ced

prog

eny

viru

s tit

ers

in th

e lu

ng

(1.1

± 0

.3 lo

g 10 p

fu) a

nd e

nhan

ced

surv

ival

of m

ice

afte

r in

fect

ion

with

a 5

-fol

d M

LD50

of t

he h

uman

influ

enza

A v

irus

st

rain

A/P

uert

o Ri

co/8

/34

(H1N

1) u

p to

50%

. Fur

ther

mor

e, tr

eatm

ent o

f mic

e w

ith V

L-01

redu

ced

the

cyto

kine

rele

ase

of IL

-alp

ha/b

eta,

IL-6

, MIP

-1 b

eta,

RA

NT

ES a

nd T

NF-

alph

a in

duce

d by

LPS

or

high

ly p

atho

gen

avia

n H

5N1

influ

enza

A

vir

us. T

he p

rese

nt d

ata

dem

onst

rate

s an

ant

ivir

al e

ffect

of V

L-01

in v

itro

and

in v

ivo

and

the

abili

ty to

redu

ce in

flu-

enza

vir

us in

duce

d cy

toki

nes

and

chem

okin

es.

Haa

sbac

h et

al.

2011

nF-

kapp

a B

inhi

bito

rPy

rone

poly

ene

C-g

luco

side

A n

ew p

yron

epol

yene

C-g

luco

side

, nam

ed is

o-D

8646

-2-6

(1) t

oget

her

with

the

know

n re

late

d co

mpo

und

D86

46-2

-6 (2

), w

as is

olat

ed fr

om th

e sp

onge

-ass

ocia

ted

fung

us E

pico

ccum

sp.

JJY4

0. T

hey

show

ed N

F-ka

ppa

B in

hibi

tory

and

ant

i-in

fluen

za A

vir

al (H

1N1)

act

iviti

es.

Peng

et a

l. 20

12


169

Prot

ease

inhi

bito

rsA

prot

inin

Leup

eptin

Cam

osta

t

Effo

rts

to d

evel

op n

ew a

ntiv

iral

che

mot

hera

peut

ic a

ppro

ache

s ar

e fo

cusi

ng o

n co

mpo

unds

that

targ

et e

ither

influ

enza

vi

rus

repl

icat

ion

itsel

f or

host

fact

or(s

) tha

t are

cri

tical

to in

fluen

za re

plic

atio

n. H

ost p

rote

ase

med

iate

d in

fluen

za

hem

aggl

utin

in (H

A) c

leav

age

is c

ritic

al fo

r ac

tivat

ion

of v

irus

infe

ctiv

ity a

nd a

s su

ch is

a c

hem

othe

rape

utic

targ

et. I

nflu

-en

za p

atho

gene

sis

invo

lves

a “v

icio

us c

ycle

” in

whi

ch h

ost p

rote

ases

act

ivat

e pr

ogen

y vi

rus

whi

ch in

turn

am

plifi

es re

p-lic

atio

n an

d st

imul

ates

furt

her

prot

ease

act

iviti

es w

hich

may

be

detr

imen

tal t

o th

e in

fect

ed h

ost.

Apr

otin

in, a

58

amin

o ac

id p

olyp

eptid

e pu

rifie

d fr

om b

ovin

e lu

ng th

at is

one

of a

fam

ily o

f hos

t-ta

rget

ed a

ntiv

iral

s th

at in

hibi

t ser

ine

prot

ease

s re

spon

sibl

e fo

r in

fluen

za v

irus

act

ivat

ion.

Thi

s dr

ug a

nd s

imila

r ag

ents

, suc

h as

leup

eptin

and

cam

osta

t, su

ppre

ss v

irus

H

A c

leav

age

and

limit

repr

oduc

tion

of h

uman

and

avi

an in

fluen

za v

irus

es w

ith a

sin

gle

argi

nine

in th

e H

A c

leav

age

site

. Si

te-d

irec

ted

stru

ctur

al m

odifi

catio

ns o

f apr

otin

in a

re p

ossi

ble

to in

crea

se it

s in

trac

ellu

lar

targ

etin

g of

cle

avag

e of

hig

hly

viru

lent

H5

and

H7

hem

aggl

utin

ins

poss

essi

ng m

ulti-

argi

nine

/lys

ine

clea

vage

site

. An

addi

tiona

l mec

hani

sm o

f act

ion

for

seri

ne p

rote

ase

inhi

bito

rs is

to ta

rget

a n

umbe

r of

hos

t med

iato

rs o

f inf

lam

mat

ion

and

dow

n re

gula

te th

eir

leve

ls in

vi

rus-

infe

cted

hos

ts. A

prot

inin

is a

gen

eric

dru

g ap

prov

ed fo

r in

trav

enou

s us

e in

hum

ans

to tr

eat p

ancr

eatit

is a

nd li

mit

post

-ope

rativ

e bl

eedi

ng. A

s an

ant

iinflu

enza

l com

poun

d, a

prot

inin

mig

ht b

e de

liver

ed b

y tw

o ro

utes

: (i)

a sm

all-

part

icle

ae

roso

l has

bee

n ap

prov

ed in

Rus

sia

for

loca

l res

pira

tory

app

licat

ion

in m

ild-t

o-m

oder

ate

influ

enza

and

(ii)

a pr

opos

ed

intr

aven

ous

adm

inis

trat

ion

for

seve

re in

fluen

za to

pro

vide

bot

h an

ant

ivir

al e

ffect

and

a d

ecre

ase

in s

yste

mic

pat

holo

gy

and

infla

mm

atio

n.

Zhi

rnov

et a

l. 20

11

Prot

ease

inhi

bito

rsC

amos

tat m

esila

teN

afam

osta

t mes

ilate

Six

nucl

eosi

de a

nalo

gues

, tw

o su

lfate

d po

lysa

ccha

ride

s, a

nd fo

ur p

rote

ase

inhi

bito

rs w

ere

eval

uate

d in

vitr

o as

inhi

bito

rs

of in

fluen

za v

irus

repl

icat

ion.

Fou

r gu

anos

ine

anal

ogue

s (m

izor

ibin

e, r

ibav

irin

, pyr

azof

urin

, and

5-e

thyn

yl-1

-bet

a-D

-ri

bofu

rano

sylim

idaz

ole-

4-ca

rbox

amid

e), t

he s

ulfa

ted

poly

sacc

hari

de d

extr

an s

ulfa

te (m

olec

ular

wei

ght 5

00 0

00),

and

two

prot

ease

inhi

bito

rs (c

amos

tat m

esila

te a

nd n

afam

osta

t mes

ilate

) wer

e in

hibi

tory

to th

e re

plic

atio

n of

str

ains

of i

nflu

-en

za v

irus

type

s A

and

B a

t con

cent

ratio

ns d

own

to 0

.3 µ

g/m

l. O

f the

se s

even

com

poun

ds, r

ibav

irin

, cam

osta

t mes

ilate

, an

d na

fam

osta

t mes

ilate

wer

e ef

ficac

ious

in b

oth

redu

cing

the

viru

s tit

er a

nd in

crea

sing

the

surv

ival

rat

e of

influ

enza

vi

rus-

infe

cted

chi

ck e

mbr

yos.

For

cam

osta

t mes

ilate

, the

ED

50 (r

equi

red

to im

prov

e th

e su

rviv

al r

ate

of in

fluen

za

viru

s-in

fect

ed c

hick

em

bryo

s by

50%

) was

0.8

0 µg

/g, a

nd it

s se

lect

ivity

inde

x, b

ased

on

the

ratio

of t

he 5

0% to

xic

dose

(r

equi

red

to re

duce

the

viab

ility

of c

hick

em

bryo

s by

50%

) to

ED50

, was

280

. Cam

osta

t mes

ilate

des

erve

s fu

rthe

r ex

plo-

ratio

n fo

r its

pot

entia

l in

the

trea

tmen

t of i

nflu

enza

vir

us in

fect

ion.

Hos

oya

et a

l. 19

93

Prot

ease

inhi

bito

rsN

afam

osta

t mes

ilate

Cam

osta

t mes

ilate

Gab

exat

e m

esila

teA

prot

inin

Chi

cken

em

bryo

s

We

stud

ied

the

effe

cts

of e

ight

pro

teas

e in

hibi

tors

on

the

mul

ticyc

le re

plic

atio

ns o

f var

ious

ort

hom

yxov

irus

es a

nd

para

myx

ovir

uses

. Am

ong

the

com

poun

ds, n

afam

osta

t mes

ilate

, cam

osta

t mes

ilate

, gab

exat

e m

esila

te, a

nd a

prot

inin

, w

hich

are

wid

ely

used

in th

e tr

eatm

ent o

f pan

crea

titis

, inh

ibite

d in

fluen

za v

irus

A a

nd B

repl

icat

ion

at c

once

ntra

tions

th

at w

ere

sign

ifica

ntly

low

er th

an th

eir

cyto

toxi

c th

resh

olds

in v

itro.

Non

e of

the

prot

ease

inhi

bito

rs h

ad a

ctiv

ity a

gain

st

resp

irat

ory

sync

ytia

l vir

us, m

easl

es v

irus

, or

para

influ

enza

vir

us ty

pe 3

at t

he h

ighe

st c

once

ntra

tions

test

ed. C

amos

tat

mes

ilate

was

foun

d to

be

the

mos

t sel

ectiv

e in

hibi

tor.

Its

50%

effe

ctiv

e co

ncen

trat

ion

for

influ

enza

vir

us A

repl

icat

ion

was

2.2

µg/

ml,

and

the

sele

ctiv

ity in

dex,

whi

ch w

as b

ased

on

the

ratio

of t

he 5

0% in

hibi

tory

con

cent

ratio

n fo

r ho

st

cell

prol

ifera

tion

to th

e 50

% e

ffect

ive

conc

entr

atio

n fo

r in

fluen

za v

irus

A re

plic

atio

n, w

as 6

80. W

hen

the

in o

vo a

nti-

vira

l act

ivity

of t

he c

ompo

unds

was

test

ed b

y us

ing

chic

ken

embr

yos,

cam

osta

t mes

ilate

at a

dos

e of

10

µg/g

mar

kedl

y re

duce

d th

e he

mag

glut

inin

tite

rs o

f inf

luen

za v

irus

es A

and

B.

Hos

oya

et a

l. 19

92


170

Hae

mag

glut

inin

fo

ldin

gG

luta

thio

ne d

eriv

ativ

e

Aim

: The

aim

of t

his

stud

y w

as to

det

erm

ine

whe

ther

GSH

-C4,

a h

ydro

phob

ic g

luta

thio

ne d

eriv

ativ

e, a

ffect

s in

vitr

o an

d in

viv

o in

fluen

za v

irus

infe

ctio

n by

inte

rfer

ing

with

redo

x-se

nsiti

ve in

trac

ellu

lar

path

way

s in

volv

ed in

the

mat

urat

ion

of v

iral

hem

aggl

utin

in (H

A).

Resu

lts: G

SH-C

4 st

rong

ly in

hibi

ted

influ

enza

A v

irus

repl

icat

ion

in c

ultu

red

cells

and

in

leth

ally

infe

cted

mic

e, w

here

it a

lso

redu

ced

lung

dam

age

and

mor

talit

y. In

cel

l-cu

lture

stu

dies

, GSH

-C4

arre

sted

vir

al

HA

fold

ing;

the

disu

lfide

-ric

h gl

ycop

rote

in re

mai

ned

in th

e en

dopl

asm

ic re

ticul

um a

s a

redu

ced

mon

omer

inst

ead

of

unde

rgoi

ng o

ligom

eriz

atio

n an

d ce

ll pl

asm

a-m

embr

ane

inse

rtio

n. H

A m

atur

atio

n de

pend

s on

the

host

-cel

l oxi

dore

duc-

tase

, pro

tein

dis

ulfid

e is

omer

ase

(PD

I), w

hose

act

ivity

in in

fect

ed c

ells

is p

roba

bly

faci

litat

ed b

y vi

rus-

indu

ced

glu-

tath

ione

dep

letio

n. B

y co

rrec

ting

this

def

icit

, GSH

-C4

incr

ease

d le

vels

of r

educ

ed P

DI a

nd in

hibi

ted

esse

ntia

l dis

ulfid

e bo

nd fo

rmat

ion

in H

A. H

ost-

cell

glyc

opro

tein

exp

ress

ion

in u

ninf

ecte

d ce

lls w

as u

naffe

cted

by

glut

athi

one,

whi

ch th

us

appe

ars

to a

ct e

xclu

sive

ly o

n gl

utat

hion

e-de

plet

ed c

ells

. Inn

ovat

ion:

All

curr

ently

app

rove

d an

ti-in

fluen

za d

rugs

targ

et

esse

ntia

l vir

al s

truc

ture

s, a

nd th

eir

effic

acy

is li

mite

d by

toxi

city

and

by

the

alm

ost i

nevi

tabl

e se

lect

ion

of d

rug-

resi

stan

t vi

ral m

utan

ts. G

SH-C

4 in

hibi

ts in

fluen

za v

irus

repl

icat

ion

by m

odul

atin

g re

dox-

sens

itive

pat

hway

s in

infe

cted

cel

ls,

with

out p

rodu

cing

toxi

city

in u

ninf

ecte

d ce

lls o

r an

imal

s. N

ovel

ant

i-in

fluen

za d

rugs

that

targ

et in

trac

ellu

lar

path

way

s es

sent

ial f

or v

iral

repl

icat

ion

(“ce

ll-ba

sed

appr

oach

”) o

ffer

two

impo

rtan

t pot

entia

l adv

anta

ges:

they

are

mor

e di

ffi-

cult

for

the

viru

s to

ada

pt to

and

thei

r ef

ficac

y sh

ould

not

be

depe

nden

t on

viru

s ty

pe, s

trai

n, o

r an

tigen

ic p

rope

rtie

s.

Con

clus

ion:

Red

ox-s

ensi

tive

host

-cel

l pat

hway

s ex

ploi

ted

for

vira

l rep

licat

ion

are

prom

isin

g ta

rget

s fo

r ef

fect

ive

anti-

influ

enza

str

ateg

ies.

Sgar

bant

i et a

l. 20

11

Prot

easo

me

inhi

bito

rsW

e ha

ve d

emon

stra

ted

that

influ

enza

A v

irus

(IAV

) RN

A s

ynth

esis

dep

ends

on

the

ubiq

uitin

-pro

teas

ome

syst

em. I

AV

repl

icat

ion

was

redu

ced

both

by

prot

easo

me

inhi

bito

rs a

nd in

E36

ts20

cel

ls, w

hich

con

tain

the

ther

mol

abile

ubi

quiti

n-ac

tivat

ing

enzy

me

E1. W

hile

vir

us e

ntry

was

not

affe

cted

in E

36ts

20 c

ells

, the

pro

teas

ome

inhi

bito

r M

G13

2 re

tain

ed

vira

l par

ticle

s in

the

cyto

plas

m. A

dditi

on-r

emov

al e

xper

imen

ts o

f MG

132

in c

ombi

natio

n w

ith b

afilo

myc

in A

1, a

wel

l-es

tabl

ishe

d in

hibi

tor

of IA

V e

ntry

and

fusi

on, s

how

ed th

at M

G13

2 af

fect

ed IA

V in

fect

ion

at a

pos

tfus

ion

step

. Thi

s w

as

conf

irm

ed b

y th

e la

ck o

f inh

ibiti

on o

f IAV

ent

ry b

y pr

otea

som

e in

hibi

tors

in a

vir

us-l

ike

part

icle

fusi

on a

ssay

.

Wid

jaja

et a

l. 20

10

Hos

t fac

tor

targ

etin

gG

enom

e-w

ide

RNA

in

terf

eren

ce sc

reen

ing

295

cellu

lar c

ofac

tors

Influ

enza

A v

irus

is a

n RN

A v

irus

that

enc

odes

up

to 1

1 pr

otei

ns a

nd th

is s

mal

l cod

ing

capa

city

dem

ands

that

the

viru

s us

e th

e ho

st c

ellu

lar

mac

hine

ry fo

r m

any

aspe

cts

of it

s lif

e cy

cle.

Kno

wle

dge

of th

ese

host

cel

l req

uire

men

ts n

ot o

nly

info

rms

us o

f the

mol

ecul

ar p

athw

ays

expl

oite

d by

the

viru

s bu

t als

o pr

ovid

es fu

rthe

r ta

rget

s th

at c

ould

be

purs

ued

for

antiv

iral

dru

g de

velo

pmen

t. H

ere

we

use

an in

tegr

ativ

e sy

stem

s ap

proa

ch, b

ased

on

geno

me-

wid

e RN

A in

terf

er-

ence

scr

eeni

ng, t

o id

entif

y 29

5 ce

llula

r co

fact

ors

requ

ired

for

earl

y-st

age

influ

enza

vir

us re

plic

atio

n. W

ithin

this

gro

up,

thos

e in

volv

ed in

kin

ase-

regu

late

d si

gnal

ling,

ubi

quiti

natio

n an

d ph

osph

atas

e ac

tivity

are

the

mos

t hig

hly

enri

ched

, an

d 18

1 fa

ctor

s as

sem

ble

into

a h

ighl

y si

gnifi

cant

hos

t-pa

thog

en in

tera

ctio

n ne

twor

k. M

oreo

ver,

219

of th

e 29

5 fa

ctor

s w

ere

conf

irm

ed to

be

requ

ired

for

effic

ient

wild

-typ

e in

fluen

za v

irus

gro

wth

, and

furt

her

anal

ysis

of a

sub

set o

f gen

es

show

ed 2

3 fa

ctor

s ne

cess

ary

for

vira

l ent

ry, i

nclu

ding

mem

bers

of t

he v

acuo

lar

AT

Pase

(vA

TPa

se) a

nd C

OPI

-pro

tein

fa

mili

es, f

ibro

blas

t gro

wth

fact

or re

cept

or (F

GFR

) pro

tein

s, a

nd g

lyco

gen

synt

hase

kin

ase

3 (G

SK3)

-bet

a. F

urth

erm

ore,

10

pro

tein

s w

ere

conf

irm

ed to

be

invo

lved

in p

ost-

entr

y st

eps

of in

fluen

za v

irus

repl

icat

ion.

The

se in

clud

e nu

clea

r im

port

com

pone

nts,

pro

teas

es, a

nd th

e ca

lciu

m/c

alm

odul

in-d

epen

dent

pro

tein

kin

ase

(CaM

kin

ase)

II b

eta

(CA

MK

2B).

Not

ably

, gro

wth

of s

win

e-or

igin

H1N

1 in

fluen

za v

irus

is a

lso

depe

nden

t on

the

iden

tifie

d ho

st fa

ctor

s, a

nd w

e sh

ow th

at

smal

l mol

ecul

e in

hibi

tors

of s

ever

al fa

ctor

s, in

clud

ing

vAT

Pase

and

CA

MK

2B, a

ntag

oniz

e in

fluen

za v

irus

repl

icat

ion.

Kon

ig e

t al.

2010


171

Tabl

e 4.

6. o

ther

ant

i-in

flue

nza

agen

ts

nuc

leop

rote

in

inhi

bito

rsN

ucle

ozin

Fluo

resc

ence

-que

nchi

ng

effec

t

Rece

nt s

tudi

es h

ave

show

n th

at N

P (n

ucle

opro

tein

), w

hich

pos

sess

es m

ultip

le fu

nctio

ns in

the

vira

l life

cyc

le, i

s a

new

po

tent

ial a

nti-

influ

enza

dru

g ta

rget

. NP

inhi

bito

rs re

liabl

y in

duce

con

form

atio

nal c

hang

es in

NPs

, and

thes

e ch

ange

s m

ay c

onfe

r in

hibi

tion

of th

e in

fluen

za v

irus

. The

six

con

serv

ed tr

ypto

phan

resi

dues

in N

P ca

n be

use

d as

an

intr

insi

c pr

obe

to m

onito

r th

e ch

ange

in fl

uore

scen

ce o

f the

tryp

toph

an re

sidu

es in

the

prot

ein

upon

bin

ding

to a

n N

P in

hibi

tor.

In th

e pr

esen

t stu

dy, w

e fo

und

that

the

fluor

esce

nce

of re

com

bina

nt N

P pr

otei

ns w

as q

uenc

hed

follo

win

g th

e bi

ndin

g of

ava

ilabl

e N

P in

hibi

tors

(suc

h as

nuc

leoz

in) i

n a

conc

entr

atio

n- a

nd ti

me-

depe

nden

t man

ner,

whi

ch s

ugge

sts

that

the

inhi

bito

r in

duce

d co

nfor

mat

iona

l cha

nges

in th

e N

Ps. T

he m

inim

al fl

uore

scen

ce-q

uenc

hing

effe

ct a

nd w

eak

bind

ing

cons

tant

of n

ucle

ozin

to th

e sw

ine-

orig

in in

fluen

za v

irus

H1N

1pdm

09 (S

OIV

) NP

reve

aled

that

the

SOIV

is re

sist

ant

to n

ucle

ozin

. We

have

use

d th

e flu

ores

cenc

e-qu

ench

ing

prop

erty

of t

rypt

opha

ns in

NPs

that

wer

e bo

und

to li

gand

s in

a

96-w

ell-

plat

e-ba

sed

drug

scr

een

to a

sses

s th

e ab

ility

of p

rom

isin

g sm

all m

olec

ules

to in

tera

ct w

ith N

Ps a

nd h

ave

iden

ti-fie

d on

e ne

w a

nti-

influ

enza

dru

g, C

SV0C

0010

18, w

ith a

hig

h SI

val

ue. T

his

conv

enie

nt m

etho

d fo

r dr

ug s

cree

ning

may

fa

cilit

ate

the

deve

lopm

ent o

f ant

ivir

al d

rugs

that

targ

et v

irus

es o

ther

than

the

influ

enza

vir

us, s

uch

as H

IV a

nd H

BV.

Hun

g et

al.

2012

nuc

leop

rote

in

inhi

bito

rsN

ucle

ozin

The

influ

enza

vir

us n

ucle

opro

tein

(NP)

is a

n em

ergi

ng ta

rget

for

anti-

influ

enza

dru

g de

velo

pmen

t. N

ucle

ozin

(1) a

nd

its c

lose

ly re

late

d de

riva

tives

had

bee

n id

entif

ied

as N

P in

hibi

tors

dis

play

ing

anti-

influ

enza

act

ivity

. Util

izin

g 1

as a

lead

m

olec

ule,

we

succ

essf

ully

des

igne

d an

d sy

nthe

size

d a

seri

es o

f 1H

-1,2

,3-t

riaz

ole-

4-ca

rbox

amid

e de

riva

tives

as

new

ant

i-in

fluen

za A

age

nts.

One

of t

he m

ost p

oten

t com

poun

ds, 3

b, in

hibi

ted

the

repl

icat

ion

of v

ario

us H

3N2

and

H1N

1 in

flu-

enza

A v

irus

str

ains

with

IC50

val

ues

rang

ing

from

0.5

to 4

.6 µ

M. C

ompo

und

3b a

lso

stro

ngly

inhi

bite

d th

e re

plic

atio

n of

H5N

1 (R

G14

), am

antid

ine-

resi

stan

t A/W

SN/3

3 (H

1N1)

, and

ose

ltam

ivir

-res

ista

nt A

/WSN

/193

3 (H

1N1,

274

Y) v

irus

st

rain

s w

ith IC

50 v

alue

s in

sub

-mu

M r

ange

s. F

urth

er c

ompu

tatio

nal s

tudi

es a

nd m

echa

nism

inve

stig

atio

n su

gges

ted

that

3b

mig

ht d

irec

tly ta

rget

influ

enza

vir

us A

nuc

leop

rote

in to

inhi

bit i

ts n

ucle

ar a

ccum

ulat

ion.

Che

ng e

t al.

2012

Inhi

bito

rs o

f non

-st

ruct

ural

pro

tein

The

inna

te im

mun

e sy

stem

gua

rds

agai

nst v

irus

infe

ctio

n th

roug

h a

vari

ety

of m

echa

nism

s in

clud

ing

mob

iliza

tion

of th

e ho

st in

terf

eron

sys

tem

, whi

ch a

ttac

ks v

iral

pro

duct

s m

ainl

y at

a p

ostt

rans

crip

tiona

l lev

el. T

he in

fluen

za v

irus

N

S1 p

rote

in is

a m

ultif

unct

iona

l fac

ilita

tor

of v

irus

repl

icat

ion,

one

of w

hose

act

ions

is to

ant

agon

ize

the

inte

rfer

on

resp

onse

. Sin

ce N

S1 is

requ

ired

for

effic

ient

vir

us re

plic

atio

n, it

was

reas

oned

that

che

mic

al in

hibi

tors

of t

his

prot

ein

coul

d be

use

d to

furt

her

unde

rsta

nd v

irus

-hos

t int

erac

tions

and

als

o se

rve

as p

oten

tial n

ew a

ntiv

iral

age

nts.

A y

east

-ba

sed

assa

y w

as d

evel

oped

to id

entif

y co

mpo

unds

that

phe

noty

pica

lly s

uppr

ess

NS1

func

tion.

Sev

eral

suc

h co

mpo

unds

ex

hibi

ted

sign

ifica

nt a

ctiv

ity s

peci

fical

ly a

gain

st in

fluen

za A

vir

us in

cel

l cul

ture

but

had

no

effe

ct o

n th

e re

plic

atio

n of

an

othe

r RN

A v

irus

, res

pira

tory

syn

cytia

l vir

us. I

nter

estin

gly,

cel

ls la

ckin

g an

inte

rfer

on re

spon

se w

ere

drug

resi

stan

t, su

gges

ting

that

the

com

poun

ds b

lock

inte

ract

ions

bet

wee

n N

S1 a

nd th

e in

terf

eron

sys

tem

. Acc

ordi

ngly

, the

com

poun

ds

reve

rsed

the

inhi

bitio

n of

bet

a in

terf

eron

mRN

A in

duct

ion

duri

ng in

fect

ion,

whi

ch is

kno

wn

to b

e ca

used

by

NS1

. In

addi

tion,

the

com

poun

ds b

lock

ed th

e ab

ility

of N

S1 p

rote

in to

inhi

bit d

oubl

e-st

rand

ed R

NA

-dep

ende

nt a

ctiv

atio

n of

a

tran

sfec

ted

beta

inte

rfer

on p

rom

oter

con

stru

ct. T

he e

ffect

s of

the

com

poun

ds w

ere

spec

ific

to N

S1, b

ecau

se th

ey

had

no e

ffect

on

the

abili

ty o

f the

sev

ere

acut

e re

spir

ator

y sy

ndro

me

coro

navi

rus

papa

inlik

e pr

otea

se p

rote

in to

blo

ck

beta

inte

rfer

on p

rom

oter

act

ivat

ion.

The

se d

ata

dem

onst

rate

that

the

func

tion

of N

S1 c

an b

e m

odul

ated

by

chem

ical

in

hibi

tors

and

that

suc

h in

hibi

tors

will

be

usef

ul a

s pr

obes

of b

iolo

gica

l fun

ctio

n an

d as

sta

rtin

g po

ints

for

clin

ical

dru

g de

velo

pmen

t.

Basu

et a

l. 20

09


172

Func

tion

of n

S1

prot

ein

Inac

tivat

ion

of N

F-ka

ppa

B Pr

even

tion

of in

terf

eron

in

duct

ion

The

alph

a/be

ta in

terf

eron

(IFN

-alp

ha/b

eta)

syst

em re

pres

ents

one

of t

he fi

rst l

ines

of d

efen

se a

gain

st v

irus

infe

ctio

ns. A

s a

resu

lt, m

ost v

irus

es e

ncod

e IF

N a

ntag

onis

tic fa

ctor

s whi

ch e

nhan

ce v

iral r

eplic

atio

n in

thei

r hos

ts. W

e ha

ve p

revi

ously

show

n th

at a

reco

mbi

nant

influ

enza

A v

irus

lack

ing

the

NS1

gen

e (d

elN

S1) o

nly

repl

icat

es e

ffici

ently

in IF

N-a

lpha

/bet

a -d

efici

ent

syst

ems.

Con

sist

ent w

ith th

is o

bser

vatio

n, w

e fo

und

that

infe

ctio

n of

tiss

ue c

ultu

re c

ells

with

del

NS1

vir

us, b

ut n

ot w

ith w

ild-

type

influ

enza

A v

irus

, ind

uced

hig

h le

vels

of m

RNA

synt

hesi

s fro

m IF

N-a

lpha

/bet

a ge

nes,

incl

udin

g IF

N-b

eta.

It is

kno

wn

that

tran

sact

ivat

ion

of th

e IF

N-b

eta

prom

oter

dep

ends

on

NF-

kapp

aB a

nd se

vera

l oth

er tr

ansc

ript

ion

fact

ors.

Inte

rest

ingl

y, ce

lls in

fect

ed w

ith d

elN

S1 v

irus

show

ed h

igh

leve

ls of

NF-

kapp

aB a

ctiv

atio

n co

mpa

red

with

thos

e in

fect

ed w

ith w

ild-t

ype

viru

s. Ex

pres

sion

of d

omin

ant-

nega

tive

inhi

bito

rs o

f the

NF-

kapp

aB p

athw

ay d

urin

g de

INS1

vir

us in

fect

ion

prev

ente

d th

e tr

ansa

ctiv

atio

n of

the

IFN

-bet

a pr

omot

er, d

emon

stra

ting

a fu

nctio

nal l

ink

betw

een

NF-

kapp

aB a

ctiv

atio

n an

d IF

N-a

lpha

/bet

a sy

nthe

sis i

n de

INS1

vir

us-in

fect

ed c

ells

. Mor

eove

r, ex

pres

sion

of t

he N

S1 p

rote

in p

reve

nted

vir

us- a

nd/o

r dou

ble-

stra

nded

RN

A (d

sRN

A)-

med

iate

d ac

tivat

ion

of th

e N

F-ka

ppaB

pat

hway

and

of I

FN-b

eta

synt

hesi

s. Th

is in

hibi

tory

pro

pert

y of

the

NS1

pro

tein

of i

nflue

nza

A v

irus

was

dep

ende

nt o

n its

abi

lity

to b

ind

dsRN

A, s

uppo

rtin

g a

mod

el in

whi

ch b

indi

ng o

f NS1

to

dsRN

A g

ener

ated

dur

ing

influ

enza

vir

us in

fect

ion

prev

ents

the

activ

atio

n of

the

IFN

syst

em. N

S1-m

edia

ted

inhi

bitio

n of

the

NF-

kapp

aB p

athw

ay m

ay th

us p

lay

a ke

y ro

le in

the

path

ogen

esis

of i

nflue

nza

A v

irus

.

Wan

g et

al.

2000

Rn

A in

terf

eren

ceSh

ort i

nter

feri

ng R

NA

sVi

ral m

RNA

deg

rada

tion

Hig

hly

path

ogen

ic a

vian

influ

enza

(HPA

I) H

5N1

viru

s is e

ndem

ic in

man

y re

gion

s aro

und

the

wor

ld a

nd re

mai

ns a

sign

ifi-

cant

pan

dem

ic th

reat

. To

date

H5N

1 ha

s cla

imed

alm

ost 3

00 h

uman

live

s wor

ldw

ide,

with

a m

orta

lity

rate

of 6

0% a

nd h

as

caus

ed th

e de

ath

or c

ullin

g of

hun

dred

s of m

illio

ns o

f pou

ltry

sinc

e its

initi

al o

utbr

eak

in 1

997.

We

have

des

igne

d m

ulti-

func

tiona

l RN

A in

terf

eren

ce (R

NA

i)-ba

sed

ther

apeu

tics t

arge

ting

H5N

1 th

at d

egra

de v

iral

mRN

A v

ia th

e RN

Ai p

athw

ay

whi

le a

t the

sam

e tim

e au

gmen

ting

the

host

ant

ivir

al re

spon

se b

y in

duci

ng h

ost t

ype

I int

erfe

ron

(IFN

) pro

duct

ion.

Mor

eo-

ver,

we

have

iden

tified

two

fact

ors c

ritic

al fo

r max

imis

ing

the

imm

unos

timul

ator

y pr

oper

ties o

f sho

rt in

terf

erin

g (s

i) RN

As

in c

hick

en c

ells

(i) m

ode

of sy

nthe

sis a

nd (i

i) nu

cleo

side

sequ

ence

to a

ugm

ent t

he re

spon

se to

vir

us. Th

e 5-

bp n

ucle

osid

e se

quen

ce 5

’-UG

UG

U-3

’ is a

key

det

erm

inan

t in

indu

cing

hig

h le

vels

of e

xpre

ssio

n of

IFN

-alp

ha, -

beta

, -la

mbd

a an

d in

terl

euki

n 1-

beta

in c

hick

en c

ells

. Pos

ition

ing

of th

is 5

’-UG

UG

U-3

’ mot

if at

the

5’- e

nd o

f the

sens

e st

rand

of s

iRN

As,

but

no

t the

3’-

end,

resu

lted

in a

rapi

d an

d en

hanc

ed in

duct

ion

of ty

pe I

IFN

. An

anti-

H5N

1 av

ian

influ

enza

siRN

A d

irec

ted

agai

nst t

he P

B1 g

ene

(PB1

-225

7) ta

gged

with

5’-U

GU

GU

-3’ i

nduc

ed ty

pe I

IFN

ear

lier a

nd to

a g

reat

er e

xten

t com

pare

d to

a

non-

tagg

ed P

B1-2

257.

Tes

ted

agai

nst H

5N1

in v

itro,

the

tagg

ed P

B1-2

257

was

mor

e eff

ectiv

e th

an n

on-t

agge

d PB

1-22

57.

Thes

e da

ta d

emon

stra

te th

e ab

ility

of a

n im

mun

ostim

ulat

ory

mot

if to

impr

ove

the

perf

orm

ance

of a

n RN

Ai-

base

d an

tivi-

ral,

a fin

ding

that

may

influ

ence

the

desi

gn o

f fut

ure

RNA

i-bas

ed a

nti-i

nflue

nza

ther

apeu

tics.

Stew

art e

t al.

2011

Rn

A in

terf

eren

ceSh

ort i

nter

feri

ng R

NA

sSi

lenc

e ge

ne e

xpre

ssio

nV

iral

mRN

A d

egra

datio

n

RNA

inte

rfer

ence

(RN

Ai)

is a

pow

erfu

l too

l to

sile

nce

gene

exp

ress

ion.

Sm

all i

nter

feri

ng R

NA

(siR

NA

)-in

duce

d RN

A d

egra

-da

tion

has b

een

rece

ntly

use

d as

an

antiv

irus

age

nt to

inhi

bit s

peci

fic v

irus

repl

icat

ion.

Her

e, w

e sh

owed

that

seve

ral s

iRN

As

spec

ific

for c

onse

rved

regi

ons o

f infl

uenz

a vi

rus m

atri

x (M

2) a

nd n

ucle

ocap

sid

prot

ein

(NP)

gen

es c

ould

effe

ctiv

ely

inhi

bit

expr

essi

on o

f the

cor

resp

ondi

ng v

iral p

rote

in. W

e al

so e

valu

ated

the

antiv

iral p

oten

tial o

f the

se si

RNA

s tar

getin

g M

2 an

d N

P of

H5N

1 av

ian

influ

enza

vir

us (A

IV),

whi

ch a

re e

ssen

tial t

o vi

ral r

eplic

atio

n. W

e in

vest

igat

ed th

e in

hibi

tory

effe

ct o

f M2-

spe-

cific

siRN

As a

nd N

P-sp

ecifi

c si

RNA

s on

influ

enza

A v

irus

(H5N

1, H

1N1

and

H9N

2) re

plic

atio

n in

Mad

in-D

arby

can

ine

kidn

ey (M

DC

K) c

ells

and

BALB

/c m

ice.

The

resu

lts sh

owed

that

trea

tmen

t with

thes

e si

RNA

s cou

ld sp

ecifi

cally

inhi

bit i

nflu-

enza

A v

irus

repl

icat

ion

in M

DC

K c

ells

(0.5

1–1.

63 T

CID

50 re

duct

ion

in v

irus

tite

rs),

and

deliv

ery

of p

S-M

48 a

nd p

S-N

P138

3 si

gnifi

cant

ly re

duce

d lu

ng v

irus

tite

rs in

the

infe

cted

mic

e (1

6–50

-fol

d re

duct

ion

in lu

ng v

irus

tite

rs) a

nd p

artia

lly p

rote

cted

th

e m

ice

from

leth

al in

fluen

za v

irus

cha

lleng

e (a

surv

ival

rate

of 4

/8 fo

r H1N

1 vi

rus-

infe

cted

mic

e an

d 2/

8 fo

r H5N

1 vi

rus

infe

cted

mic

e). M

oreo

ver,

the

trea

tmen

t of p

S-M

48 a

nd p

S-N

P138

3 co

uld

supp

ress

repl

icat

ion

of d

iffer

ent s

ubty

pes o

f infl

u-en

za A

vir

uses

, inc

ludi

ng a

H5N

1 hi

ghly

pat

hoge

nic

avia

n is

olat

e st

rain

. The

resu

lts p

rovi

ded

a ba

sis f

or fu

rthe

r dev

elop

men

t of

siRN

A fo

r pro

phyl

axis

and

ther

apy

of in

fluen

za v

irus

infe

ctio

n in

hum

ans a

nd a

nim

als.

Zho

u et

al.

2007


173

Rn

A in

terf

eren

ceSh

ort i

nter

feri

ng R

NA

ssi

RNA

-res

ista

nt v

irus

esH

1-sh

ort h

airp

in R

NA

RNA

inte

rfer

ence

(RN

Ai)

prov

ides

a p

ower

ful n

ew m

eans

to in

hibi

t vir

al in

fect

ion

spec

ifica

lly. H

owev

er, t

he s

elec

tion

of s

iRN

A-r

esis

tant

vir

uses

is a

maj

or c

once

rn in

the

use

of R

NA

i as

antiv

iral

ther

apeu

tics.

In th

is s

tudy

, we

cond

ucte

d a

lent

ivir

al v

ecto

r w

ith a

H1-

shor

t hai

rpin

RN

A (s

hRN

A) e

xpre

ssio

n ca

sset

te to

del

iver

sm

all i

nter

feri

ng R

NA

s (s

iRN

As)

in

to m

amm

alia

n ce

lls. U

sing

this

vec

tor

that

als

o ex

pres

ses

enha

nced

gre

en fl

uore

scen

ce p

rote

in (E

GFP

) as

surr

ogat

e m

arke

r, st

able

shR

NA

-exp

ress

ing

cell

lines

wer

e su

cces

sful

ly e

stab

lishe

d an

d th

e in

hibi

tion

effic

ienc

ies

of r

atio

nally

de

sign

ed s

iRN

As

targ

etin

g to

con

serv

ed re

gion

s of

influ

enza

A v

irus

gen

ome

wer

e as

sess

ed. T

he re

sults

sho

wed

that

a

siRN

A ta

rget

ing

influ

enza

M2

gene

(siM

2) p

oten

tly in

hibi

ted

vira

l rep

licat

ion.

The

siM

2 w

as n

ot o

nly

effe

ctiv

e fo

r H

1N1

viru

s bu

t als

o fo

r hi

ghly

pat

hoge

nic

avia

n in

fluen

za v

irus

H5N

1. In

add

ition

to it

s M

2 in

hibi

tion,

the

siM

2 al

so

inhi

bite

d N

P m

RNA

acc

umul

atio

n an

d pr

otei

n ex

pres

sion

. A lo

ng te

rm in

hibi

tion

effe

ct o

f the

siM

2 w

as d

emon

stra

ted

and

the

emer

genc

e of

siR

NA

-res

ista

nt m

utan

ts in

influ

enza

qua

sisp

ecie

s w

as n

ot o

bser

ved.

Tak

en to

geth

er, o

ur s

tudy

su

gges

ted

that

M2

gene

mig

ht b

e an

opt

imal

RN

Ai t

arge

t for

ant

ivir

al th

erap

y. T

hese

find

ings

pro

vide

use

ful i

nfor

ma-

tion

for

the

deve

lopm

ent o

f RN

Ai-

base

d pr

ophy

laxi

s an

d th

erap

y fo

r hu

man

influ

enza

vir

us in

fect

ion.

Sui e

t al.

2009

Alk

aloi

ds fr

om

Am

aryl

lidac

eae

Lyco

rine

Hae

man

tham

ine

Influ

enza

A v

irus

es o

ccas

iona

lly c

ause

larg

e ep

idem

ics

and

kill

thou

sand

s ev

ery

year

. Whi

le li

ttle

is k

now

n ab

out t

he

mec

hani

sm o

f cel

l fus

ion

in d

isea

ses,

esp

ecia

lly in

fluen

za v

irus

infe

cted

and

pro

tect

ion

from

Am

aryl

lidac

eae

Alk

aloi

ds.

The

two

com

poun

ds ly

cori

ne (A

A1)

and

hae

man

tham

ine

(AA

3) w

ere

obta

ined

from

bul

bs o

f L. r

adia

te, e

xhib

ited

anti-

influ

enza

act

ivity

aft

er in

fluen

za v

irus

ent

ry c

ells

. The

two

com

poun

ds w

ere

inve

stig

ated

for

in v

itro

disp

layi

ng d

iffer

-en

t lev

els

of re

sist

ance

to p

ro-a

popt

otic

stim

uli.

Seve

n in

fluen

za v

irus

es w

ere

used

, A/C

K/G

D/1

78/0

4 (H

5N, 1

78),

A/

DK

/GD

/212

/04

(H5N

1, 2

12),

A/s

win

e/G

D/1

66/0

6 (H

3N2,

166

), A

/CK

/HN

/170

/03

(H1N

1, 1

70),

A/P

uert

o Ri

co/8

/34

(H1N

1, P

R8),

A/C

k/G

D/4

00/0

7 (H

9N2,

400

), A

/Ck/

GD

/228

/04

(H9N

2, 2

28),

the

two

com

poun

ds e

xhib

ited

pote

ncy

in th

e si

ngle

-dig

it m

icro

mol

ar r

ange

. The

stu

dies

als

o sh

owed

that

AA

1 an

d A

A3

exer

ted

thei

r in

vitr

o an

ti-in

fluen

za

activ

ity th

roug

h cy

tost

atic

rat

her

than

cyt

otox

ic e

ffect

s. M

any

viru

ses

inte

ract

with

the

host

cel

ls to

cha

nge

thei

r ow

n gr

owth

whi

ch th

ey fa

vor

the

spee

d. T

he c

ells

infe

cted

with

vir

us, g

row

th o

f MD

CK

cel

ls w

as s

low

ed d

own

by a

rres

ting

cell

cycl

e at

G1/

S ph

ase.

With

the

com

poun

d tr

eate

d, th

e gr

owth

cyc

le w

as d

ecre

ased

in S

pha

se. W

ith H

5N1

influ

enza

vi

rus

trea

ted,

the

cyto

skel

eton

of c

ells

was

cha

nged

whi

le w

ith th

e co

mpo

und

trea

ted

the

prot

ectio

n of

cyt

oske

leto

n w

as

obvi

ousl

y pr

otec

ted.

The

dat

a sh

owed

diff

eren

ces

betw

een

drug

trea

ted

cells

and

vir

us in

fect

ed c

ells

, pro

vide

d a

basi

s to

fu

rthe

r ex

plor

e ce

ll fu

sion

and

ant

i-in

fluen

za m

echa

nism

of t

he tw

o co

mpo

unds

.

He

et a

l. 20

12

Poly

sacc

hari

des

from

se

awee

dsSu

lfate

d fu

cans

Het

erog

lycu

rona

ns

To e

xplo

re th

e po

lysa

ccha

ride

s fr

om s

elec

ted

seaw

eeds

of A

tlant

ic C

anad

a an

d to

eva

luat

e th

eir

pote

ntia

l ant

i-in

fluen

za

viru

s ac

tiviti

es, p

olys

acch

arid

es w

ere

isol

ated

from

sev

eral

Atla

ntic

Can

adia

n se

awee

ds, i

nclu

ding

thre

e re

d al

gae

(Pol

y-si

phon

ia la

nosa

, Fur

cella

ria

lum

bric

alis

, and

Pal

mar

ia p

alm

ata)

, tw

o br

own

alga

e (A

scop

hyllu

m n

odos

um a

nd F

ucus

ve

sicu

losu

s), a

nd o

ne g

reen

alg

a (U

lva

lact

uca)

by

sequ

entia

l ext

ract

ion

with

col

d w

ater

, hot

wat

er, a

nd a

lkal

i sol

utio

ns.

The

se p

olys

acch

arid

es w

ere

anal

yzed

for

mon

osac

char

ide

com

posi

tion

and

othe

r ge

nera

l che

mic

al p

rope

rtie

s, a

nd th

ey

wer

e ev

alua

ted

for

anti-

influ

enza

vir

us a

ctiv

ities

. Tot

al s

ugar

con

tent

s in

thes

e po

lysa

ccha

ride

s ra

nged

from

15.

4% (i

n U

. lac

tuca

) to

91.4

% (i

n F.

lum

bric

alis

); su

lfatio

n le

vel w

as a

s hi

gh a

s 17

.6%

in a

pol

ysac

char

ide

from

U. l

actu

ca, w

here

as

it co

uld

not b

e de

tect

ed in

an

alik

ali-

extr

act f

rom

P. p

alm

aria

. For

pol

ysac

char

ides

from

red

seaw

eeds

, the

mai

n su

gar

units

wer

e su

lfate

d ga

lact

ans

(aga

r or

car

rage

enan

) for

P. l

anos

a, F

. lum

bric

alis

, and

xyl

ans

for

P. p

alm

ata.

In b

row

n se

awee

ds, t

he p

olys

acch

arid

es la

rgel

y co

ntai

ned

sulfa

ted

fuca

ns, w

here

as th

e po

lysa

ccha

ride

s in

gre

en s

eaw

eed

wer

e m

ainl

y co

mpo

sed

of h

eter

ogly

curo

nans

. Scr

eeni

ng fo

r an

tivir

al a

ctiv

ity a

gain

st in

fluen

za A

/PR/

8/34

(H1N

1) v

irus

re

veal

ed th

at b

row

n al

gal p

olys

acch

arid

es w

ere

part

icul

arly

effe

ctiv

e. S

eaw

eeds

from

Atla

ntic

Can

ada

are

a go

od s

ourc

e of

mar

ine

poly

sacc

hari

des

with

pot

entia

l ant

ivir

al p

rope

rtie

s.

Jiao

et a

l. 20

12


174

Car

rage

enan

s fr

om r

ed

alga

eK

appa

-car

rage

enan

ol

igos

acch

arid

es

Car

rage

enan

s, th

e su

lpha

ted

gala

ctan

s der

ived

from

red

alga

e, a

re a

ttra

ctin

g in

crea

sing

inte

rest

in d

evel

opin

g po

tent

ial

anti-

vira

l dru

gs. I

n th

is st

udy,

low

mol

ecul

ar w

eigh

t kap

pa-c

arra

geen

an o

ligos

acch

arid

es (K

CO

) and

thei

r sul

phat

ed d

eriv

-at

ives

(KC

O-S

) wer

e pr

epar

ed, a

nd th

eir a

nti-i

nflue

nza

A v

irus

(IAV

) pro

pert

ies w

ere

inve

stig

ated

. The

resu

lts in

dica

ted

that

KC

O a

nd K

CO

-S c

ould

effe

ctiv

ely

inhi

bit I

AV m

ultip

licat

ion

in M

DC

K c

ells

in a

dos

e-de

pend

ent m

anne

r. Fu

rthe

r-m

ore,

a st

ruct

ure-

activ

ity re

latio

nshi

p st

udy

show

ed th

at th

e de

gree

of s

ulph

atio

n an

d m

olec

ular

wei

ght w

ere

the

mai

n fa

ctor

s tha

t infl

uenc

ed th

e an

ti-IA

V a

ctiv

ity o

f kap

pa-c

arra

geen

an o

ligos

acch

arid

e. Th

e m

ost a

ctiv

e ka

ppa-

carr

agee

nan

oli-

gosa

ccha

ride

had

a su

lpha

te c

onte

nt o

f 0.8

–1.0

mol

e/m

ole

of d

isac

char

ide

and

a m

olec

ular

wei

ght o

f 1–3

kD

a. In

add

ition

, K

CO

and

KC

O-S

cou

ld si

gnifi

cant

ly im

prov

e su

rviv

al a

nd d

ecre

ase

pulm

onar

y vi

ral t

itres

in IA

V-i

nfec

ted

mic

e. M

oreo

ver,

the

antiv

iral

effe

ct o

f KC

O a

nd K

CO

-S d

oes n

ot se

em to

be

depe

nden

t on

the

inte

rfer

on sy

stem

. In

conc

lusi

on, c

arra

-ge

enan

olig

osac

char

ide

and

its su

lpha

ted

deri

vativ

e ha

ve g

ood

inhi

bito

ry a

ctio

ns o

n IA

V re

plic

atio

n in

vitr

o an

d in

viv

o.

Wan

g et

al.

2012

Subs

tanc

es fr

om M

osla

di

anth

era

Phen

olic

sesq

uite

rpen

esA

rom

atic

com

poun

ds

Ethn

opha

rmac

olog

ical

rele

vanc

e: M

osla

dia

nthe

ra a

s an

arom

atic

her

b is

use

d in

folk

med

icin

e fo

r the

trea

tmen

t of c

ough

, co

lds,

feve

r, br

onch

itis,

nasa

l con

gest

ion

and

head

ache

. Aim

of t

he st

udy:

To

char

acte

rize

che

mic

al c

ompo

sitio

ns a

nd to

ev

alua

te th

e an

ti-in

fluen

za e

ffect

s of e

ssen

tial o

ils o

f M. d

iant

hera

(MD

EO) i

n in

fluen

za v

irus

A (I

VA) i

nfec

ted

mic

e. M

ater

i-al

s and

met

hods

: MD

EO w

as o

btai

ned

by h

ydro

dist

illat

ion

and

anal

ysed

by

gas c

hrom

atog

raph

y-m

ass s

pect

rom

etry

(G

C-M

S). I

CR

mic

e w

ere

trea

ted

with

MD

EO fo

r 5 c

onse

cutiv

e da

ys a

t dos

es o

f 90–

360

mg/

kg a

fter p

ost-

infe

cted

. Lev

els o

f Se

rum

IL-4

and

IFN

-gam

ma

wer

e as

saye

d by

ELI

SA. L

evel

s of M

OD

, SO

D, T

AO

C a

nd G

SH-P

x in

lung

tiss

ue w

ere

dete

r-m

ined

by

colo

rim

etri

c m

etho

d. R

esul

ts: G

C-M

S an

alys

is re

veal

ed th

e pr

esen

ce o

f 29

com

pone

nts t

hat a

ccou

nt fo

r 97.

74%

of

phen

olic

sesq

uite

rpen

es a

nd a

rom

atic

com

poun

ds. Th

e m

ajor

com

poun

ds w

ere

elem

icin

(16.

51%

), th

ymol

(14.

77%

), be

ta-

cary

ophy

llene

(14.

49%

), is

o-el

emic

in (9

.22%

), as

aron

e (6

.09%

) and

alp

ha-c

aryo

phyl

lene

(5.2

6%).

It ha

d si

gnifi

cant

effe

cts o

n de

crea

sing

lung

vira

l tite

rs, i

nhib

iting

pne

umon

ia, r

educ

ing

leve

ls of

seru

m IF

N-g

amm

a an

d IL

-4, a

nd e

nhan

cing

ant

ioxi

dant

ac

tivity

in th

e lu

ng ti

ssue

of I

VA in

fect

ed m

ice.

Con

clus

ions

: MPE

cou

ld e

xhib

it th

erap

eutic

al e

ffect

s in

IVA

infe

cted

mic

e as

a su

ppre

ssor

of I

VA re

plic

atio

n an

d in

flam

mat

ory

med

iato

rs a

nd a

pro

mot

er o

f ant

ioxi

dant

pot

entia

ls. Th

eref

ore,

MD

EO

coul

d pr

ovid

e a

safe

and

effe

ctiv

e th

erap

eutic

can

dida

te fo

r tre

atm

ent o

f infl

uenz

a an

d its

subs

eque

nt v

iral p

neum

onia

.

Wu

et a

l. 20

12

Inhi

bito

rs fr

om h

erba

l ex

trac

tBe

rber

ine

Obj

ectiv

e: T

o ex

plor

e th

e po

tent

ial e

ffect

s of b

erbe

rine

on in

fluen

za v

irus i

nfec

tion

both

in v

itro

and

in v

ivo.

Met

hods

: In

vitr

o an

ti-in

fluen

za v

irus a

ssay

s wer

e pe

rfor

med

by

cyto

path

ogen

ic e

ffect

and

neu

ram

inid

ase

assa

ys in

Mad

in D

arby

can

ine

kidn

ey

cells

. In

vivo

ant

i-infl

uenz

a vi

rus a

ssay

s wer

e pe

rfor

med

on

the

vira

l pne

umon

ia m

odel

of m

ice.

The

num

bers

of m

ice

that

die

d w

ithin

day

2 to

day

14

post

infe

ctio

n w

ere

reco

rded

to c

alcu

late

the

mor

talit

y. O

n da

ys 2

, 4, a

nd 6

, the

vira

l tite

rs in

the

lung

s w

ere

dete

rmin

ed b

y he

mag

glut

inat

ion

assa

y; h

emat

oxyl

in/e

osin

stai

ning

was

use

d to

ass

ess t

he p

atho

geni

c ch

ange

s of l

ung

tis-

sues

; the

con

cent

ratio

ns o

f tum

or n

ecro

sis fa

ctor

-alp

ha (T

NF-

alph

a) a

nd m

onoc

yte

spec

ific

chem

oattr

acta

nt m

olec

ule

(MC

P-1)

w

ere

mea

sure

d by

radi

o im

mun

oass

ay o

r enz

yme-

linke

d im

mun

osor

bent

ass

ay; t

he c

once

ntra

tions

of n

itric

oxi

de (N

O) a

nd

indu

cibl

e ni

tric

oxi

de sy

nthe

tase

(iN

OS)

wer

e de

tect

ed b

y co

lorim

etric

met

hod;

reve

rse

tran

scrip

tion

poly

mer

ase

chai

n re

actio

n w

as u

sed

to d

etec

t the

mRN

A le

vel o

f TN

F-al

pha

and

MC

P-1.

Res

ults

: Ber

berin

e sh

owed

inhi

bito

ry e

ffect

s on

cyto

path

ogen

ic

effec

ts a

nd n

eura

min

idas

e ac

tivity

of v

irus,

with

the

ther

apeu

tic in

dex

9.69

. In

vivo

, ber

berin

e de

crea

sed

mic

e m

orta

lity

from

90

% to

55%

, red

uced

viru

s tite

rs in

the

lung

s on

day

2 po

stin

fect

ion

(P <

0.0

5). Th

e lu

ng h

istol

ogy

scor

es w

ere

1.50

±0.

67, 4

.50

± 1.

00, a

nd 5

.50

± 1.

00 in

the

berb

erin

e gr

oup

on d

ays 2

, 4, a

nd 6

, res

pect

ivel

y, w

hich

wer

e sig

nific

antly

redu

ced

com

pare

d to

2.

17 ±

0.2

2, 6

.83

± 0.

44, a

nd 8

.50

± 0.

33 in

the

infe

cted

gro

up (P

< 0

.05)

. The

prod

uctio

ns o

f NO

and

iNO

S w

ere

repr

esse

d by

be

rber

ine

com

pare

d w

ith th

ose

in th

e in

fect

ed g

roup

(P <

0.0

1). Th

e tr

ansc

riptio

n an

d ex

pres

sion

of T

NF-

alp

ha w

ere

inhi

bite

d by

ber

berin

e on

day

4 (P

< 0

.01)

and

day

6 (P

< 0

.05)

, and

thos

e of

MC

P-1

wer

e in

hibi

ted

on d

ay 6

(P <

0.0

1) c

ompa

red

with

the

infe

cted

gro

up. C

oncl

usio

ns: B

erbe

rine

exhi

bite

d an

tivira

l effe

cts o

n th

e in

fluen

za v

irus b

oth

in v

itro

and

in v

ivo.

The

poss

ible

th

erap

eutic

mec

hani

sm o

f ber

berin

e on

influ

enza

-indu

ced

vira

l pne

umon

ia m

ight

be

inhi

bitin

g th

e vi

rus i

nfec

tion,

as w

ell a

s im

prov

ing

the

path

ogen

ic c

hang

es b

y re

pres

sing

infla

mm

ator

y su

bsta

nces

rele

ase.

Wu

et a

l. 20

11


175

nan

obod

ies

Targ

etin

g of

H

5N1h

emag

glut

inin

We

asse

ssed

the

prot

ectiv

e po

tent

ial o

f mon

oval

ent a

nd b

ival

ent N

anob

odie

s (A

blyn

x) a

gain

st c

halle

nge

with

this

vir

us.

Thes

e N

anob

odie

s wer

e de

rive

d fr

om ll

amas

and

targ

et H

5N1

hem

aggl

utin

in. I

ntra

nasa

l adm

inis

trat

ion

of N

anob

odie

s eff

ectiv

ely

cont

rolle

d ho

mol

ogou

s infl

uenz

a A

vir

us re

plic

atio

n. A

dmin

istr

atio

n of

Nan

obod

ies b

efor

e ch

alle

nge

stro

ngly

re

duce

d H

5N1

viru

s rep

licat

ion

in th

e lu

ngs a

nd p

rote

cted

mic

e fr

om m

orbi

dity

and

mor

talit

y af

ter a

leth

al c

halle

nge

with

H

5N1

viru

s. Th

e bi

vale

nt N

anob

ody

was

at l

east

60-

fold

mor

e eff

ectiv

e th

an th

e m

onov

alen

t Nan

obod

y in

con

trol

ling

viru

s re

plic

atio

n. In

add

ition

, Nan

obod

y th

erap

y af

ter c

halle

nge

stro

ngly

redu

ced

vira

l rep

licat

ion

and

sign

ifica

ntly

del

ayed

tim

e to

dea

th. E

pito

pe m

appi

ng re

veal

ed th

at th

e V

HH

Nan

obod

y bi

nds t

o an

tigen

ic si

te B

in H

5 he

mag

glut

inin

. Bec

ause

Nan

o-bo

dies

are

smal

l, st

able

, and

sim

ple

to p

rodu

ce, t

hey

are

a pr

omis

ing,

nov

el th

erap

eutic

age

nt a

gain

st in

fluen

za.

Iban

ez e

t al.

2011

nan

obod

ies

M2

prot

ein

targ

etin

gN

eutr

aliz

ing

antib

odie

s aga

inst

the

high

ly c

onse

rved

M2

ion

chan

nel i

s tho

ught

to o

ffer b

road

pro

tect

ion

agai

nst i

nflue

nza

A

viru

ses.

Her

e, w

e sc

reen

ed sy

nthe

tic C

amel

sing

le-d

omai

n an

tibod

y (V

HH

) lib

rari

es a

gain

st n

ativ

e M

2 io

n ch

anne

l pro

tein

. O

ne o

f the

isol

ated

VH

Hs,

M2-

7A, s

peci

fical

ly b

ound

to M

2-ex

pres

sed

cell

mem

bran

e as

wel

l as i

nflue

nza

A v

irio

n, in

hibi

ted

repl

icat

ion

of b

oth

aman

tadi

ne-s

ensi

tive

and

resi

stan

t infl

uenz

a A

vir

uses

in v

itro,

and

pro

tect

ed m

ice

from

a le

thal

influ

enza

vi

rus c

halle

nge.

Mor

eove

r, M

2-7A

show

ed b

lock

ing

activ

ity fo

r pro

ton

influ

x th

roug

h M

2 io

n ch

anne

l. Th

ese

piec

es o

f evi

-de

nce

colle

ctiv

ely

dem

onst

rate

for t

he fi

rst t

ime

that

a n

eutr

aliz

ing

antib

ody

agai

nst M

2 w

ith b

road

spec

ifici

ty is

ach

ieva

ble,

an

d M

2-7A

may

hav

e po

tent

ial f

or c

ross

pro

tect

ion

agai

nst a

num

ber o

f var

iant

s and

subt

ypes

of i

nflue

nza

A v

irus

es.

Wei

et a

l. 20

11

Hum

anis

ed m

onoc

lona

l an

tibo

dies

Hae

mag

glut

inin

ta

rget

ing

Hum

aniz

ed m

onoc

lona

l ant

ibod

ies (

mA

bs) t

hat n

eutr

aliz

e H

5N1

viru

s cou

ld b

e us

ed a

s pro

phyl

axis

and

trea

tmen

t to

aid

in th

e co

ntai

nmen

t of s

uch

a pa

ndem

ic. M

etho

ds: N

eutr

aliz

ing

mA

bs a

gain

st H

5 he

mag

glut

inin

wer

e hu

man

ized

and

in

trod

uced

into

C57

BL/6

mic

e (1

, 5, o

r 10

mg/

kg b

odyw

eigh

t) o

ne d

ay p

rior

to-,

one

day

post

- and

thre

e da

ys p

ost-

leth

al

chal

leng

e w

ith H

5N1

A/V

ietn

am/1

203/

04 v

irus

. Effi

cacy

was

det

erm

ined

by

obse

rvat

ion

of w

eigh

t los

s as w

ell a

s sur

viva

l. Re

sults

: Tw

o m

Abs

neu

tral

izin

g fo

r ant

igen

ical

ly v

aria

nt H

5N1

viru

ses,

A/V

ietn

am/1

203/

04 a

nd A

/Hon

g K

ong/

213/

03

wer

e id

entifi

ed a

nd h

uman

ized

with

out l

oss o

f spe

cific

ity. B

oth

antib

odie

s exh

ibite

d pr

ophy

lact

ic e

ffica

cy in

mic

e, h

ow-

ever

, VN

04-2

-huG

1 pe

rfor

med

bet

ter r

equi

ring

onl

y 1

mg/

kg b

odyw

eigh

t for

com

plet

e pr

otec

tion.

Whe

n us

ed to

trea

t in

fect

ion

VN

04-2

-huG

1 w

as a

lso

com

plet

ely

prot

ectiv

e, e

ven

whe

n in

trod

uced

thre

e da

ys p

ost i

nfec

tion,

alth

ough

hig

her

dose

of a

ntib

ody

was

requ

ired

. Con

clus

ion:

Pro

phyl

axis

and

trea

tmen

t usi

ng n

eutr

aliz

ing

hum

aniz

ed m

Abs

is e

ffica

ciou

s ag

ains

t let

hal c

halle

nge

with

A/V

ietn

am/1

203/

04, p

rovi

ding

pro

of o

f pri

ncip

le fo

r the

use

of p

assi

ve a

ntib

ody

ther

apy

as a

co

ntai

nmen

t opt

ion

in th

e ev

ent o

f pan

dem

ic in

fluen

za.

Han

son

et a

l. 20

06

Hum

an m

onoc

lona

l an

tibo

dies

Pre-

exis

ting

neut

raliz

ing

antib

ody

prov

ides

the

first

line

of d

efen

ce a

gain

st p

atho

gens

in g

ener

al. F

or in

fluen

za v

irus

, ann

ual

vacc

inat

ions

are

giv

en to

mai

ntai

n pr

otec

tive

leve

ls of

ant

ibod

y ag

ains

t the

cur

rent

ly c

ircul

atin

g st

rain

s. H

ere

we

repo

rt th

at

afte

r boo

ster

vac

cina

tion

ther

e w

as a

rapi

d an

d ro

bust

influ

enza

-spe

cific

IgG

+ ant

ibod

y- se

cret

ing

plas

ma

cell

(ASC

) res

pons

e th

at p

eake

d at

app

roxi

mat

ely

day

7 an

d ac

coun

ted

for u

p to

6%

of p

erip

hera

l blo

od B

cel

ls. Th

ese

ASC

s cou

ld b

e di

stin

guis

hed

from

influ

enza

- spe

cific

IgG

+ mem

ory

B ce

lls th

at p

eake

d 14

–21

days

afte

r vac

cina

tion

and

aver

aged

1%

of a

ll B

cells

. Im

por-

tant

ly, a

s muc

h as

80%

of A

SCs p

urifi

ed a

t the

pea

k of

the

resp

onse

wer

e in

fluen

za sp

ecifi

c. Th

is A

SC re

spon

se w

as c

hara

cter

-iz

ed b

y a

high

ly re

stri

cted

B-c

ell r

ecep

tor (

BC

R) re

pert

oire

that

in so

me

dono

rs w

as d

omin

ated

by

only

a fe

w B

-cel

l clo

nes.

This

pau

cicl

onal

resp

onse

, how

ever

, sho

wed

ext

ensi

ve in

trac

lona

l div

ersi

ficat

ion

from

acc

umul

ated

som

atic

mut

atio

ns. W

e us

ed th

e im

mun

oglo

bulin

var

iabl

e re

gion

s iso

late

d fr

om so

rted

sing

le A

SCs t

o pr

oduc

e ov

er 5

0 hu

man

mon

oclo

nal a

ntib

od-

ies (

mA

bs) t

hat b

ound

to th

e th

ree

influ

enza

vac

cine

stra

ins w

ith h

igh

affini

ty. Th

is st

rate

gy d

emon

stra

tes t

hat w

e ca

n ge

ner-

ate

mul

tiple

hig

h-affi

nity

mA

bs fr

om h

uman

s with

in a

mon

th a

fter v

acci

natio

n. Th

e pa

nel o

f infl

uenz

a-vi

rus-

spec

ific

hum

an

mA

bs a

llow

ed u

s to

addr

ess t

he is

sue

of o

rigi

nal a

ntig

enic

sin

(OA

S): t

he p

heno

men

on w

here

the

indu

ced

antib

ody

show

s hi

gher

affi

nity

to a

pre

viou

sly e

ncou

nter

ed in

fluen

za v

irus

stra

in c

ompa

red

with

the

viru

s str

ain

pres

ent i

n th

e va

ccin

e (1

). H

owev

er, w

e fo

und

that

mos

t of t

he in

fluen

za- v

irus

- spe

cific

mA

bs sh

owed

the

high

est a

ffini

ty fo

r the

cur

rent

vac

cine

stra

in.

Thus

, OA

S do

es n

ot se

em to

be

a co

mm

on o

ccur

renc

e in

nor

mal

, hea

lthy

adul

ts re

ceiv

ing

influ

enza

vac

cina

tion.

Wra

mm

ert e

t al.

2008


176

Tabl

e 5.

Com

bina

tion

ther

apy

of in

flue

nza

infe

ctio

ns

ose

ltam

ivir

wit

h am

anta

dine

Mou

se m

odel

In th

e pr

esen

t stu

dy, t

he e

ffect

of c

ombi

ning

ant

i-infl

uenz

a dr

ugs a

ctiv

e at

diff

eren

t ste

ps o

f the

influ

enza

vir

us re

plic

a-tio

n cy

cle,

ose

ltam

ivir

as a

neu

ram

inid

ase

(NA

) inh

ibito

r and

am

anta

dine

targ

etin

g M

2 pr

otei

n, w

as in

vest

igat

ed in

viv

o by

ora

l adm

inis

trat

ion

in a

mou

se m

odel

of a

eros

ol in

fluen

za v

irus

infe

ctio

n an

d in

vitr

o in

MD

CK

cel

ls. I

n m

ice,

dos

es

of o

selta

miv

ir a

nd a

man

tadi

ne p

rovi

ding

50–

60%

surv

ival

aga

inst

A/H

ongk

ong/

1/68

(H3N

2) o

r A/P

R/8/

34 (H

1N1)

wer

e ca

pabl

e of

con

ferr

ing

com

plet

e pr

otec

tion

whe

n us

ed si

mul

tane

ousl

y, su

gges

ting

that

incr

ease

d in

hibi

tion

of in

fluen

za

viru

s rep

licat

ion

by c

ombi

ning

ose

ltam

ivir

and

am

anta

dine

in v

itro

tran

slat

es in

to p

rote

ctio

n fr

om le

thal

infe

ctio

n of

m

ice.

The

com

bina

tion

of a

man

tadi

ne w

ith o

selta

miv

ir re

quir

ed 1

5-fo

ld le

ss o

selta

miv

ir th

an m

onot

hera

py to

con

fer

com

plet

e pr

otec

tion

agai

nst l

etha

l aer

osol

influ

enza

vir

us in

fect

ion.

Rem

arka

bly,

aman

tadi

ne-b

ased

com

bina

tion

chem

o-pr

ophy

laxi

s was

eve

n eff

ectiv

e ag

ains

t am

anta

dine

-res

ista

nt A

/PR/

8/34

influ

enza

vir

us. Th

us, c

ombi

natio

n ch

emot

hera

py

may

be

mor

e effi

caci

ous t

han

mon

othe

rapy

aga

inst

new

ly e

mer

ging

Influ

enza

A su

btyp

es.

Mas

ihi e

t al.

2007

Zan

amiv

ir w

ith

imm

uno-

mod

ulat

ors

Cel

ecox

ibG

emfib

rozi

lM

esal

azin

eM

ouse

mod

el

The

mor

talit

y of

hum

an in

fect

ion

by in

fluen

za A

/H5N

1 vi

rus c

an e

xcee

d 80

%. Th

e hi

gh m

orta

lity

and

its p

oor r

espo

nse

to

the

neur

amin

idas

e in

hibi

tor o

selta

miv

ir h

ave

been

att

ribu

ted

to u

ncon

trol

led

viru

s-in

duce

d cy

toki

ne st

orm

. We

chal

-le

nged

BA

LB/c

mic

e w

ith 1

000

LD50

of i

nflue

nza

A/V

ietn

am/1

194/

04. S

urvi

val,

body

wei

ght,

hist

opat

holo

gy, i

nflam

ma-

tory

mar

kers

, vir

al lo

ads,

T ly

mph

ocyt

e co

unts

, and

neu

tral

izin

g an

tibod

y re

spon

se w

ere

docu

men

ted

in in

fect

ed m

ice

trea

ted

indi

vidu

ally

or i

n co

mbi

natio

n w

ith z

anam

vir,

cele

coxi

b, g

emfib

rozi

l, an

d m

esal

azin

e. T

o im

itate

the

real

-life

sce-

nari

o, tr

eatm

ent w

as in

itiat

ed a

t 48

h af

ter v

iral

cha

lleng

e. Th

ere

wer

e si

gnifi

cant

impr

ovem

ents

in su

rviv

al ra

te (P

= 0

.02)

, su

rviv

al ti

me

(P <

0.0

2), a

nd in

flam

mat

ory

mar

kers

(P <

0.0

1) in

the

grou

p tr

eate

d-w

ith a

trip

le c

ombi

natio

n of

zan

amiv

ir,

cele

coxi

b, a

nd m

esal

azin

e w

hen

com

pare

d w

ith z

anam

ivir

alo

ne. Z

anam

ivir

with

or w

ithou

t im

mun

omod

ulat

ors r

educ

ed

vira

l loa

d to

a si

mila

r ext

ent.

Insi

gnifi

cant

pro

long

atio

n of

surv

ival

was

obs

erve

d w

hen

indi

vidu

al a

gent

s wer

e us

ed

alon

e. S

igni

fican

tly h

ighe

r lev

els o

f CD

4+ and

CD

8+ T ly

mph

ocyt

es a

nd le

ss p

ulm

onar

y in

flam

mat

ion

wer

e al

so fo

und

in

the

grou

p re

ceiv

ing

trip

le th

erap

y. Z

anam

ivir

alo

ne re

duce

d vi

ral l

oad

but n

ot in

flam

mat

ion

and

mor

talit

y. Th

e su

rviv

al

bene

fits o

f add

ing

cele

coxi

b an

d m

esal

azin

e to

zan

amiv

ir c

ould

be

caus

ed b

y th

eir s

yner

gist

ic e

ffect

s in

redu

cing

cyt

okin

e dy

sfun

ctio

n an

d pr

even

ting

apop

tosi

s. C

ombi

natio

ns o

f a n

eura

min

idas

e in

hibi

tor w

ith th

ese

imm

unom

odul

ator

s sho

uld

be c

onsi

dere

d in

rand

omiz

ed c

ontr

olle

d tr

eatm

ent t

rial

s of p

atie

nts s

uffer

ing

from

H5N

1 in

fect

ion.

Zhe

ng e

t al.

2008

ose

ltam

ivir

wit

h co

rtic

oste

roid

sM

ethy

lpre

dnis

olon

eH

ydro

cort

ison

eLu

ng in

jury

acu

te

resp

irat

ory

dist

ress

sy

ndro

me

PURP

OSE

: Dur

ing

the

2009

H1N

1 in

fluen

za A

vir

us p

ande

mic

, a m

inor

ity o

f pat

ient

s dev

elop

ed ra

pidl

y pr

ogre

ssiv

e pn

eum

onia

lead

ing

to a

cute

lung

inju

ry (A

LI)-

acut

e re

spir

ator

y di

stre

ss sy

ndro

me

(ARD

S). A

rece

nt m

eta-

anal

ysis

pro

-vi

des s

uppo

rt fo

r pro

long

ed c

ortic

oste

roid

trea

tmen

t in

ALI

-ARD

S. W

e pr

ospe

ctiv

ely

eval

uate

d th

e re

spon

se to

ose

ltam

i-vi

r and

pro

long

ed c

ortic

oste

roid

trea

tmen

t in

patie

nts w

ith A

LI-A

RDS

and

susp

ecte

d H

1N1

influ

enza

. MET

HO

DS:

Fr

om Ju

ne 2

4 th

roug

h 12

July

200

9, 1

3 pa

tient

s with

susp

ecte

d H

1N1

pneu

mon

ia a

nd A

LI-A

RDS

wer

e ad

mitt

ed to

the

inte

nsiv

e ca

re u

nit (

ICU

) of a

tert

iary

car

e ho

spita

l. H

1N1

influ

enza

was

con

firm

ed w

ith re

al-t

ime

reve

rse

tran

scri

ptas

e-po

lym

eras

e ch

ain

reac

tion

assa

y in

eig

ht p

atie

nts.

Ose

ltam

ivir

and

cor

ticos

tero

id tr

eatm

ent w

ere

initi

ated

con

com

itant

ly

at IC

U a

dmis

sion

; tho

se w

ith se

vere

ARD

S re

ceiv

ed m

ethy

lpre

dnis

olon

e (1

mg/

kg/d

ay),

and

othe

rs re

ceiv

ed h

ydro

cor-

tison

e (3

00 m

g/da

y) fo

r a d

urat

ion

of 2

1 ±

6 da

ys. R

ESU

LTS:

Pat

ient

s with

and

with

out c

onfir

med

H1N

1 in

fluen

za h

ad

sim

ilar d

isea

se se

veri

ty a

t pre

sent

atio

n an

d a

com

para

ble

resp

onse

to tr

eatm

ent.

By d

ay 7

of t

reat

men

t, pa

tient

s exp

eri-

ence

d a

sign

ifica

nt im

prov

emen

t in

lung

inju

ry a

nd m

ultip

le o

rgan

dys

func

tion

scor

es (P

< 0

.001

). Tw

elve

pat

ient

s (92

%)

impr

oved

lung

func

tion,

wer

e ex

tuba

ted,

and

dis

char

ged

aliv

e fr

om th

e IC

U. H

ospi

tal l

engt

h of

stay

and

mor

talit

y w

ere

18.7

± 9

.6 d

ays a

nd 1

5%, r

espe

ctiv

ely.

Surv

ivor

s wer

e di

scha

rged

hom

e w

ithou

t oxy

gen

supp

lem

enta

tion.

CO

NC

LUSI

ON

S:

In A

RDS

patie

nts,

with

and

with

out c

onfir

med

H1N

1 in

fluen

za, p

rolo

nged

low

-to-

mod

erat

e do

se c

ortic

oste

roid

trea

t-m

ent w

as w

ell t

oler

ated

and

ass

ocia

ted

with

sign

ifica

nt im

prov

emen

t in

lung

inju

ry a

nd m

ultip

le o

rgan

dys

func

tion

scor

es a

nd a

low

hos

pita

l mor

talit

y. Th

ese

findi

ngs p

rovi

de th

e ra

tiona

le fo

r dev

elop

ing

a ra

ndom

ized

tria

l.

Qui

spe-

Laim

e et

al

. 201

0


177

Lani

nam

ivir

oct

anoa

te

wit

h ar

tific

ial

surf

acta

nt

Mou

se m

odel

Back

grou

nd: P

atie

nts

with

influ

enza

vir

us in

fect

ion

can

deve

lop

seve

re p

neum

onia

and

acu

te re

spir

ator

y di

stre

ss

synd

rom

e (A

RDS)

whi

ch h

ave

a hi

gh m

orta

lity.

Influ

enza

vir

us in

fect

ion

is tr

eate

d w

orld

wid

e m

ainl

y by

neu

ram

inid

ase

inhi

bito

rs (N

AIs

). H

owev

er, m

onot

hera

py w

ith N

AIs

is in

suff

icie

nt fo

r se

vere

pne

umon

ia s

econ

dary

to in

fluen

za v

irus

in

fect

ion.

We

prev

ious

ly d

emon

stra

ted

that

mic

e in

fect

ed w

ith a

leth

al d

ose

of in

fluen

za v

irus

dev

elop

diff

use

alve

olar

da

mag

e (D

AD

) with

alv

eola

r co

llaps

e si

mila

r to

that

see

n in

ARD

S in

hum

ans.

Add

ition

ally

, pul

mon

ary

surf

acta

nt p

ro-

tein

s w

ere

grad

ually

incr

ease

d in

mou

se s

erum

, sug

gest

ing

a de

crea

se in

pul

mon

ary

surf

acta

nt in

the

lung

. The

refo

re,

the

pres

ent s

tudy

exa

min

ed w

heth

er c

ombi

natio

n th

erap

y of

NA

I with

exo

geno

us a

rtifi

cial

sur

fact

ant a

ffect

s m

orta

l-ity

of i

nflu

enza

vir

us-i

nfec

ted

mic

e. M

etho

dolo

gy/P

rinc

ipal

Fin

ding

s: B

ALB

/c m

ice

wer

e in

ocul

ated

with

sev

eral

vir

al

dose

s of

influ

enza

A/P

uert

o Ri

co/8

/34

(PR8

) vir

us (H

1N1)

. The

mic

e w

ere

addi

tiona

lly a

dmin

iste

red

exog

enou

s ar

tifi-

cial

sur

fact

ant i

n th

e pr

esen

ce o

r ab

senc

e of

a n

ew

up to

20

days

aft

er in

ocul

atio

n. V

iral

tite

r an

d cy

toki

ne/c

hem

okin

e le

vels

in th

e lu

ngs,

lung

wei

ght,

path

olog

ical

ana

lysi

s, a

nd b

lood

O2 a

nd C

O2 p

ress

ures

wer

e ev

alua

ted.

Infe

cted

mic

e tr

eate

d w

ith c

ombi

natio

n th

erap

y of

lani

nam

ivir

oct

anoa

te w

ith a

rtifi

cial

sur

fact

ant s

how

ed a

sig

nific

antly

hig

her

surv

ival

rat

e co

mpa

red

with

thos

e th

at re

ceiv

ed la

nina

miv

ir o

ctan

oate

mon

othe

rapy

(P =

0.0

03).

How

ever

, vir

us ti

ter,

lung

wei

ght a

nd c

ytok

ine/

chem

okin

e re

spon

ses

wer

e no

t diff

eren

t bet

wee

n th

e gr

oups

. His

topa

thol

ogic

al e

xam

inat

ion,

a

hydr

osta

tic lu

ng te

st a

nd b

lood

gas

ana

lysi

s sh

owed

pos

itive

resu

lts in

the

com

bina

tion

ther

apy

grou

p. C

oncl

usio

ns/

Sign

ifica

nce:

Com

bina

tion

ther

apy

of la

nina

miv

ir o

ctan

oate

with

art

ifici

al s

urfa

ctan

t red

uces

leth

ality

in m

ice

infe

cted

w

ith in

fluen

za v

irus

, and

eve

ntua

lly s

uppr

esse

s D

AD

form

atio

n an

d pr

eser

ves

lung

func

tion.

Thi

s co

mbi

natio

n co

uld

be e

ffect

ive

for

prev

entio

n of

sev

ere

pneu

mon

ia s

econ

dary

to in

fluen

za v

irus

infe

ctio

n in

hum

ans,

whi

ch is

not

im

prov

ed b

y N

AI m

onot

hera

py.

Fuku

shi e

t al.

2012

ose

ltam

ivir

wit

h ke

toti

fen

Mas

t cel

l deg

ranu

latio

n in

hibi

tor

In v

itro/

in v

ivo

test

s

Alth

ough

an

impo

rtan

t rol

e fo

r m

ast c

ells

in s

ever

al v

iral

infe

ctio

ns h

as b

een

dem

onst

rate

d, it

s ro

le in

the

inva

sion

of

hig

hly

path

ogen

ic H

5N1

influ

enza

vir

us is

unk

now

n. In

the

pres

ent s

tudy

, we

dem

onst

rate

that

mas

t cel

ls w

ere

activ

ated

sig

nific

antly

by

H5N

1 vi

rus

(A/c

hick

en/H

enan

/1/2

004)

infe

ctio

n bo

th in

viv

o an

d in

vitr

o. M

ast c

ells

cou

ld

poss

ibly

inte

nsify

the

lung

inju

ry th

at re

sults

from

H5N

1 in

fect

ion

by re

leas

ing

proi

nfla

mm

ator

y m

edia

tors

, inc

lud-

ing

hist

amin

e, tr

ypta

se, a

nd g

amm

a in

terf

eron

(IFN

-gam

ma)

. Lun

g le

sion

s an

d ap

opto

sis

indu

ced

by H

5N1

infe

ctio

n w

ere

redu

ced

dram

atic

ally

by

trea

tmen

t with

ket

otife

n, w

hich

is a

mas

t cel

l deg

ranu

latio

n in

hibi

tor.

A c

ombi

natio

n of

ke

totif

en a

nd th

e ne

uram

inid

ase

inhi

bito

r os

elta

miv

ir p

rote

cted

100

% o

f the

mic

e fr

om d

eath

pos

tinfe

ctio

n. In

con

-cl

usio

n, o

ur d

ata

sugg

est t

hat m

ast c

ells

pla

y a

cruc

ial r

ole

in th

e ea

rly

stag

es o

f H5N

1 in

fluen

za v

irus

infe

ctio

n an

d pr

ovid

e a

new

app

roac

h to

com

bat h

ighl

y pa

thog

enic

influ

enza

vir

us in

fect

ion.

Hu

et a

l. 20

12

dua

l-ta

rget

ed

bifu

ncti

onal

ant

i-in

fluen

za d

rugs

Ant

i-infl

amm

ator

y eff

ect

Caff

eic

acid

-zan

amiv

ir

conj

ugat

eIn

viv

o/in

vitr

o te

sts

Influ

enza

ther

apy

with

a s

ingl

e ta

rget

ed c

ompo

und

is o

ften

lim

ited

in e

ffic

acy

due

to th

e ra

pidl

y de

velo

ped

drug

resi

st-

ance

. Mor

eove

r, th

e un

cont

rolle

d vi

rus-

indu

ced

cyto

kine

s co

uld

caus

e th

e hi

gh m

orta

lity

of h

uman

infe

cted

by

H5N

1 av

ian

influ

enza

vir

us. I

n th

is s

tudy

, we

expl

ored

the

nove

l dua

l-ta

rget

ed b

ifunc

tiona

l ant

i-in

fluen

za d

rugs

form

ed b

y co

njug

atio

n w

ith a

nti-

infla

mm

ator

y ag

ents

. In

part

icul

ar, t

he c

affe

ic a

cid

(CA

)-be

arin

g za

nam

ivir

(ZA

) con

juga

tes

ZA

-7-C

A (1

) and

ZA

-7-C

A-a

mid

e (7

) sho

wed

sim

ulta

neou

s in

hibi

tion

of in

fluen

za v

irus

neu

ram

inid

ase

and

supp

res-

sion

of p

ro-i

nfla

mm

ator

y cy

toki

nes.

The

se Z

A c

onju

gate

s pr

ovid

ed re

mar

kabl

e pr

otec

tion

of c

ells

and

mic

e ag

ains

t in

fluen

za in

fect

ions

. Int

rana

sal a

dmin

istr

atio

n of

low

dos

age

(<1.

2 m

u m

ol/k

g/da

y) o

f ZA

con

juga

tes

exhi

bite

d m

uch

grea

ter

effe

ct th

an th

e co

mbi

natio

n th

erap

y w

ith Z

A a

nd th

e an

ti-in

flam

mat

ory

agen

ts in

pro

tect

ion

of th

e le

thal

ly

infe

cted

mic

e by

H1N

1 or

H5N

1 in

fluen

za v

irus

es.

Liu

et a

l. 20

12


178

neu

ram

inid

ase

inhi

bito

rs w

ith

rim

anta

dine

Add

itive

and

syne

rgis

tic

effec

tsIn

vitr

o te

sts

The

re is

insu

ffici

ent i

nfor

mat

ion

abou

t com

bina

tion

ther

apy

with

app

rove

d an

ti-in

fluen

za a

gent

s. W

e te

sted

com

bina

-tio

ns th

at p

aire

d a

neur

amin

idas

e (N

A) i

nhib

itor (

zana

miv

ir, o

selta

miv

ir c

arbo

xyla

te, o

r per

amiv

ir) w

ith ri

man

tadi

ne

agai

nst i

nfec

tion

of M

DC

K c

ells

with

H1N

1 an

d H

3N2

subt

ypes

of i

nflue

nza

A v

irus

and

cha

ract

eriz

ed th

eir m

ode

of

inte

ract

ion.

Whe

n re

duct

ion

of e

xtra

cellu

lar v

irus

was

ana

lyze

d by

indi

vidu

al re

gres

sion

mod

els a

nd th

ree-

dim

ensi

onal

re

pres

enta

tions

of t

he d

ata,

all

thre

e co

mbi

natio

ns sh

owed

add

itive

and

syne

rgis

tic e

ffect

s with

no

cyto

toxi

city

. Max

imum

sy

nerg

y ag

ains

t A/N

ew C

aled

onia

/20/

99 (H

1N1)

vir

us in

fect

ion

was

obs

erve

d w

ith <

2.5

µM

rim

anta

dine

pai

red

with

low

co

ncen

trat

ions

of N

A in

hibi

tors

. All

com

bina

tions

redu

ced

the

extr

acel

lula

r yie

ld o

f A/P

anam

a/20

07/9

9 (H

3N2)

influ

enza

vi

rus s

yner

gist

ical

ly. H

owev

er, o

ur fi

ndin

gs w

ere

diffe

rent

for t

he c

ell-a

ssoc

iate

d vi

rus y

ield

. At s

ome

drug

con

cent

ratio

ns,

the

yiel

d of

cel

l-ass

ocia

ted

viru

s was

inhi

bite

d an

tago

nist

ical

ly. Th

eref

ore,

the

met

hod

of a

naly

sis c

an b

e a

cruc

ial f

acto

r in

eval

uatin

g th

e in

tera

ctio

ns o

f dru

gs w

ith d

iffer

ent m

echa

nism

s. W

e hy

poth

esiz

e th

at a

ssay

s bas

ed o

n ce

ll-as

soci

ated

vir

us

yiel

d m

ay u

nder

estim

ate

the

effica

cies

of d

rug

com

bina

tions

that

incl

ude

an N

A in

hibi

tor.

Take

n to

geth

er, o

ur re

sults

su

gges

t tha

t reg

imen

s tha

t com

bine

NA

inhi

bito

rs a

nd ri

man

tadi

ne e

xert

syne

rgis

tic a

nti-

influ

enza

effe

cts i

n vi

tro.

Thes

e fin

ding

s pro

vide

bas

elin

e in

form

atio

n fo

r the

rape

utic

test

ing

of th

e dr

ug c

ombi

natio

ns in

viv

o.

Gov

orko

va e

t al.

2004

Trip

le-c

ombi

nati

on

anti

vira

l dru

g th

erap

yA

man

tadi

neRi

bavi

rin

Ose

ltam

ivir

Adu

lt pa

tient

s

A re

cent

in v

itro

stud

y sh

owed

that

the

thre

e co

mpo

unds

of a

ntiv

iral

dru

gs w

ith d

iffer

ent m

echa

nism

s of a

ctio

n (a

man

ta-

dine

, rib

avir

in, a

nd o

selta

miv

ir) c

ould

resu

lt in

syne

rgis

tic a

ntiv

iral

act

ivity

aga

inst

influ

enza

vir

us. H

owev

er, n

o cl

inic

al

stud

ies h

ave

eval

uate

d th

e effi

cacy

and

safe

ty o

f com

bina

tion

antiv

iral

ther

apy

in p

atie

nts w

ith se

vere

influ

enza

illn

ess.

A

tota

l of 2

45 a

dult

patie

nts w

ho w

ere

criti

cally

ill w

ith c

onfir

med

pan

dem

ic in

fluen

za A

/H1N

1 20

09 (p

H1N

1) v

irus

infe

c-tio

n an

d w

ere

adm

itted

to o

ne o

f the

inte

nsiv

e ca

re u

nits

of 2

8 ho

spita

ls in

Kor

ea w

ere

revi

ewed

. Pat

ient

s who

requ

ired

ve

ntila

tor s

uppo

rt a

nd re

ceiv

ed e

ither

trip

le-c

ombi

natio

n an

tivir

al d

rug

(TC

AD

) the

rapy

or o

selta

miv

ir m

onot

hera

py

wer

e an

alyz

ed. A

tota

l of 1

27 p

atie

nts w

ere

incl

uded

in o

ur a

naly

sis.

Am

ong

them

, 24

patie

nts r

ecei

ved

TCA

D th

erap

y, an

d 10

3 pa

tient

s rec

eive

d os

elta

miv

ir m

onot

hera

py. Th

e 14

-day

mor

talit

y w

as 1

7% in

the

TCA

D g

roup

and

35%

in th

e os

elta

miv

ir g

roup

(P =

0.0

8), a

nd th

e 90

-day

mor

talit

y w

as 4

6% in

the

TCA

D g

roup

and

59%

in th

e os

elta

miv

ir g

roup

(P

= 0

.23)

. Non

e of

the

toxi

citie

s att

ribu

tabl

e to

ant

ivir

al d

rugs

occ

urre

d in

eith

er g

roup

of o

ur st

udy,

incl

udin

g he

mol

ytic

an

emia

and

hep

atic

toxi

citie

s rel

ated

to th

e us

e of

riba

viri

n. L

ogis

tic re

gres

sion

ana

lysi

s ind

icat

ed th

at th

e od

ds ra

tio fo

r th

e as

soci

atio

n of

TC

AD

with

90-

day

mor

talit

y w

as 0

.58

(95%

con

fiden

ce in

terv

al, 0

.24

to 1

.42;

P =

0.2

4). A

lthou

gh th

is

stud

y w

as re

tros

pect

ive

and

did

not p

rovi

de v

irol

ogic

out

com

es, o

ur re

sults

sugg

est t

hat t

he tr

eatm

ent o

utco

me

of th

e tr

iple

com

bina

tion

of a

man

tadi

ne, r

ibav

irin

, and

ose

ltam

ivir

was

com

para

ble

to th

at o

f ose

ltam

ivir

mon

othe

rapy

.

Kim

et a

l. 20

11

Trip

le-c

ombi

nati

on

anti

vira

l dru

g th

erap

yA

man

tadi

neRi

bavi

rin

Ose

ltam

ivir

Dos

e-de

pend

ent

prot

ectio

nSy

nerg

istic

effe

ctBr

oad-

spec

trum

act

ivity

Mou

se m

odel

The

lim

ited

effic

acy

of e

xist

ing

antiv

iral

ther

apie

s fo

r in

fluen

za –

cou

pled

with

wid

espr

ead

base

line

antiv

iral

resi

stan

ce

– hi

ghlig

hts

the

urge

nt n

eed

for

mor

e ef

fect

ive

ther

apy.

We

desc

ribe

a tr

iple

com

bina

tion

antiv

iral

dru

g (T

CA

D) r

egi-

men

com

pose

d of

am

anta

dine

, ose

ltam

ivir,

and

rib

avir

in th

at is

hig

hly

effic

acio

us a

t red

ucin

g m

orta

lity

and

wei

ght l

oss

in m

ouse

mod

els

of in

fluen

za in

fect

ion.

TC

AD

ther

apy

was

sup

erio

r to

dua

l and

sin

gle

drug

regi

men

s in

mic

e in

fect

ed

with

dru

g-su

scep

tible

, low

pat

hoge

nic

A/H

5N1

(A/D

uck/

MN

/152

5/81

) and

am

anta

dine

-res

ista

nt 2

009

A/H

1N1

influ

-en

za (A

/Cal

iforn

ia/0

4/09

). Tr

eatm

ent w

ith T

CA

D a

fford

ed >

90%

sur

viva

l in

mic

e in

fect

ed w

ith b

oth

viru

ses,

whe

reas

tr

eatm

ent w

ith d

ual a

nd s

ingl

e dr

ug re

gim

ens

resu

lted

in 0

% to

60%

sur

viva

l. Im

port

antly

, am

anta

dine

had

no

activ

ity

as m

onot

hera

py a

gain

st th

e am

anta

dine

-res

ista

nt v

irus

, but

dem

onst

rate

d do

se-d

epen

dent

pro

tect

ion

in c

ombi

natio

n w

ith o

selta

miv

ir a

nd r

ibav

irin

, ind

icat

ive

that

am

anta

dine

’s ac

tivity

had

bee

n re

stor

ed in

the

cont

ext o

f TC

AD

ther

apy.

Furt

herm

ore,

TC

AD

ther

apy

prov

ided

sur

viva

l ben

efit

whe

n tr

eatm

ent w

as d

elay

ed u

ntil

72 h

ours

pos

t-in

fect

ion,

w

here

as o

selta

miv

ir m

onot

hera

py w

as n

ot p

rote

ctiv

e af

ter

24 h

ours

pos

t-in

fect

ion.

The

se fi

ndin

gs d

emon

stra

te in

viv

o ef

ficac

y of

TC

AD

ther

apy

and

conf

irm

pre

viou

s re

port

s of

the

syne

rgy

and

broa

d sp

ectr

um a

ctiv

ity o

f TC

AD

ther

apy

agai

nst s

usce

ptib

le a

nd re

sist

ant i

nflu

enza

str

ains

in v

itro.

Ngu

yen

et a

l. 20

12


179

Com

bina

tion

of

favi

pira

vir

and

osel

tam

ivir

Mou

se m

odel

Favi

pira

vir

(T-7

05 [6

-flu

oro-

3-hy

drox

y-2-

pyra

zine

carb

oxam

ide]

) and

ose

ltam

ivir

wer

e co

mbi

ned

to tr

eat i

nflu

enza

vi

rus

A/N

WS/

33 (H

1N1)

, A/V

icto

ria/

3/75

(H3N

2), a

nd A

/Duc

k/M

N/1

525/

81 (H

5N1)

infe

ctio

ns. T

-705

alo

ne in

hibi

ted

viru

ses

in c

ell c

ultu

re a

t 1.4

to 4

.3 µ

M. O

selta

miv

ir in

hibi

ted

thes

e th

ree

viru

ses

in c

ells

at 3

.7, 0

.02,

and

0.1

6 µM

and

in

neu

ram

inid

ase

assa

ys a

t 0.9

4, 0

.46,

and

2.3

1 nM

, res

pect

ivel

y. O

ral t

reat

men

ts w

ere

give

n tw

ice

daily

to m

ice

for

5 to

7

days

sta

rtin

g, g

ener

ally

, 24

h af

ter

infe

ctio

n. S

urvi

val r

esul

ting

from

5 d

ays

of o

selta

miv

ir tr

eatm

ent (

0.1

and

0.3

mg/

kg/d

ay) w

as s

igni

fican

tly b

ette

r in

com

bina

tion

with

20

mg/

kg o

f bod

y w

eigh

t/da

y of

T-7

05 a

gain

st th

e H

1N1

infe

ctio

n.

Trea

tmen

t of t

he H

3N2

infe

ctio

n re

quir

ed 5

0 m

g/kg

/day

of o

selta

miv

ir fo

r 7

days

to a

chie

ve 6

0% p

rote

ctio

n; 2

5 m

g/kg

/da

y w

as in

effe

ctiv

e. T

-705

was

≥ 7

0% p

rote

ctiv

e at

50

to 1

00 m

g/kg

/day

but

inac

tive

at 2

5 m

g/kg

/day

. The

com

bina

tion

of in

hibi

tors

(25

mg/

kg/d

ay e

ach)

incr

ease

d su

rviv

al to

90%

. The

H5N

1 in

fect

ion

was

not

ben

efite

d by

trea

tmen

t with

os

elta

miv

ir (≤

100

mg/

kg/d

ay fo

r 7

days

). T-

705

was

30

to 7

0% p

rote

ctiv

e at

25

to 1

00 m

g/kg

/day

. Sur

viva

l im

prov

ed

slig

htly

with

com

bina

tion

trea

tmen

ts. I

ncre

ased

act

ivity

was

see

n ag

ains

t H5N

1 in

fect

ion

by s

tart

ing

trea

tmen

ts 2

h

befo

re in

fect

ion.

Ose

ltam

ivir

was

inef

fect

ive

at ≤

40

mg/

kg/d

ay. T

-705

was

100

% p

rote

ctiv

e at

40

and

80 m

g/kg

/day

and

in

activ

e at

20

mg/

kg/d

ay. C

ombi

ning

inef

fect

ive

dose

s (2

0 m

g/kg

/day

of T

-705

and

10

to 4

0 m

g/kg

/day

of o

selta

miv

ir)

affo

rded

60

to 8

0% p

rote

ctio

n an

d im

prov

ed b

ody

wei

ghts

dur

ing

infe

ctio

n. T

hus,

syn

ergi

stic

resp

onse

s w

ere

achi

eved

w

ith lo

w d

oses

of T

-705

com

bine

d w

ith o

selta

miv

ir. T

hese

com

poun

ds m

ay b

e vi

able

can

dida

tes

for

com

bina

tion

trea

t-m

ent o

f hum

an in

fluen

za in

fect

ions

.

Smee

et a

l. 20

10

ose

ltam

ivir

wit

h za

nam

ivir

Tran

smis

sion

in

hous

ehol

ds

Back

grou

nd: T

he e

ffect

iven

ess

of n

eura

min

idas

e in

hibi

tors

to re

duce

tran

smis

sion

whe

n us

ed a

s tr

eatm

ent i

n in

flu-

enza

-inf

ecte

d pa

tient

s re

mai

ns d

ebat

ed. M

etho

ds: I

n a

pres

peci

fied

anal

ysis

of a

blin

ded

rand

omiz

ed c

ontr

olle

d tr

ial

on th

e ef

ficac

y of

ose

ltam

ivir

-zan

amiv

ir c

ombi

natio

n th

erap

y ve

rsus

ose

ltam

ivir

and

zan

amiv

ir m

onot

hera

py c

on-

duct

ed d

urin

g th

e 20

08–2

009

seas

onal

influ

enza

epi

dem

ic, w

e co

mpa

red

the

rate

of s

econ

dary

illn

ess

in h

ouse

hold

co

ntac

ts o

f inf

luen

za-p

ositi

ve in

dex

patie

nts

betw

een

arm

s. S

econ

dary

illn

ess

was

def

ined

as

occu

rren

ce in

con

tact

s of

feve

r pl

us c

ough

with

in 7

day

s fr

om r

ando

miz

atio

n of

inde

x pa

tient

s. A

naly

ses

wer

e co

nduc

ted

acco

rdin

g to

the

dela

y be

twee

n pa

tient

s’ on

set o

f sym

ptom

s an

d in

terv

entio

n. R

esul

ts: A

tota

l of 5

43 h

ouse

hold

con

tact

s of

267

inde

x pa

tient

s w

ere

incl

uded

, of w

hich

466

had

follo

w-u

p as

sess

men

t. A

sec

onda

ry il

lnes

s w

as re

port

ed in

58

(12.

5%) c

on-

tact

s w

ith n

o si

gnifi

cant

diff

eren

ce b

etw

een

arm

s ov

eral

l (P

= 0.

07).

Whe

n th

e an

alys

is w

as li

mite

d to

the

232

cont

acts

of

136

inde

x pa

tient

s w

ith fi

rst t

reat

men

t int

ake

with

in 2

4 h

of o

nset

of s

ympt

oms,

a lo

wer

rat

e of

sec

onda

ry il

lnes

s w

as re

port

ed in

the

com

bina

tion

ther

apy

arm

(2 o

f 56

[4%

]) th

an in

the

osel

tam

ivir

arm

(14

of 8

1 [1

7%];

P =

0.01

4) a

nd

the

zana

miv

ir a

rm (1

4 of

95

[15%

]; P

= 0.

031)

. Mul

tivar

iate

ana

lysi

s ac

coun

ting

for

intr

a-ho

useh

old

corr

elat

ion

con-

firm

ed th

ese

findi

ngs.

Con

clus

ions

: Our

ana

lysi

s su

gges

ts a

gre

ater

effe

ctiv

enes

s of

the

com

bina

tion

ther

apy

to re

duce

tr

ansm

issi

bilit

y w

hen

give

n to

the

inde

x pa

tient

with

in 2

4 h

of o

nset

of s

ympt

oms.

As

the

findi

ng w

as o

btai

ned

from

a

subg

roup

ana

lysi

s, it

sho

uld

be in

terp

rete

d w

ith c

autio

n.

Car

rat e

t al.

2012


180

Tabl

e 6.

Infl

uenz

a dr

ug r

esis

tanc

e

Mec

hani

sm o

f dru

g re

sist

ance

Neu

ram

inid

ase

mut

ants

Tam

iflu

Rele

nza

New

mut

ants

of h

uman

influ

enza

vir

us (A

/H1N

1) e

xhib

it re

sist

ance

to a

ntiv

iral

dru

gs. T

he m

echa

nism

whe

reby

they

de

velo

p in

sens

itivi

ty to

thes

e m

edic

atio

ns is

, how

ever

, not

yet

com

plet

ely

unde

rsto

od. A

cry

stal

logr

aphi

c st

ruct

ure

of

A/H

1N1

neur

amin

idas

e ha

s be

en p

ublis

hed

rece

ntly

. Usi

ng m

olec

ular

dyn

amic

sim

ulat

ions

, it i

s no

w p

ossi

ble

to c

har-

acte

rize

at t

he a

tom

ic le

vel t

he m

echa

nism

that

und

erlie

s th

e lo

ss o

f bin

ding

aff

inity

of t

he d

rugs

. In

this

stu

dy, f

ree-

ener

gy p

ertu

rbat

ion

was

use

d to

eva

luat

e th

e re

lativ

e bi

ndin

g fr

ee e

nerg

ies

of T

amifl

u an

d Re

lenz

a w

ith H

274Y

, N29

4S,

and

Y252

H n

eura

min

idas

e m

utan

ts. O

ur re

sults

dem

onst

rate

a re

mar

kabl

e co

rrel

atio

n be

twee

n th

eore

tical

and

exp

eri-

men

tal d

ata,

whi

ch q

uant

itativ

ely

conf

irm

s th

at th

e m

utan

ts a

re re

sist

ant t

o Ta

mifl

u bu

t are

stil

l str

ongl

y in

hibi

ted

by

Rele

nza.

The

sim

ulat

ions

furt

her

reve

al th

e ke

y in

tera

ctio

ns th

at g

over

n th

e af

finity

of t

he tw

o dr

ugs

for

each

mut

ant.

Thi

s in

form

atio

n is

env

isio

ned

to p

rove

use

ful f

or th

e de

sign

of n

ovel

neu

ram

inid

ase

inhi

bito

rs a

nd fo

r th

e ch

arac

teri

-za

tion

of n

ew p

oten

tial m

utan

ts.

Verg

ara-

Jaqu

e et

al

. 201

2

Res

ista

nce

to z

anam

ivir

an

d os

elta

miv

irM

olec

ular

mar

kers

of

resi

stan

cePy

rose

quen

cing

We

repo

rt h

ere

the

desi

gn o

f a p

yros

eque

ncin

g ap

proa

ch fo

r th

e de

tect

ion

of m

olec

ular

mar

kers

of r

esis

tanc

e to

the

neur

amin

idas

e in

hibi

tors

zan

amiv

ir a

nd o

selta

miv

ir in

influ

enza

vir

uses

of t

ype

B. P

rim

ers

wer

e de

sign

ed to

ana

lyze

the

sequ

ence

s at

eig

ht a

min

o ac

id p

ositi

ons

E119

, R15

2, D

198.

122

2, S

250,

H27

4, R

371,

and

G40

2 (u

nive

rsal

A/N

2 nu

mbe

r-in

g) in

the

neur

amin

idas

e (N

A) w

hich

hav

e be

en p

revi

ousl

y fo

und

to b

e as

soci

ated

with

resi

stan

ce o

r re

duce

d su

scep

ti-bi

lity

to o

selta

miv

ir a

nd/o

r za

nam

ivir

in th

e N

A in

hibi

tion

assa

y. In

add

ition

, the

des

igne

d pr

imer

s co

uld

be u

tiliz

ed to

th

e di

stin

guis

h be

twee

n th

e N

As

of in

fluen

za B

vir

uses

from

the

two

maj

or li

neag

es (V

icto

ria

and

Yam

agat

a) th

at h

ave

co-c

ircu

late

d gl

obal

ly in

rece

nt y

ears

, thu

s pr

ovid

ing

a va

luab

le to

ol fo

r vi

rus

stra

in s

urve

illan

ce.

Sheu

et a

l. 20

10

Res

ista

nce

to

osel

tam

ivir

Five

yea

rs o

f non

-pr

escr

iptio

n os

elta

miv

irN

ew Z

eala

nd

In 2

007

New

Zea

land

(NZ

) bec

ame

the

first

cou

ntry

to m

ake

osel

tam

ivir

(Tam

iflu)

ava

ilabl

e of

f-pr

escr

iptio

n. T

his

stud

y in

vest

igat

ed th

e ex

tent

of p

harm

acis

t sup

ply

of o

selta

miv

ir o

ver

5 ye

ars,

incl

udin

g du

ring

the

influ

enza

A(H

1N1)

pan

-de

mic

, and

the

impa

ct o

f pha

rmac

ist s

uppl

y of

ose

ltam

ivir

on

influ

enza

vir

us o

selta

miv

ir s

usce

ptib

ility

, per

sona

l sto

ck-

pilin

g an

d in

fluen

za v

acci

ne u

ptak

e. R

ando

mly

sel

ecte

d co

mm

unity

pha

rmac

ies

in N

Z re

port

ed o

selta

miv

ir p

rovi

sion

by

pres

crip

tion

and

thro

ugh

phar

mac

ist s

uppl

y fr

om 1

Janu

ary

2007

to 1

5 Se

ptem

ber

2011

. Ose

ltam

ivir

resi

stan

ce d

ata

on

influ

enza

vir

uses

isol

ated

dur

ing

influ

enza

sur

veill

ance

from

200

8 to

201

1 w

ere

obta

ined

, alo

ng w

ith in

fluen

za v

acci

ne

upta

ke d

ata

from

200

5 to

201

1 an

d in

fluen

za d

etec

tion

data

. Sev

enty

of 8

5 el

igib

le p

harm

acie

s co

mpl

eted

the

stud

y (8

2 re

spon

se r

ate)

. Mos

t sup

plie

s of

ose

ltam

ivir

thro

ugho

ut th

e 5

year

s w

ere

disp

ense

d ag

ains

t a p

resc

ript

ion

rath

er th

an

phar

mac

ist s

uppl

ied,

with

pha

rmac

ist s

uppl

y re

spon

sibl

e fo

r 11

of s

uppl

ies

duri

ng th

e pa

ndem

ic y

ears

(200

9–20

10)

vers

us 2

7 an

d 31

dur

ing

2007

and

200

8, re

spec

tivel

y. P

harm

acis

t-su

pplie

d os

elta

miv

ir d

id n

ot a

ppea

r to

be

asso

ciat

ed

with

the

deve

lopm

ent o

f res

ista

nce,

with

iden

tifie

d lik

ely

stoc

kpili

ng o

r w

ith a

dec

line

in in

fluen

za im

mun

izat

ion.

Pha

r-m

acis

t sup

plie

s la

rgel

y m

atch

ed th

e tim

ing

of in

fluen

za in

the

com

mun

ity a

nd p

eake

d in

June

200

9, a

s di

d pr

escr

iptio

n su

pplie

s. F

ive

year

s of

non

-pre

scri

ptio

n os

elta

miv

ir in

NZ

has

resu

lted

in n

o si

gnifi

cant

cha

nge

in th

e de

velo

pmen

t of

resi

stan

ce o

r ra

tes

of in

fluen

za im

mun

izat

ion.

Sup

plie

s re

mai

ned

mod

est a

nd s

igni

fican

t con

sum

er s

tock

pilin

g th

roug

h ph

arm

acis

t sup

ply

has

not o

ccur

red,

eve

n du

ring

the

influ

enza

A(H

1N1)

pdm

09 p

ande

mic

in 2

009

and

2010

. Pha

rma-

cist

s co

uld

be b

ette

r ut

ilize

d in

ens

urin

g fa

st d

istr

ibut

ion

of a

ntiv

iral

s to

influ

enza

suf

fere

rs d

urin

g a

pand

emic

Gau

ld e

t al.

2012


181

Res

ista

nce

to

aman

tadi

ne

Am

anta

dine

trea

ted

child

ren

Sequ

enci

ng

Clin

ical

sam

ples

from

15

aman

tadi

ne-t

reat

ed c

hild

ren

wer

e co

llect

ed s

eria

lly-b

efor

e, d

urin

g, a

nd/o

r af

ter

trea

tmen

t and

w

ere

stud

ied

to d

eter

min

e th

e ac

tual

pre

vale

nce,

tim

ing,

and

clin

ical

impl

icat

ions

of M

2 m

utat

iona

l eve

nts.

Aft

er v

iral

RN

A e

xtra

ctio

n an

d re

vers

e-tr

ansc

ript

ase

poly

mer

ase

chai

n re

actio

n am

plifi

catio

n of

the

vira

l RN

A e

ncod

ing

the

M2

prot

ein,

the

prod

ucts

wer

e cl

oned

into

pla

smid

s, a

nd th

eir

sequ

ence

s w

ere

dete

rmin

ed. F

ive

mut

atio

ns k

now

n to

con

fer

aman

tadi

ne re

sist

ance

in c

linic

al s

ampl

es w

ere

iden

tifie

d in

12

(80%

) of 1

5 ev

alua

ble

patie

nts,

and

9 p

atie

nts

had

> 1

(2–4

) mut

ant v

irus

. The

pat

tern

of e

mer

genc

e of

mut

ant s

trai

ns w

as c

lari

fied

from

the

stud

y of

6 p

atie

nts

with

at l

east

4

seri

al s

ampl

es. A

lthou

gh v

irus

es w

ith M

2 m

utat

ions

tend

ed to

bec

ome

the

dom

inan

t pop

ulat

ions

, in

2 ca

ses,

wild

-ty

pe v

irus

es b

ecam

e do

min

ant a

fter

dec

reas

ing

to lo

w le

vels

. The

se re

sults

sug

gest

that

resi

stan

t vir

uses

em

erge

in a

m

uch

high

er p

ropo

rtio

n of

am

anta

dine

-tre

ated

pat

ient

s th

an h

as b

een

sugg

este

d by

pre

viou

s st

udie

s.

Shir

aish

i et a

l. 20

03

Res

ista

nce

to

aman

tadi

ne

Am

ino

acid

subs

titut

ions

In tw

o in

fluen

za s

easo

ns d

urin

g w

hich

H1N

1 an

d H

3N2

coci

rcul

ated

, res

ista

nce

was

mor

e fr

eque

nt in

H3N

2 st

rain

s th

an in

H1N

1 st

rain

s af

ter

aman

tadi

ne tr

eatm

ent.

Pred

omin

ant a

min

o ac

id s

ubst

itutio

ns in

M2

prot

ein

occu

rred

at

posi

tion

31 (s

erin

e to

asp

arag

ine)

in H

3N2

stra

ins

and

at p

ositi

on 2

7 (v

alin

e to

ala

nine

) in

H1N

1 st

rain

s.

Saito

et a

l. 20

03

Res

ista

nce

to

adam

anta

nes

281

influ

enza

isol

ates

Aus

tral

iaEu

rope

Asi

a

The

ada

man

tane

s (a

man

tadi

ne a

nd r

iman

tadi

ne) w

ere

the

initi

al a

ntiv

iral

s lic

ense

d fo

r us

e ag

ains

t inf

luen

za A

vir

uses

an

d ha

ve b

een

used

in s

ome

coun

trie

s to

con

trol

sea

sona

l inf

luen

za a

nd h

ave

also

bee

n st

ockp

iled

for

pote

ntia

l pan

-de

mic

use

. Whi

le h

igh

rate

s of

resi

stan

ce h

ave

been

obs

erve

d in

rece

nt y

ears

with

A(H

3) v

irus

es, t

he r

ates

of r

esis

t-an

ce w

ith A

(H1)

vir

uses

has

var

ied

wid

ely.

In th

is s

tudy

we

anal

ysed

281

hum

an in

fluen

za A

vir

uses

isol

ated

in 2

007

that

wer

e re

ferr

ed to

the

WH

O C

olla

bora

ting

Cen

tre

for

Refe

renc

e an

d Re

sear

ch in

Mel

bour

ne, m

ainl

y fr

om A

us-

tral

ia a

nd th

e su

rrou

ndin

g re

gion

s, fo

r ev

iden

ce o

f res

ista

nce

to a

dam

anta

nes

and

a su

bset

of t

hese

was

exa

min

ed fo

r re

sist

ance

to th

e ne

uram

inid

ase

inhi

bito

rs (N

Is).

We

foun

d th

at th

e ra

tes

of a

cdam

anta

ne re

sist

ance

in A

(H3)

vir

uses

co

ntin

ued

to in

crea

se in

mos

t cou

ntri

es in

200

7 bu

t a d

istin

ct v

aria

tion

was

see

n w

ith A

(H1)

resi

stan

ce le

vels

. A(H

1)

viru

ses

from

Aus

tral

ia, N

ew Z

eala

nd a

nd E

urop

e ha

d lo

w r

ates

of r

esis

tanc

e (2

–9%

) whe

reas

vir

uses

from

a n

umbe

r of

Sou

th E

ast (

SE) A

sian

cou

ntri

es h

ad h

igh

rate

s of

resi

stan

ce (3

3–10

0%).

Thi

s di

ffere

nce

can

be a

ttri

bute

d to

the

spre

ad o

f A/B

risb

ane/

59/2

007-

like

viru

ses

to m

any

part

s of

the

wor

ld w

ith th

e ex

cept

ion

of S

E A

sia

whe

re A

/Hon

g K

ong/

2652

/200

6-lik

e vi

ruse

s co

ntin

ue to

pre

dom

inat

e. W

hen

thes

e tw

o A

(H1)

sub

grou

ps w

ere

com

pare

d fo

r th

eir

in

vitr

o se

nsiti

vity

to th

e ot

her

clas

s of

influ

enza

ant

ivir

al d

rugs

, the

neu

ram

inid

ase

inhi

bito

rs, n

o di

ffere

nce

was

see

n be

twee

n th

e gr

oups

with

bot

h sh

owin

g no

rmal

leve

ls o

f sen

sitiv

ity to

thes

e dr

ugs,

The

find

ing

of re

duci

ng A

(H1)

resi

st-

ance

rat

es in

Aus

tral

ia a

nd r

isin

g le

vels

in S

E A

sia

in 2

007,

reve

rses

the

tren

d se

en in

200

6 w

hen

A(H

1) re

sist

ance

leve

ls

wer

e ri

sing

in A

ustr

alia

and

els

ewhe

re b

ut re

mai

ned

low

in m

ost o

f SE

Asi

a.

Barr

et a

l. 20

08

Res

ista

nce

to

adam

anta

nes

and

neur

amin

idas

e in

hibi

tors

Cen

tral

/Sou

th A

mer

ica

Back

grou

nd R

ecen

t inf

luen

za a

ntiv

iral

resi

stan

ce s

tudi

es re

veal

an

alar

min

g in

crea

se in

bot

h ad

aman

tane

s an

d ne

u-ra

min

idas

e in

hibi

tors

(NA

Is) r

esis

tant

vir

al s

trai

ns w

orld

wid

e, p

artic

ular

ly in

Asi

a, E

urop

e an

d th

e U

nite

d St

ates

. O

bjec

tives

In th

is s

tudy

, we

have

eva

luat

ed in

fluen

za v

irus

resi

stan

ce in

Cen

tral

and

Sou

th A

mer

ica.

Met

hods

Influ

enza

vi

ruse

s, is

olat

ed fr

om s

ympt

omat

ic p

atie

nts

thro

ugho

ut C

entr

al a

nd S

outh

Am

eric

a in

200

5–20

08 w

ere

anal

yzed

for

inhi

bito

r re

sist

ance

. The

M2

and

NA

gen

es o

f inf

luen

za v

irus

es w

ere

sequ

ence

d an

d re

sist

ance

was

infe

rred

by

com

-pa

riso

n w

ith p

ublis

hed

sequ

ence

s an

d kn

own

resi

stan

t mut

atio

ns. R

esul

ts O

ur re

sults

indi

cate

that

: (i)

resi

stan

ce to

ad

aman

tane

s w

as s

een

in th

e m

ajor

ity (9

5.5%

) of t

he in

fluen

za A

/H3N

2 is

olat

es b

ut o

nly

in o

ne is

olat

e of

the

influ

enza

A

/H1N

1 vi

ruse

s; (i

i) re

sist

ance

to N

AIs

beg

an to

be

dete

cted

in A

/H1N

1 is

olat

es fr

om C

entr

al A

mer

ica

in 2

008;

and

(ii

i) no

ne o

f the

influ

enza

B v

irus

es a

naly

zed

wer

e re

sist

ant t

o N

AIs

. Con

clus

ions

The

se fi

ndin

gs s

ugge

st a

lim

ited

effe

c-tiv

enes

s of

influ

enza

inhi

bito

rs d

ue to

the

dete

ctio

n of

resi

stan

ce a

mon

g A

/H1

and

A/H

3 vi

ruse

s.

Gar

cia

et a

l. 20

09


182

Res

ista

nce

to

adam

anta

nes

Pyro

sequ

enci

ng

Back

grou

nd a

dam

anta

nes

have

bee

n us

ed to

trea

t inf

luen

za A

vir

us in

fect

ions

for

man

y ye

ars.

Stu

dies

hav

e sh

own

a lo

w

inci

denc

e of

resi

stan

ce to

thes

e dr

ugs

amon

g ci

rcul

atin

g in

fluen

za v

irus

es; h

owev

er, t

heir

use

is r

isin

g w

orld

wid

e an

d dr

ug re

sist

ance

has

bee

n re

port

ed a

mon

g in

fluen

za A

(H5N

1) v

irus

es is

olat

ed fr

om p

oultr

y an

d hu

man

bei

ngs

in A

sia.

W

e so

ught

to a

sses

s ad

aman

tane

resi

stan

ce a

mon

g in

fluen

za A

vir

uses

isol

ated

dur

ing

the

past

dec

ade

from

cou

ntri

es

part

icip

atin

g in

WH

O’s

glob

al in

fluen

za s

urve

illan

ce n

etw

ork.

Met

hods

We

anal

ysed

dat

a fo

r in

fluen

za fi

eld

isol

ates

th

at w

ere

obta

ined

wor

ldw

ide

and

subm

itted

to th

e W

HO

Col

labo

ratin

g C

ente

r fo

r In

fluen

za a

t the

US

Cen

ters

for

Dis

-ea

se C

ontr

ol a

nd P

reve

ntio

n be

twee

n O

ct 1

, 199

4, a

nd M

ar 3

1, 2

005.

We

used

pyr

oseq

uenc

ing,

con

firm

ator

y se

quen

ce

anal

ysis

, and

phe

noty

pic

test

ing

to d

etec

t dru

g re

sist

ance

am

ong

circ

ulat

ing

influ

enza

A H

3N2

(n =

652

4), H

1N1

(n =

58

9), a

nd H

1N2

(n =

83)

vir

uses

. Fin

ding

s M

ore

than

700

0 in

fluen

za A

fiel

d is

olat

es w

ere

scre

ened

for

spec

ific

ami-

noac

id s

ubst

itutio

ns in

the

M2

gene

kno

wn

to c

onfe

r dr

ug re

sist

ance

. Dur

ing

the

deca

de o

f sur

veill

ance

a s

igni

fican

t in

crea

se in

dru

g re

sist

ance

was

not

ed, f

rom

0.4

% in

199

4–19

95 to

12.

3% in

200

3–20

04. T

his

incr

ease

in th

e pr

opor

tion

of re

sist

ant v

irus

es w

as w

eigh

ted

heav

ily b

y th

ose

obta

ined

from

Asi

a w

ith 6

1% o

f res

ista

nt v

irus

es is

olat

ed s

ince

200

3 be

ing

from

peo

ple

in A

sia.

Inte

rpre

tatio

n O

ur d

ata

rais

e co

ncer

ns a

bout

the

appr

opri

ate

use

of a

dam

anta

nes

and

draw

at

tent

ion

to th

e im

port

ance

of t

rack

ing

the

emer

genc

e an

d sp

read

of d

rug-

resi

stan

t inf

luen

za A

vir

uses

.

Brig

ht e

t al.

2005

Vira

l fitn

ess

and

tran

smis

sibi

lity

Neu

ram

inid

ase

mut

atio

nsO

selta

miv

ir c

arbo

xyla

te

Lim

ited

antiv

iral

com

poun

ds a

re a

vaila

ble

for

the

cont

rol o

f inf

luen

za, a

nd th

e em

erge

nce

of re

sist

ant v

aria

nts

wou

ld

furt

her

narr

ow th

e op

tions

for

defe

nse.

The

H27

5Y n

eura

min

idas

e (N

A) m

utat

ion,

whi

ch c

onfe

rs re

sist

ance

to

osel

tam

ivir

car

boxy

late

, has

bee

n id

entif

ied

amon

g th

e se

ason

al H

1N1

and

2009

pan

dem

ic in

fluen

za v

irus

es; h

owev

er,

thos

e H

275Y

resi

stan

t var

iant

s de

mon

stra

ted

dist

inct

epi

dem

iolo

gica

l out

com

es in

hum

ans.

Spe

cific

ally

, dom

inan

ce

of th

e H

275Y

var

iant

ove

r th

e os

elta

miv

ir-s

ensi

tive

viru

ses

was

onl

y re

port

ed fo

r a

seas

onal

H1N

1 va

rian

t dur

ing

2008

–200

9. H

ere,

we

syst

emat

ical

ly a

naly

ze th

e ef

fect

of t

he H

275Y

NA

mut

atio

n on

vir

al fi

tnes

s an

d tr

ansm

issi

bil-

ity o

f A(H

1N1)

pdm

09 a

nd s

easo

nal H

1N1

influ

enza

vir

uses

. The

NA

gen

es fr

om A

(H1N

1)pd

m09

A/C

alifo

rnia

/04/

09

(CA

04),

seas

onal

H1N

1 A

/New

Cal

edon

ia/2

0/19

99 (N

ewC

al),

and

A/B

risb

ane/

59/2

007

(Bri

sban

e) w

ere

indi

vidu

-al

ly in

trod

uced

into

the

gene

tic b

ackg

roun

d of

CA

04. T

he H

275Y

mut

atio

n le

d to

redu

ced

NA

enz

yme

activ

ity, a

n in

crea

sed

Km

for

3’-s

ialy

lact

ose

or 6

’-sia

lyla

ctos

e, a

nd d

ecre

ased

infe

ctiv

ity in

muc

in-s

ecre

ting

hum

an a

irw

ay e

pith

elia

l ce

lls c

ompa

red

to th

e os

elta

miv

ir-s

ensi

tive

wild

-typ

e co

unte

rpar

ts. A

tten

uate

d pa

thog

enic

ity in

bot

h RG

-CA

04(N

A-

H27

5Y) a

nd R

G-C

A04

x B

risb

ane(

NA

-H27

5Y) v

irus

es w

as o

bser

ved

in fe

rret

s co

mpa

red

to R

G-C

A04

vir

us, a

lthou

gh

the

tran

smis

sibi

lity

was

min

imal

ly a

ffect

ed. I

n pa

ralle

l exp

erim

ents

usi

ng re

com

bina

nt B

risb

ane

viru

ses

diffe

ring

by

hem

aggl

utin

in a

nd N

A, c

ompa

rabl

e di

rect

con

tact

and

resp

irat

ory

drop

let t

rans

mis

sibi

litie

s w

ere

obse

rved

am

ong

RG-

New

Cal

(HA

,NA

), RG

-New

Cal

(HA

,NA

-H27

5Y),

RG-B

risb

ane(

HA

,NA

-H27

5Y),

and

RG-N

ewC

al(H

A) ×

Bri

sban

e(N

A-

H27

5Y) v

irus

es. O

ur re

sults

dem

onst

rate

that

, des

pite

the

H27

5Y m

utat

ion

lead

ing

to a

min

or re

duct

ion

in v

iral

fitn

ess,

th

e tr

ansm

issi

on p

oten

tials

of t

hree

diff

eren

t ant

igen

ic s

trai

ns c

arry

ing

this

mut

atio

n w

ere

com

para

ble

in th

e na

ive

ferr

et m

odel

.

Won

g et

al.

2012

Rim

anta

dine

-res

ista

nce

mut

atio

nsG

row

th c

hara

cter

istic

s an

d vi

rule

nce

The

influ

ence

of r

iman

tadi

ne-r

esis

tanc

e m

utat

ions

on

the

viru

lenc

e of

hum

an H

3N2

viru

ses

in fe

rret

s w

as e

xam

ined

. T

he s

imila

ritie

s in

vir

ulen

ce o

f the

dru

g-re

sist

ant m

utan

ts w

ith s

ingl

e am

ino

acid

sub

stitu

tions

at t

hree

diff

eren

t loc

a-tio

ns, 2

7, 3

0, a

nd 3

1, w

ithin

the

M2

sequ

ence

and

thei

r co

rres

pond

ing

sens

itive

wild

-typ

e is

olat

es c

ontr

aste

d w

ith d

if-fe

renc

es in

vir

ulen

ce b

etw

een

the

thre

e pa

irs

of v

irus

es. T

hese

dat

a pr

ovid

e fu

rthe

r ev

iden

ce th

at r

iman

tadi

ne-r

esis

tant

vi

ruse

s th

at e

mer

ge d

urin

g tr

eatm

ent o

f pat

ient

s w

ith th

e dr

ug a

re u

nalte

red

both

in th

eir

grow

th c

hara

cter

istic

s an

d vi

rule

nce.

Swee

t et a

l. 19

91


183

Res

ista

nce

to

neur

amin

idas

e in

hibi

tors

Con

serv

ed re

sidu

esVi

ral fi

tnes

sN

eura

min

idas

e ac

tivity

Neu

ram

inid

ase

inhi

bito

rs (N

AIs

) are

ant

ivir

als d

esig

ned

to ta

rget

con

serv

ed re

sidu

es a

t the

neu

ram

inid

ase

(NA

) enz

yme

activ

e si

te in

influ

enza

A a

nd B

vir

uses

. The

cons

erve

d re

sidu

es th

at in

tera

ct w

ith N

AIs

are

und

er se

lect

ive

pres

sure

, but

on

ly a

few

hav

e be

en li

nked

to re

sist

ance

. In

the

A/W

uhan

/359

/95

(H3N

2) re

com

bina

nt v

irus

bac

kgro

und,

we

char

acte

r-iz

ed se

ven

char

ged,

con

serv

ed N

A re

sidu

es (1

1118

, R37

1, E

227,

R15

2, R

224,

E27

6, a

nd D

151)

that

dir

ectly

inte

ract

with

th

e N

Als

but

hav

e no

t bee

n re

port

ed to

con

fer r

esis

tanc

e to

NA

Is. Th

ese

NA

resi

dues

wer

e re

plac

ed w

ith a

min

o ac

ids

that

pos

sess

side

cha

ins h

avin

g si

mila

r pro

pert

ies t

o m

aint

ain

thei

r ori

gina

l cha

rge.

The

NA

mut

atio

ns w

e in

trod

uced

si

gnifi

cant

ly d

ecre

ased

NA

act

ivity

com

pare

d to

that

of t

he A

/Wuh

an/3

59/9

5 re

com

bina

nt w

ild-t

ype

and

R292

K (a

n N

A

mut

atio

n fr

eque

ntly

repo

rted

to c

onfe

r res

ista

nce)

vir

uses

, whi

ch w

ere

anal

yzed

for c

ompa

riso

n. H

owev

er, t

he re

com

bi-

nant

vir

uses

diff

ered

in re

plic

atio

n effi

cien

cy w

hen

we

seri

ally

pas

sage

d th

em in

vitr

o; th

e gr

owth

of t

he R

118K

and

E22

7D

viru

ses w

as m

ost i

mpa

ired

. The

R224

K E

276D

, and

R37

1K m

utat

ions

con

ferr

ed re

sist

ance

to b

oth

zana

miv

ir a

nd o

selta

mi-

vir,

whi

le th

e D

151E

mut

atio

n re

duce

d su

scep

tibili

ty to

ose

ltam

ivir

onl

y (s

imila

r to

10-f

old)

and

the

R152

K m

utat

ion

did

not a

lter s

usce

ptib

ility

to e

ither

dru

g. B

ecau

se th

e R2

24K

mut

atio

n w

as g

enet

ical

ly u

nsta

ble

and

the

emer

genc

e of

the

R371

K m

utat

ion

in th

e N

2 su

btyp

e is

stat

istic

ally

unl

ikel

y, ou

r res

ults

sugg

est t

hat o

nly

the

E276

D m

utat

ion

is li

kely

to

emer

ge u

nder

sele

ctiv

e pr

essu

re. Th

e re

sults

of o

ur st

udy

may

hel

p to

opt

imiz

e th

e de

sign

of N

AIs

.

Yen

et a

l. 20

06

Viru

s tr

ansm

issi

onRe

sist

ance

to

neur

amin

idas

e in

hibi

tors

Thre

e ty

pe A

influ

enza

vir

uses

, eac

h of

whi

ch h

as a

dis

tinct

neu

ram

inid

ase-

gene

mut

atio

n an

d is

resi

stan

t to

the

neur

ami-

nida

se in

hibi

tor o

selta

miv

ir, h

ave

been

isol

ated

. Pre

viou

sly,

in th

e fe

rret

mod

el, a

n R2

92K

mut

ant o

f a ty

pe A

(H3N

2) v

irus

w

as n

ot tr

ansm

itted

und

er c

ondi

tions

in w

hich

the

wild

-typ

e vi

rus w

as tr

ansm

itted

. This

mod

el w

as u

sed

to in

vest

igat

e w

heth

er th

e E1

19V

mut

ant o

f a ty

pe A

(H3N

2) v

irus

and

the

H27

4Y m

utan

t of a

type

A (H

1N1)

vir

us w

ould

be

tran

smit-

ted

unde

r sim

ilar c

ircu

mst

ance

s. B

oth

mut

ant v

irus

es w

ere

tran

smitt

ed, a

lthou

gh th

e H

274Y

mut

ant r

equi

red

a 10

0-fo

ld-

high

er d

ose

for i

nfec

tion

of d

onor

ferr

ets a

nd w

as tr

ansm

itted

mor

e sl

owly

than

was

the

wild

type

. Bot

h th

e m

utan

t and

th

e w

ild-t

ype

viru

ses r

etai

ned

thei

r gen

otyp

ic c

hara

cter

istic

s.

Her

loch

er e

t al.

2004

Res

ista

nce

to

neur

amin

idas

e in

hibi

tors

and

ad

aman

tane

sC

ompu

tatio

nal 3

D

stru

ctur

esRe

sidu

e m

utat

ions

The

neur

amin

idas

e (N

A) a

nd M

2 pr

oton

cha

nnel

of i

nflue

nza

viru

s are

the

drug

-tar

getin

g pr

otei

ns, b

ased

on

whi

ch se

vera

l dr

ugs w

ere

deve

lope

d. H

owev

er th

ese

once

pow

erfu

l dru

gs e

ncou

nter

ed d

rug-

resi

stan

t pro

blem

to th

e H

5N1

and

H1N

1 flu

. To

addr

ess t

his p

robl

em, t

he c

ompu

tatio

nal 3

D st

ruct

ures

of N

A a

nd M

2 pr

otei

ns o

f 200

9-H

1N1

influ

enza

vir

us w

ere

built

usi

ng th

e m

olec

ular

mod

elin

g te

chni

que

and

com

puta

tiona

l che

mis

try

met

hod.

Bas

ed o

n th

e m

odel

s the

stru

ctur

e fe

atur

es o

f NA

and

M2

prot

eins

wer

e an

alyz

ed, t

he d

ocki

ng st

ruct

ures

of d

rug-

prot

ein

com

plex

es w

ere

com

pute

d, a

nd th

e re

sidu

e m

utat

ions

wer

e an

nota

ted.

The

resu

lts m

ay h

elp

to so

lve

the

drug

-res

ista

nt p

robl

em a

nd st

imul

ate

desi

gnin

g m

ore

effec

tive

drug

s aga

inst

200

9-H

1N1

influ

enza

pan

dem

ic.

Du

et a

l. 20

10

Mol

ecul

ar m

arke

rs o

f re

sist

ance

Pyro

sequ

enci

ngM

2 bl

ocke

rsN

eura

min

idas

e in

hibi

tors

In th

e pr

esen

t stu

dy, w

e de

scri

be h

ow a

pyr

oseq

uenc

ing

met

hod

can

be u

sed

to ra

pidl

y de

tect

est

ablis

hed

mol

ecul

ar m

ark-

ers o

f res

ista

nce

to M

2 bl

ocke

rs a

nd N

A in

hibi

tors

in in

fluen

za A

(H5N

1) v

irus

es. Th

e re

sidu

es L

26, V

27, A

30, S

31, a

nd

G34

in th

e M

2 pr

otei

n w

ere

targ

eted

for p

yros

eque

ncin

g. Th

e N

A re

sidu

es fo

r pyr

oseq

uenc

ing

anal

ysis

incl

uded

the

esta

b-lis

hed

mar

kers

of d

rug

resi

stan

ce (H

274

and

N29

4), a

s wel

l as r

esid

ues o

f les

s cer

tain

rele

vanc

e (V

116,

I117

, Q13

6, K

150,

an

d I2

22).

A si

ngle

pai

r of p

yro-

reve

rse

tran

scri

ptio

n (R

T)-

PCR

prim

ers w

as d

esig

ned

to a

llow

am

plifi

catio

n of

an

appr

oxi-

mat

ely

600-

nucl

eotid

e-lo

ng a

mpl

icon

of t

he N

A g

enes

of H

5N1

viru

ses f

rom

var

ious

cla

des/

subc

lade

s ass

ocia

ted

with

in

fect

ions

in h

uman

s. Th

e se

nsiti

vity

of t

he a

ssay

was

dem

onst

rate

d by

the

succ

essf

ul p

yros

eque

ncin

g of

RN

A e

xtra

cted

fr

om sa

mpl

es o

f ser

ially

dilu

ted

(10–5

to 1

0–7 v

irus

stoc

ks w

ith in

itial

con

cent

ratio

ns ra

ngin

g fr

om 1

05 to 1

08 PFU

/ml.

The

mar

kers

of r

esis

tanc

e w

ere

dete

cted

in sa

mpl

es w

ith th

resh

old

cycl

e va

lues

rang

ing

from

32

to 3

7, a

s det

erm

ined

by

real

-tim

e RT

-PC

R. Th

e py

rose

quen

cing

app

roac

h m

ay p

rovi

de a

val

uabl

e to

ol fo

r rap

id d

etec

tion

of m

arke

rs o

f dru

g re

sist

ance

in

H5N

1 vi

ruse

s and

faci

litat

e th

e el

ucid

atio

n of

the

role

of s

uch

chan

ges i

n na

tura

l and

acq

uire

d dr

ug re

sist

ance

.

Dey

de e

t al.

2009


184

Iden

tific

atio

n of

the

pote

ntia

l res

ista

nce

site

sC

ompu

ter-

aide

d m

etho

dO

selta

miv

irZ

anam

ivir

Mol

ecul

ar b

asis

of d

rug

resi

stan

ce

The

out

brea

k an

d hi

gh s

peed

glo

bal s

prea

d of

the

new

str

ain

of in

fluen

za A

(H1N

1) v

irus

in 2

009

pose

s a

seri

ous

thre

at

to th

e ge

nera

l pop

ulat

ion

and

gove

rnm

ents

. At p

rese

nt, t

he m

ost e

ffect

ive

drug

s fo

r th

e tr

eatm

ent o

f 200

9 in

fluen

za A

(H

1N1)

vir

us a

re n

eura

min

idas

e in

hibi

tors

: mai

nly

osel

tam

ivir

and

zan

amiv

ir. T

he u

se o

f the

se tw

o in

hibi

tors

will

un

doub

tedl

y in

crea

se, a

nd th

eref

ore

it is

mor

e lik

ely

that

dru

g-re

sist

ant i

nflu

enza

str

ains

will

ari

se. T

he id

entif

icat

ion

of th

e po

tent

ial r

esis

tanc

e si

tes

for

thes

e dr

ugs

in a

dvan

ce a

nd th

e un

ders

tand

ing

of c

orre

spon

ding

mol

ecul

ar b

asis

to

caus

e dr

ug re

sist

ance

are

no

doub

t ver

y im

port

ant t

o fig

ht a

gain

st th

e ne

w re

sist

ant i

nflu

enza

str

ains

. In

this

stu

dy,

first

, the

com

plex

es o

f neu

ram

inid

ase

with

the

subs

trat

e si

alic

aci

d an

d tw

o in

hibi

tors

ose

ltam

ivir

and

zan

amiv

ir w

ere

obta

ined

by

fittin

g th

em to

the

3D s

truc

ture

of 2

009

influ

enza

A (H

1N1)

neu

ram

inid

ase

obta

ined

by

hom

olog

y m

ode-

ling.

By

usin

g th

ese

com

plex

es a

s th

e in

itial

str

uctu

res,

mol

ecul

ar d

ynam

ics

sim

ulat

ion

and

mol

ecul

ar m

echa

nics

gen

er-

aliz

ed B

orn

surf

ace

area

(MM

-GBS

A) c

alcu

latio

ns w

ere

perf

orm

ed to

iden

tify

the

resi

dues

with

sig

nific

ant c

ontr

ibut

ion

to th

e bi

ndin

g of

sub

stra

te a

nd in

hibi

tors

. By

anal

yzin

g th

e di

ffere

nce

of in

tera

ctio

n pr

ofile

s of

sub

stra

te a

nd in

hibi

tors

, th

e po

tent

ial d

rug

resi

stan

ce s

ites

for

two

inhi

bito

rs w

ere

iden

tifie

d. P

arts

of t

he id

entif

ied

site

s ha

ve b

een

veri

fied

to

conf

er re

sist

ance

to o

selta

miv

ir a

nd z

anam

ivir

for

influ

enza

vir

us o

f the

pas

t flu

epi

dem

ic. T

he id

entif

ied

pote

ntia

l re

sist

ance

site

s in

this

stu

dy w

ill b

e us

eful

for

the

deve

lopm

ent o

f new

effe

ctiv

e dr

ugs

agai

nst t

he d

rug

resi

stan

ce a

nd

avoi

d th

e si

tuat

ion

of h

avin

g no

effe

ctiv

e dr

ugs

to tr

eat n

ew m

utan

t inf

luen

za s

trai

ns.

Liu

et a

l. 20

10

Prev

alen

ce o

f am

anta

dine

-res

ista

nt

viru

ses

M2

chan

nel m

utat

ions

Gen

ome

sequ

enci

ngJa

pan

Back

grou

nd: T

he p

reva

lenc

e of

am

anta

dine

-res

ista

nt in

fluen

za A

/H3N

2 vi

ruse

s (b

elon

ging

to th

e N

-lin

eage

), po

sses

s-in

g an

S31

N m

utat

ion

in th

e M

2 pr

otei

n an

d S1

93F

and

D22

5N s

ubst

itutio

ns in

thei

r H

A1

subu

nit,

has

sign

ifica

ntly

in

crea

sed

wor

ldw

ide

sinc

e 20

05. T

he a

im o

f thi

s st

udy

was

to c

lari

fy th

e ge

nom

ic e

vent

s co

ntri

butin

g to

the

evol

utio

n an

d co

ntin

uity

of t

he N

-lin

eage

am

anta

dine

-res

ista

nt v

irus

es. M

etho

ds: T

he fu

llgen

ome

sequ

ence

of A

/H3N

2 is

olat

es,

incl

udin

g bo

th a

man

tadi

ne-r

esis

tant

and

am

anta

dine

-sen

sitiv

e vi

ruse

s, c

olle

cted

in Ja

pan

betw

een

2006

and

200

8, w

as

dete

rmin

ed a

nd p

hylo

gene

tical

ly c

ompa

red

with

isol

ates

obt

aine

d fr

om th

e da

taba

se. R

esul

ts: O

n th

e ba

sis

of th

e fu

ll ge

nom

e se

quen

ce a

naly

sis,

the

N-l

inea

ge c

ould

be

furt

her

divi

ded

into

thre

e ge

netic

ally

rela

ted

clad

es: N

i (A

/Wis

cons

in/6

7/20

05-l

ike

aman

tadi

ne-r

esis

tant

vir

uses

from

yea

rs 2

005–

2007

), N

2 (a

man

tadi

ne-s

ensi

tive

viru

ses

from

200

7) a

nd N

3 (A

/Bri

sban

e/10

/200

7-lik

e am

anta

dine

-res

ista

nt v

irus

es fr

om 2

007

and

2008

). T

he 2

006/

2007

sea

son

show

ed c

ocir

cula

tion

of a

ntig

enic

var

iant

s of

am

anta

dine

-res

ista

nt v

irus

es o

f cla

des

Ni a

nd N

3 in

add

ition

to th

e N

2-se

nsiti

ve v

irus

es. I

n th

e 20

07/2

008

seas

on, t

he c

lade

N3

aman

tadi

ne-r

esis

tant

line

age

dom

inat

ed a

nd re

plac

ed o

ther

st

rain

s. P

hylo

gene

tic a

naly

sis

of e

ach

indi

vidu

al s

egm

ent s

ugge

sted

that

N2

and

N3

wer

e ge

nera

ted

from

two

inde

pend

-en

t rea

ssor

tmen

t eve

nts

invo

lvin

g cl

ade

Ni v

irus

es a

nd p

re-N

-lin

eage

str

ains

. Con

clus

ions

: Our

dat

a sh

ow th

at s

ever

al

reas

sort

men

t eve

nts

have

con

trib

uted

to th

e ev

olut

ion

of a

man

tadi

ne-r

esis

tant

A/H

3N2

stra

ins

and,

con

sequ

ently

, to

the

succ

essf

ul s

prea

d of

this

line

age.

Alth

ough

am

anta

dine

resi

stan

ce is

cau

sed

by s

ingl

e am

ino

acid

mut

atio

ns in

the

M2

prot

ein,

gen

ome-

wid

e ad

just

men

t inv

olvi

ng m

ultip

le g

enes

app

ears

to b

e ne

cess

ary

to o

btai

n ef

ficie

nt re

plic

atio

n an

d tr

ansm

issi

on o

f res

ista

nt v

irus

es. S

uch

adju

stm

ents

are

att

aina

ble

thro

ugh

reas

sort

men

t of s

egm

ents

am

ong

diffe

r-en

t vir

us li

neag

es.

Zar

aket

et a

l. 20

10


185

Prev

alen

ce o

f ant

ivir

al

resi

stan

ceO

selta

miv

irZ

anam

ivir

Am

anta

dine

Arg

entin

a20

05–2

008

Obj

ectiv

e. T

o de

scri

be th

e vi

rolo

gica

l cha

ract

eris

tics

of th

e in

fluen

za s

trai

ns c

ircu

latin

g in

Arg

entin

a in

200

5–20

08

and

to a

sses

s th

e pr

eval

ence

of a

ntiv

iral

resi

stan

ce. M

etho

ds. O

n th

e ba

sis

of th

eir

geog

raph

ical

spr

ead

and

prev

alen

ce,

influ

enza

Lam

bda

and

B is

olat

es g

row

n in

Mad

in-D

arby

can

ine

kidn

ey c

ells

wer

e se

lect

ed a

fter

ant

igen

ic a

nd g

enom

ic

char

acte

riza

tion

to b

e an

alyz

ed fo

r an

tivir

al re

sist

ance

by

enzy

mat

ic a

ssay

and

pyr

oseq

uenc

ing.

Am

anta

dine

sus

cep-

tibili

ty w

as e

valu

ated

by

pyro

sequ

enci

ng fo

r kn

own

resi

stan

ce m

arke

rs o

n 45

str

ains

of i

nflu

enza

A. S

usce

ptib

ility

to

osel

tam

ivir

and

zan

amiv

ir w

as e

valu

ated

by

enzy

mat

ic a

ssay

of 6

7 in

fluen

za L

ambd

a an

d 46

influ

enza

B s

trai

ns, s

ome

of w

hich

wer

e fu

rthe

r an

alyz

ed b

y se

quen

cing

the

neur

amin

idas

e ge

ne. R

esul

ts. R

esis

tanc

e to

am

anta

dine

was

obs

erve

d on

ly o

n A

(H3N

2) s

trai

ns (2

9/33

); al

l of t

hem

car

ried

the

mut

atio

n S3

1N in

thei

r M

2 se

quen

ce. O

selta

miv

ir re

sist

-an

ce w

as o

bser

ved

in 1

2 (3

4.3%

) of t

he 3

5 A

(H1N

1) s

trai

ns fr

om 2

008;

all

of th

em c

arri

ed th

e m

utat

ion

H27

5Y in

thei

r ne

uram

inid

ase

sequ

ence

. All

thes

e vi

ruse

s re

mai

ned

sens

itive

to z

anam

ivir.

Con

clus

ions

. Thi

s st

udy

desc

ribe

s a

high

in

cide

nce

of a

man

tadi

ne-r

esis

tant

influ

enza

A(H

3N2)

vir

uses

sin

ce 2

006

and

an u

npre

cede

nted

incr

ease

in o

selta

miv

ir

resi

stan

ce d

etec

ted

only

in in

fluen

za A

(H1N

1) v

irus

es is

olat

ed in

200

8. In

fluen

za A

and

B v

irus

es w

ere

mor

e se

nsiti

ve

to o

selta

miv

ir th

an to

zan

amiv

ir, a

nd in

fluen

za A

vir

uses

wer

e m

ore

sens

itive

to b

oth

neur

amin

idas

e in

hibi

tors

than

the

influ

enza

B v

irus

es. T

he n

atio

nal d

ata

gene

rate

d an

d an

alyz

ed in

this

stu

dy m

ay h

elp

incr

ease

kno

wle

dge

abou

t inf

lu-

enza

ant

ivir

al d

rug

resi

stan

ce, w

hich

is a

pro

blem

of g

loba

l con

cern

.

Pont

orie

ro e

t al.

2011

Res

ista

nce

to

haem

aggl

utin

in

inhi

bito

rsSt

achy

flin

In si

lico

scre

enin

g

In th

is s

tudy

we

perf

orm

ed m

olec

ular

dyn

amic

s si

mul

atio

ns o

n a

spik

e pr

otei

n on

the

vira

l env

elop

, hem

aggl

utin

in

for

the

wild

type

and

thre

e ki

nds

of m

utan

ts u

sing

a m

odel

sys

tem

con

sist

ing

of a

trim

eric

hem

aggl

utin

com

plex

, vir

al

lipid

mem

bran

e. s

olva

tion

wat

ers,

and

ions

. A

nat

ural

pro

duct

sta

chyf

lin, w

hich

sho

ws

a hi

gh le

vel o

f ant

ivir

al a

ctiv

ity

spec

ific

to s

ome

subt

ypes

of i

nflu

enza

vir

uses

, was

exa

min

ed o

n bi

ndin

g to

the

wild

-typ

e he

mag

glut

inin

was

cla

rifie

d.

Nex

t, 8

com

poun

ds w

ere

sele

cted

from

a c

hem

ical

dat

abas

e by

in s

ilico

scr

eeni

ng, c

onsi

deri

ng th

e fin

ding

s fr

om th

e si

mul

atio

ns. I

nhib

itory

act

iviti

es to

sup

pres

s th

e pr

olife

ratio

n of

influ

enza

vir

us w

ere

mea

sure

d by

cel

l-ba

sed

antiv

iral

as

says

, and

che

mic

al s

caffo

lds

wer

e fo

und

to b

e po

tent

for

an in

hibi

tor.

Mor

e th

an 3

0 de

riva

tives

bea

ring

eith

er o

f the

se

two

chem

ical

sca

ffold

s w

ere

synt

hesi

zed,

and

cel

l cul

ture

ass

ays

wer

e ca

rrie

d ou

t of e

valu

ate

the

com

poun

d po

tenc

y. Se

vera

l der

ivat

ives

dis

play

ed a

hig

h co

mpo

und

pote

ncy,

and

50%

effe

ctiv

e co

ncen

trat

ions

of t

wo

synt

hesi

zed

com

-po

unds

wer

e be

low

1 µ

M.

Yana

gita

et a

l. 20

12

Res

ista

nce

to

phos

phor

o-th

ioat

e ol

igon

ucle

otid

e

In a

pre

viou

s st

udy

a 15

-mer

pho

spho

roth

ioat

e ol

igon

ucle

otid

e (S

-ON

) der

ived

from

the

pack

agin

g si

gnal

in th

e 5’

end

of

seg

men

t 1 (P

B2) o

f inf

luen

za A

vir

us (d

esig

nate

d 5-

15b)

pro

ved

mar

kedl

y in

hibi

tory

to v

irus

repl

icat

ion.

Her

e w

e in

vest

igat

ed w

heth

er a

nalo

gous

inhi

bito

ry S

-ON

s ta

rget

ing

the

5’ e

nd o

f seg

men

ts 2

(PB1

) and

3 (P

A) c

ould

be

iden

ti-fie

d an

d w

heth

er v

iral

resi

stan

ce to

S-O

Ns

can

be d

evel

oped

. Sim

ilar

to o

ur e

arlie

r re

sult,

20-

mer

S-O

Ns

repr

oduc

ing

the

5’ e

nds

of s

egm

ents

2 o

r 3

(com

plem

enta

ry to

the

3’-c

odin

g re

gion

s of

PB1

and

PA

, res

pect

ivel

y) e

xert

ed a

pow

erfu

l an

tivir

al a

ctiv

ity a

gain

st a

var

iety

of i

nflu

enza

A v

irus

sub

type

s in

MO

CK

cel

ls. S

eria

l pas

sage

of t

he A

/Tai

wan

/1/8

6 H

1N1

stra

in in

the

pres

ence

of S

-ON

5-1

5b o

r its

ant

isen

se a

s 5-

15b

anal

ogue

sho

wed

that

mut

ant v

irus

es w

ith re

duce

d su

scep

tibili

ty to

the

S-O

N c

ould

inde

ed b

e ge

nera

ted,

alth

ough

the

resi

stan

t vir

uses

dis

play

ed re

duce

d re

plic

ativ

e fit

-ne

ss. S

eque

ncin

g th

e re

sist

ant v

irus

es id

entif

ied

mut

atio

ns in

the

PB1,

PB2

, PA

and

M1

gene

s. In

trod

uctio

n of

thes

e ch

ange

s in

to th

e A

/PR/

8/34

H1N

1 st

rain

by

reve

rse

gene

tics,

sug

gest

ed th

at a

ltera

tions

to R

NA

func

tion

in th

e pa

ck-

agin

g re

gion

s of

seg

men

ts 2

and

3 w

ere

impo

rtan

t in

deve

lopi

ng re

sist

ance

to S

-ON

inhi

bitio

n. H

owev

er, m

any

of

the

othe

r se

quen

ce c

hang

es in

duce

d by

S-O

N tr

eatm

ent w

ere

mar

kedl

y de

lete

riou

s to

vir

us fi

tnes

s. W

e co

nclu

de th

at

pack

agin

g si

gnal

s in

the

influ

enza

A v

irus

pol

ymer

ase

segm

ents

pro

vide

feas

ible

targ

ets

for

nucl

eic

acid

-bas

ed a

ntiv

iral

s th

at m

ay b

e di

ffic

ult f

or th

e vi

rus

to e

vade

thro

ugh

resi

stan

ce m

utat

ions

.

Gia

nnec

chin

i et

al. 2

011

Antiviral agents targeting the influenza virus: a review ...vri.cz/docs/vetmed/58-3-113.pdf ·...

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