Novel Targets for the Development of Anti-Herpes Compounds

7/29/2019 Novel Targets for the Development of Anti-Herpes Compounds

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Infectious Disorders - Drug Targets 2007, 7, 11-18 11

1871-5265/07 $50.00+.00 2007 Bentham Science Publishers Ltd.

Novel Targets for the Development of Anti-Herpes Compounds

A. Greco*,1, J-J. Diaz

1, D. Thouvenot

2and F. Morfin

2,3

1Universit de Lyon, Lyon, F-69003, France; Universit Lyon 1, Lyon, F-69003, France; CNRS, UMR5534, Centre de Gntique

Molculaire et Cellulaire, 16 rue Dubois, Villeurbanne, F-69622, France,2Laboratoire de Virologie Est, Hospices Civils de Lyon and

UMR5537 CNRS - Universit Lyon 1, 8 avenue Rockefeller, F-69373 Lyon cedex 08, France,3Universit Lyon 1, Institut des Sciences

Pharmaceutiques et Biologiques, 8 avenue Rockefeller, F-69373 Lyon cedex 08, France

Abstract: Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are members of the Herpesviridae family.HSV infections have been known since ancient times and are one of the most common communicable diseases in humans. Althoughinfections are often subclinical, HSV can cause mild to severe diseases, especially in immunocompromised patients. Herpes simplexviruses establish latency in the nuclei of neuronal cells and may reactivate, with or without symptoms, throughout the host's lifetime.

Over one third of the world's population suffer from recurrent HSV infections several times a year and are thus capable of transmittingHSV by close personal contact.

There are few drugs licensed for the treatment of HSV infections. Most target the viral DNA polymerase, and indeed acyclovir remainsthe reference treatment some thirty years after its discovery! Extensive clinical use of this drug has led to the emergence of resistant viral

strains, mainly in immunocompromised patients. This highlights the crucial need for the development of new anti-herpes drugs that caninhibit infection by both wild-type viruses and drug-resistant strains.

Over the last few years, significant efforts have been made to set up a range of strategies for the identification of potential new anti-viraldrugs. One alternative is to develop drugs with different mechanisms of action. The present article reviews potential viral and cellular

targets that are now known to be involved in HSV infection and for which specific inhibitors with anti-HSV activity, at least in cellculture, have been identified.

Keywords: HSV, resistance, antiviral drugs, viral target, cellular target.

INTRODUCTION

Herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) areresponsible for a variety of clinical manifestations in humans. Theyare distributed throughout the world and are transmitted frominfected hosts to susceptible individuals by close personal contact.Transmission is broadly favored by the considerable number ofasymptomatic infections, associated with viral shedding. Herpesinfections are generally localized on the face and trunk in the caseof HSV-1 and in the genital sphere in the case of HSV-2, althoughan increasing proportion of genital infections are associated withHSV-1 [1].

Herpes simplex viruses (HSV) possess an envelope. The central

electron-dense core containing the linear double-stranded DNAgenome is surrounded by an icosahedral capsid and a tegumentlayer. The outer envelope presents spike structures corresponding toviral glycoproteins [2]. During the entry step, viral membrane

proteins bind to cellular receptors, prior to fusion of the twomembranes. Nucleocapsids containing the viral genome arereleased into the cytoplasm and transported to nuclear pores. ViralDNA is then released into the nucleus. The viral replication cycle isrelatively short (18 to 24 hours) and results in cytolysis. The viralDNA encodes at least 80 genes whose expression is sequentiallyand temporally regulated by complex mechanisms. Viral genes can

be divided into imme-diate-early, early and late genes, according totheir expression kinetics. Immediate-early proteins are involved inthe regulation of the synthesis of early and late proteins. Early

proteins participate in viral DNA replication, and late proteins formthe structural components of the viral particles. Viral replication

and encapsidation take place in the nucleus of the infected cell.Capsids containing the newly replicated viral DNA leave thenucleus and new virus particles are released into the extracellularspace via a number of complex processes, including budding ofnucleocapsids through the inner nuclear membrane, transport of

*Address correspondence to this author at the Universit de Lyon, Lyon, F-69003, France; Universit Lyon 1, Lyon, F-69003, France; CNRS,

UMR5534, Centre de Gntique Molculaire et Cellulaire, 16 rue Dubois,Villeurbanne, F-69622, France; Tel: +33 472 447926; Fax: +33 472 432685;

E-mail: [email protected]

mature virus particles through the cytoplasm by exocytic vesiclesand fusion with the plasma membrane.

The HSV-1 DNA polymerase (pol) has been extensivelystudied as a target for antiviral drugs. Indeed, some of the manynucleotide and pyrophosphate analogues that have been tested fortheir ability to inhibit viral replication have led to the developmentof antiviral drugs - notably acyclovir (ACV) and foscarnet.Acyclovir is of significant therapeutic value and is considered asthe "gold standard" in HSV therapy.

However, the emergence of ACV-resistant virus strains affectsabout 5% of immunocompromised patients receiving long-term

prophylactic treatment with ACV [3]. Hence, resistance is an

important clinical problem that may lead to ineffective therapy (fora review, see [4]). One alternative is to develop antiviral drugs withdifferent mechanisms of action. In principal, all the essential viral

proteins involved in HSV replication can be considered as potentialtargets for chemo-therapy. These include any key protein in anystep in the viral cycle, from HSV attachment to the host cell to therelease of newly synthesized virions. Most current investigationaldrugs target viral enzymes involved in key steps of the viral DNAreplication, since HSV-1 or HSV-2 encode several viral proteinsinvolved in this function. The latter include DNA-metabolizingenzymes - ribonucleotide reductase (RR), thymidine kinase anddUTPase - and seven proteins that are strictly required for HSVDNA replication, since mutations in the corresponding genesabrogate viral replication. The seven proteins correspond to theorigin-binding protein UL9, the single stranded DNA-binding

protein ICP8, DNA pol and its accessory protein, encoded by UL30

and UL42 respectively, and the UL5, UL9 and UL52 gene productswhich form a stable complex with helicaseprimase activities.Some of these viral proteins have been reported in the literature asalternative targets for the development of new kinds of anti-herpescompounds [5-10]. In addition, new antiviral molecules directedagainst the viral DNA pol are in development; they target otherdomains of the protein than those targeted by the commerciallyavailable drugs.

Another alternative strategy in the development of new anti-herpes drugs is to target cellular proteins, since many cellularfactors are required in the different steps of HSV infection Fig.


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(1). In the last 10 years, it has been shown that cellular proteinscan indeed be relevant targets for anti-HSV drugs. The first andmost documented example is cyclin-dependent kinase 2 (cdk2) [5,11, 12].

The present review will focus on the viral and cellular functionsthat are now known to be involved in HSV infection and for whichspecific inhibitors with anti-HSV activity, at least in cell culture,have been identified.

HSV INFECTIONS

Following the primary infection of mucosal surfaces, HSV istransported by retrograde axonal transport from the infectedmucosal area and then establishes a latent infection, in thetrigeminal ganglia for HSV-1 and the sacral ganglia for HSV-2.This latent infection, in which viral DNA is retained in neuronalnuclei in the absence of viral production, persists throughout thehost's lifetime [13]. Despite the development of an immuneresponse, reactivations from this source are triggered by variousinternal or external stimuli, such as stress, menses, fever, UV-lightexposure and immunosuppression.

From a clinical standpoint, HSV can cause diseases rangingfrom mild conditions to morbid or even lethal infections, espe-cially in neonates and immunocompromised patients. Primaryinfections and reactivations are often subclinical. Asymptomaticshedding of HSV, prompted by subclinical reactivations, occursfrequently in infected individuals. HSVs cause mucocutaneouslesions presenting as vesicles or ulceration in a range of body sites.

Oropharyngeal infection often results in gingivostomatitis,which is the most common form of symptomatic, primary HSVinfection of the oropharynx. After a short incubation period (4days), progression is usually rapid (15 days) and may becomplicated by auto-inoculation at other sites such as the skin,fingers, nose, eyes, genital area, etc. Recurrent infections areusually described as herpes labialis and last for about 7 days.

Genital HSV infection is often more severe and associated withsymptoms such as pain. Each episode lasts about 3 weeks in

primary infection and 2 weeks for reactivation. Genital herpes may

be complicated by meningitis [14]. The most important differencebetween HSV-1 and HSV-2 genital infections is that HSV-1 israrely associated with reactivations, whereas HSV-2 may beassociated with 3 to 9 reactivations per year [15].

HSV-1 is involved in ocular infections and may cause keratitis(presenting as unilateral or bilateral conjunctivitis) with pre-auricular lymphadenopathy, followed by an unilateral,characteristic, dendritic ulcer which usually lasts for 3 weeks [16].In 20 to 30% of patients, these infections are associated withrecurrence within 2 years. Multiple infections can result in loss of

vision; indeed, in developed countries, ocular HSV infection is themost common cause of corneal blindness.

Neonatal herpes is a serious infection predominantly caused byHSV-2. HSV-1 can also be responsible, as transmission may occurwith a primary HSV-1 genital infection or via an oral herpes fromthe mother, her relatives or hospital staff. Literature reports ofincidence of neonatal herpes vary considerably and range from 1case per 2,500 live births in the USA [17] to 1.65 per 100,000 live

births in the UK [18]. American studies suggest that there are threetypes of disease presentations: (i) localized infections with skin, eyeand/or mouth lesions, (ii) infections associated with neurologicalsymptoms and (iii) disseminated disease (the most severe form). Inthe absence of antiviral therapy, the mortality rate reaches 80% incases of disseminated disease, warranting early antiviral therapy forall neonatal HSV infections.

Another serious form of HSV infection is encephalitis, which isconfined to the frontotemporal and parietal areas of the brain andmay progress rapidly to coma and death. HSV encephalitis is aconcern for all age groups, in both men and women. The incidenceof HSV encephalitis ranges from about 1 case per million

population per year to 1 case per 200,000 [17]. Most cases arecaused by HSV-1. In untreated patients, mortality reaches 70% andonly 2.5% of surviving patients have no neurological sequellae[17].

HSV infection may be particularly severe in immunocom-promised patients in general and those with cell-mediated immunitydeficits in particular. These patients present extensive lesions that

Fig. (1). Strategies for the development of anti-herpes therapies. In the conventional strategy, the essential viral proteins involved in HSV replication are

considered as potential targets for chemotherapy. The alternative strategy is to target cellular proteins required in the different steps of HSV infection.


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may disseminate and/or progress chronically. These infectionsrequire prophylactic and/or curative antiviral chemo-therapy.

HSV infection diagnosis relies on virus detection in mucocu-taneous samples via antigen or nucleic acid detection and culture[19, 20]. Serology can also be of interest. HSV culture is sensitiveand easy to perform; furthermore, it is required for testing HSV'ssensitivity to antiviral drugs using phenotypic methods.

Despite the fact that vaccination against HSV has beenevaluated since the 1920s, a HSV vaccine is still not available (for areview see [21]). However, there are a few drugs licensed for thetreatment of these infections. Most target the viral DNA pol, andACV remains the reference treatment.

AVAILABLE ANTI-HERPES DRUGS

A number of antiviral drugs for the treatment of HSV infectionshave been developed since the mid 1950s. The first wereidoxuridine and trifluridine, which are pyrimidine analo-gues thatare phosphorylated by viral and cellular kinases. Given theirtoxicity, these molecules could only be used for topical treatment ofHSV infections. In the 1980s, vidarabine became available as asystemic treatment for HSV encephalitis. This molecule is anadenosine analogue that is phosphorylated only by cellular kinases.

There are currently three classes of drugs licensed for the

treatment of HSV infections, and all target viral DNA replication:(i) the acyclic guanosine analogue ACV and related compounds,such as penciclovir and their prodrugs (valacyclovir and fam-ciclovir, respectively), (ii) the acyclic nucleotide analogue cidofovirand (iii) the pyrophosphate analogue foscarnet. Despite numerousattempts to improve on ACV, the latter remains the gold standardfor HSV treatment - some thirty years after its discovery!

ACV has to be phosphorylated three times before its incor-poration into viral DNA. Since the first phosphorylation is achievedby the viral TK, ACV becomes active only in infected cells. Thesecond and third phosphorylations are carried out by cellularthymidylate kinases. ACV triphosphate is a competitive inhibitor ofviral DNA pol and it is a DNA chain terminator [22]. Penciclovirhas a very similar mechanism of action.

ACV and penciclovir have very poor oral bioavailability and

synthesis of the prodrugs, valacyclovir and famciclovir, respec-tively, has improved this parameter. ACV and penciclovir havevery few side effects. Some nephrotoxicity has been reported but isuncommon and always reversible [23].

ACV-resistant HSV strains were first reported in 1982 [24-26],with most being recovered from immunocompromised patients

previously treated with ACV [27-29]. ACV resistance may beassociated with a mutation occurring spontaneously in one or bothof the two viral enzymes targeted by ACV's mecha-nism of action(TK and DNA pol). Three resistance mechanisms have beenreported: a loss of TK activity (TK-deficient virus), an alteration inTK substrate specificity (TK-altered virus) and/or an alteration inDNA pol activity [30]. The most frequent mutations occur in theviral TK gene, and 95% of ACV-resistant isolates present a TK-deficient phenotype [31]. Resistance associated with a mutation inthe viral DNA pol gene occurs rarely [32] because a functional

DNA pol is essential for viral replication (unlike viral TK).

Large-scale studies have shown that resistance is mainly aconcern for immunocompromised patients, and several reports havedescribed a mean prevalence below 1% in immunocompe-tent

populations [27, 29, 33]. Recently, another large survey reported aprevalence of 0.32% [3], i.e. a value that has not increased since thefirst studies were performed ten years previously, despite a majorincrease in ACV use. According to the literature, most ACV-resistant HSVs isolated from immuno-competent individuals have

been detected in a context of recurrent genital herpes, and theobserved prevalence ranged from 3.5% to 8.6% [34, 35]. However,in most cases, the clinical course of the infection was unaffected.

The prevalence of ACV resistance in immunocompromisedpatients is about 5%, according to several published reports [27-29].This incidence depends on the type of immunosuppression. In arecent survey, resistance was detected in 2.8% of solid organtransplant patients, 3.5% of human immunodeficiency virus-infected patients and 14% of bone marrow transplant patients [3]. Inthis latter population, the incidence of resistance was 2% forautologous transplant patients and 29% for allogeneic transplant

patients. Another recent study revealed that patients receiving bonemarrow of either autologous or allogeneic origin have about thesame risk of developing an HSV infection (9%) but that resistanceto ACV was only detected in allogeneic transplant patients, with a

prevalence of up to 30% in this population [36].

These findings indicate that the prevalence of ACV-resistantHSV is steady, relative to studies published more than 10 years ago.This stability is noted for both immunocompetent andimmunocompromised patients. Finally, as suggested by otherstudies, ACV resistance is restricted to allogeneic bone marrowtransplant patients but is a major concern for these individuals.

In immunocompromised patients, resistance is associated withclinical failure after 1 to 2 weeks of treatment. Lesions may presentas large, ulcerated and extensive. These infections are often serious,and may become chronic [37]. After healing, recurrent infections

are most often associated with an ACV-sensitive strain, althoughsome cases of recurrences due to ACV-resistant viruses have beenreported [38-41].

One way of managing ACV-resistant infection consists inimproving the patient's immune status, when possible, bydecreasing immunosuppressive treatments [42]. In addition, severalalternative antiviral drugs may be used. Most ACV-resistant HSVisolates are also resistant to penciclovir, although ACV-resistant,

penciclovir-susceptible isolates have been reported from time totime; the ACV resistance mechanism of these strains was related toeither an altered TK or a mutation in the viral DNA pol [43, 44].Foscarnet and cidofovir act directly on viral DNA pol without theneed for prior activation by viral TK, and so both of thesemolecules are active against ACV-resistant viruses which carry amutation in the TK gene [39, 45]. However, in clinical practice,these drugs may be associated with a significant level of toxicity.

Foscarnet inhibits the pyrophos-phate binding site of DNA pol.Cidofovir is a monophosphory-lated derivative of cytidine that is

phosphorylated twice by cellular kinases before being incorporatedin the DNA chain by viral DNA pol. Foscarnet-resistant clinicalisolates of HSV have been isolated, albeit rarely, from AIDS

patients [46] and bone marrow transplanted patients [47]. Foscarnetresistance is associated with viral DNA pol mutations [46].Cidofovir has been used for the treatment of ACV- and foscarnet-resistant HSV infections [48]. To date, resistance to cidofovir hasnot been reported for HSV.

POTENTIAL VIRAL TARGETS

Numerous investigations using a variety of approaches havefocused on the identification and validation of viral targets. Proteinsinvolved in viral DNA replication are still attractive targets;however, viral proteins that are essential in other steps of the viral

infection also represent potentially valuable targets as well Fig.(1), Table (1).

Attachment and Penetration

Penetration of HSV-1 into the cell involves at least five (gC,gB, gD, gH and gL) of the dozen or so of the viral envelopeglycoproteins, together with three classes of host-cell membranereceptors; herpes virus entry mediator (HVEM, a member of thetumor necrosis factor receptor family), nectin-1 and nectin-2 (twomembers of the immunoglobulin superfamily) and cell surfaceglycosaminoglycans (preferentially heparan sulfate) [49].


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At present, none of the antiherpes drugs currently licensed forthe treatment of HSV infections displays a well-demonstrated modeof action consisting of the inhibition of viral entry [50]. The lack ofmolecules with this type of mechanism of action is certainly due tothe fact that virus penetration is a very complex, incompletely-deciphered process that involves numerous viral and cellularmolecules that are difficult to target simultaneously with smallmolecule drugs. Nevertheless, many studies provide convincingevidence that certain types of molecules interfere more or lessspecifically with virus entry (by interacting either with the viral

particle or with cellular receptors or both) and thus display anti-herpes activity. Hence, these data strongly support the notion that

blocking viral entry could be used to develop new kinds of antiviralcompounds; targeting the process of virus penetration has theadvantage of developing molecules that do not need to enter the cellto be effective, thus facilitating their development during the drugdiscovery process.

To illustrate this kind of approach, one might mentioned thedevelopment and use of several microbicides against herpesinfections, although this type of molecules are not generallyclassified as antiviral compounds, due to their mode of action andusage restricted to local applications. Nevertheless, severalmicrobicides are now being tested in Phase II or III clinical trialsfor their ability to prevent virus shedding and its transmission byclose personal contact. A few of these are sulfated polymer-basedinhibitors that interact directly with viral envelope glycoproteinsand prevent viral attachment to the cell (Carraguard

TM, Emmelle

/dextrin-2-sulfate, polystyrene sulfonate PSS, PRO2000/5TM,Ushercell

TM, Virend, dendrimers) [8, 51]. In addition, various

plant-extracted compounds with chemical structures of varyingcomplexity derive their valuable antiherpes activity, at least in part,

by acting on the virus attachment and penetration stages [52, 53].

It was recently shown that a phosphorothioate oligonuc-leotidedisplays a strong virucidal activity by inhibiting virus attachment.The mechanism of action of this oligonucleotide is not welldetermined although it was proposed that it is able to induce aconformational change in gB that results in inactivation ofinfectivity [54].

Other types of molecules have been shown to interfere withviral entry with limited specificity. The lactoferrin protein andlactoferricin, a small peptide fragment from lactoferrin's N-terminaldomain, are prototypes of this family of compounds, whose

mechanism of action is currently receiving much attention.Lactoferricin interacts with cell surface heparan sulfate to blockviral entry. In contrast, lactoferrin's mechanism of action has not yet

been elucidated in detail, although it is clear that lactoferrin must belocated at the cell surface to exert its anti-HSV activity and thatlactoferrin probably interferes with viral binding to heparan sulfate[55]. Modeling of the anti-HSV activity of lactoferricin analoguesis currently being performed, in order to design more efficient anti-HSV peptides [56].

More generally, small amphiphilic peptides that are able toform beta-sheet structures display some antiviral activity - either by

binding to the virus themselves or by binding to the cell surface and

Table 1. Potential Viral and Cellular Targets for the Development of Anti Herpes Drugs. The Potential Viral and Cellular Targets

and their Inhibitors Described in the Review are Presented

Potential targets Drugs

Viral Glycoproteins CarraguardTM

Emmelle/dextrin-2-sulfate

polystyrene sulfonate PSS, PRO2000/5TM

UshercellTM, Virend

Dendrimers

Ribonucleotide reductase Thiosemicarbazone family

Peptidomimetic inhibitors

Antisens oligomers

DNA polymerase 4-hydroxyquinoline-3-carboxamide (PNU-182171 and PNU-183792);

thieno[2,3-b]pyridine carboxamide

Peptidomimetic inhibitors

Small molecules i.e. BP5

Helicase-primase complex

(UL5, UL8 and UL52)

2-amino-thiazole (T157602) (helicase domain)

Aminothiazolyl-phenyl compounds (BILS 179 BS and BILS 45 BS)

Thiazolylsulfonamide compounds (BAY 57-1293)

siRNAs (UL5)

Cellular Cyclin-dependent kinase 2 Pharmacological cdk inhibitors (olomoucine, roscovitine)

Polyamine pathway Inhibitors of SAMDC

Inhibitors of ornithine decarboxylase

Entry receptors (HVEM, extracellular

proteoglycans)

Anti-HVEM antibodies; Apolipoprotein E-derived peptides

SAMDC: S-adenosyl methionine decarboxylase; HVEM: human virus entry mediator.


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therefore interfering with virus attachment [56, 57]. However, giventhat these compounds are not very specific and frequently produce ahigh degree of hemolysis, their potential use would be restricted totopical treatments in order to reduce viral spread and duration andto ease the symptomatology of cutaneous HSV disease.

Viral DNA Replication

Ribonucleotide Reductase

Herpes simplex viruses encode their own RR, which convertsribonucleoside diphosphates to the corresponding deoxyribo-nucleotides required for viral DNA synthesis. Herpesviridae RRsare highly conserved and are composed of two homodimericsubunits, RR1 (large) and RR2 (small), whose association isessential for enzymatic activity.

For the last 20 years or so, HSV RR has been considered as apotential target for antiviral chemotherapy. The first anti-RRproducts to be identified belonged to the thiosemicarbazone family.However, the latter inhibit cellular RR as well as viral RR.Thereafter, highly potent and specific peptidomimetic viral RRinhibitors that impair association of the two subunits have beendeveloped. Given that the C terminus of RR2 is critical for thisassociation, peptides corresponding to the sequence of the last fewamino acids of this subunit have been synthesized. These peptides

inhibit HSV RR activity in vitro by inducing disso-ciation of thetwo subunits [58-60] and suppress the replication of HSV-1, HSV-2and ACV-resistant HSV strains in cell culture. They also reducedthe severity of HSV-1-induced keratitis in a murine ocular model[61, 62]. Studies conducted with a viral strain selected in vitro forits resistance to one of these peptides demonstrate that mutationsare located within the C terminus of the RR2 gene; however, viralRR is not required for HSV replication in exponentially growingcells and so drugs that prevent RR function have poor antiviralactivity in this context [63, 64].

However, recent studies have also revealed that the large (RR1)subunit of HSV-2 RR contains a protein kinase function in additionto its well known RR function, and that HSV-2 RR1 is involved inimmediate early gene transcription and virus growth [65]. Indeed,antisense oligonucleotides complementary to HSV-2 RR1 inhibitHSV-2 RR1 expression and HSV-2 replication. In addition, the

protein kinase function of HSV-2 RR1 may contribute to HSV-2reactivation, and it has been reported that antisense oligomersinhibit HSV-2 reactivation from latently infected ganglia [66-68].Hence, the RR1 protein kinase domain, required for HSV-2replication and reactivation from the latent state in neurons,represents a potential target in the field of anti-HSV-2 drugdevelopment.

DNA Polymerase

The core of the viral DNA pol from HSV-1-infected cellsconsists of a heterodimer of the DNA pol catalytic subunit and theaccessory subunit, encoded by UL30 and UL42, respectively. HSVDNA pol is still an attractive target. ACV resistance has promptedattempts to develop anti-herpes drugs that inhibit DNA pol activity

by other mechanisms.

The combination of high-throughput screening of small

molecules that specifically inhibit herpes DNA polymerases andstructure-activity relationship studies resulted in the discovery of anovel class of non-nucleoside inhibitors, the 4-hydroxy-quinoline-3-carboxamide compounds. These molecules appear to be specificfor a broad range of herpes DNA pols, including those of HSV-1and HSV-2 [69, 70]. In fact, a derivative of 8-hydroxyquinoline wasshown to prevent HSV-1 infection as early as 1976 [71]. The PNU-182171 and PNU-183792 derivatives of 4-hydroxyquinoline-3-carboxamide and the 10c and 14 deriva-tives of a novel series ofthieno[2,3-b]pyridine carboxamides have anti-HSV activity in cellculture via inhibition of viral DNA pol [72, 73]. These compoundsare competitive inhibitors of deoxynucleoside triphosphate binding

to DNA pol. However, they maintain potent antiviral activityagainst ACV-resistant HSV strains, indicating that the mechanismsof action are different. Laboratory HSV mutants selected for theirresistance to PNU-182171 are resistant to hydroxyquinoline as wellas to thieno[2,3-b]pyridine derivatives. Sequence analysis of DNA

pol from these mutants revealed that a single amino acid change ofvaline to alanine at position 823 (V823A) within the conserveddomain III is responsible for the resistance. V823 is conserved in 6

of the 8 herpes DNA polymerases. Since domain III plays a criticalrole in the interaction between the incoming nucleotide and primertemplate DNA, these inhibitors could bind to the 823 region of theDNA pol and may disrupt the pol's interaction with the DNAtemplate strand, thus interfering with the binding or incorporationof the incoming dNTP [73]. The crystal structure of the HSV-1DNA pol catalytic subunit has been published very recently. In the

proposed model based on results of co-crystal-lization and soakingexperiments, the PNU-183792 non-nucleoside inhibitor competeswith incoming nucleotides and dislocates the template from theactive site [74]. PNU-183792 does not exhibit any cytotoxicity in arange of cell lines at relevant drug concentrations, and is orally bio-available. These carboxamide derivatives are active against a broadrange of herpes viruses and their DNA pols. Preclinical animalstudies are ongoing, particularly for human cytomegalovirusinfections [73].

The accessory subunit of the DNA pol complex is encoded bythe UL42 gene. It binds directly to DNA and increases the

processivity of polymerization by (i) increasing the affinity of theDNA pol catalytic UL30 subunit for the 3' terminus of the primertemplate and (ii) decreasing the probability that the DNA pol willdissociate from the template after each catalytic cycle. Specificinteraction between DNA pol and the UL42 accessory protein isnecessary for full DNA pol function and for viral replication [75-77]. Since the UL30-UL42 interaction is critical for viralreplication, its disruption represents a potential target in the designof novel antiviral agents.

Three peptides corresponding to the last 38, 27 and 18 C-terminal residues of HSV-1 DNA pol catalytic subunit have beensynthesized. In vitro, they inhibit the UL30-UL42 interaction andUL42-dependent long-chain DNA synthesis by HSV DNA pol but

not the activity of the cellular Pol

-PCNA [78-80]. When the 27-mer peptide of the HSV-1 DNA pol C-terminus is fused with theenterotoxin B peptide, the resulting molecule is targeted into thenucleus of HSV-1-infected cells and inhibits viral replication bydisruption of the UL30-UL42 interaction [79]. This UL30-UL42interaction has been extensively characterized by muta-tional andstructural analysis [81-83]. Biophysical studies have identifiedspecific hydrogen bonds that are crucial for the inter-action. In

particular, a hydrogen bond network connects the R1229 residue ofDNA pol to the Q171 residue of UL42, which in turn bonds to theF1211 residue of DNA pol [84]. Recently, small molecules thatspecifically interfere with the UL30-UL42 interaction in HSV-1have been selected using high-throughput screening. One of these,called BP5, inhibits the interaction in vitro. In addition, BP5 also

prevents HSV-1 infection in Vero cells as efficiently as ACV,although it is more toxic. The mechanism of action is still unknown.

However, results from a HSV mutant selected for its resistance toBP5 suggest that BP5 is virus-specific. More potent and less toxicmolecules are being developed [85].

Helicase-Primase

The helicaseprimase complex comprises three viral proteinsencoded by the UL5, UL8 and UL52 gene products. It unwindsdouble-stranded viral DNA and generates primers for DNAsynthesis by the viral DNA pol. UL8 is not required for enzy-maticactivities but may be involved in recruiting HSV-1 DNA pol intothe viral DNA replication complex [86]. Therefore, the helicase-

primase complex plays an essential role in HSV DNA replicationand thus constitutes an excellent target for antiviral therapy.


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Three new types of herpes inhibitors that target the HSVhelicase-primase complex have recently been discovered. Highthroughput screening of a molecule library with detection of theHSV1 UL5/UL8/UL52 complex's helicase activity led toidentification of T157602, a 2-amino-thiazole. Genetic analysisdemonstrated that T157602 targets the helicase domain of thecomplex. It also inhibits HSV replication in cell culture [87].Viruses selected for their resistance to T157602 presented a

mutated UL5 gene [88].Another high throughput screening program coupled with lead

optimization studies, led to aminothiazolyl-phenyl-containingcompounds that act by increasing the affinity between enzyme andDNA and prevent the propagation of HSV helicase-primasecatalytic cycles. Two of these, BILS 179 BS and BILS 45 BS, aremuch more active than ACV against wild-type laboratory andclinical HSV-1 strains and ACV-resistant HSV isolates. Theantiviral activity of BILS 179 BS is HSV-specific, since it does notinhibit replication of the other members of the herpes family. BILS45 BS displays very effective oral therapy of experimental ACV-resistant HSV-1 infections in nude mice [89, 90]. Single amino acidchanges in the UL5 protein have been identified in mutant virusesselected for their resistance to a BIL 45 BS analog [91].

Thiazolylsulfonamide compounds also target the viral primase-

helicase complex and inhibit its ATPase activity [92, 93]. Of these,BAY 57-1293 has potent anti-herpes activity in vitro and is activeagainst ACV-resistant mutant strains. BAY 57-1293 is more potentthan ACV in vitro and in vivo, and is especially effective whentreatment is delayed. It has a lower resistance rate than ACV invitro. In animal models BAY 57-1293 has superior efficacy againstHSV and also shows favorable pharmacokinetic parameters. Italmost completely suppresses recurrent disease and asymptomaticvirus shedding, and no detectable HSV replication takes place in theneuronal tissues of animals treated with BAY 57-1293.

A very different approach has demonstrated that mice areprotected from lethal infection after vaginal instillation of siRNAsdesigned to inhibit the HSV-2 UL5 gene expression, thusstrengthening the latter's status as an important viral gene [94].

Hence, the viral helicase-primase complex represents a verypromising target in the development of new anti-herpes drugs.

POTENTIAL CELLULAR TARGETS

Although the viral genome encodes many functions that arenecessary for viral propagation in host cells, cellular proteins arealso required. Much effort has been devoted to identifying the latterFig. (1), Table (1).

Within the last 10 years, attention has been focused on cellularprotein kinases as antiviral targets. Recent studies have shown thatreplication of HSV requires specific cyclin-dependent kinases(cdks). These Ser/Thr kinases regulate the cell division cycle,apoptosis, transcription, differentiation and numerous neuronalfunctions. They are active when they are associated with a cyclin oranother regulatory partner.

Upon HSV infection, the cdk2/cyclin E complex is activated.Many pharmacological cdk inhibitors (PCIs) are available since the

latter have been developed as anticancer agents. Some of these,such as olomoucine and roscovitine, have potent in vitro antiviralactivity against HSV-1 and HSV-2. Roscovitine belongs to a classof specific cdk inhibitors which inhibit cdk1, 2, 5 and 7. Theinhibition of HSV-1 infection by PCIs occurs by a variety ofmechanisms. The PCIs inhibit activation of immediate-early andearly gene expression and viral DNA synthesis. Roscovitineinterferes with phosphorylation of the viral proteins ICP4 and ICP0;it inhibits ICP0's transactivating activity but not the latter's ability todisperse or degrade the cellular proteins associated with ND10nuclear structures. Moreover, roscovitine also affects replication of

HSV-1 DNA by a mechanism which remains to be elucidated [95-100].

Interestingly, cdk2 is not expressed in quiescent neurons,whereas nuclear cdk2 is induced in neurons submitted to a stressfulstimulus that induces HSV reactivation. Since HSV-1 reactivationis inhibited in the presence of a cdk2-specific inhibitor, it has beenspeculated that cdk2 is one of the factors that determine theoutcome of HSV infection in neurons [101].

Co-crystallization studies indicate that all PCIs analyzed to datetarget the ATP-binding pocket of the kinase's catalytic site (for areview, see [11]). PCIs inhibit the replication of wild-type andmulti-drug resistant strains of HSV-1 and HSV-2. Despite repeatedattempts, PCI-resistant HSV-1 strains have never been isolated.This suggests that PCIs do not act by inhibiting a single viral target.Many PCIs are being developed as antivirals and are scheduled toenter clinical trials (for a review, see [12]).

A recent report has demonstrated that the polyamine bio-synthetic pathway remains active in HSV-1 infected cells and thatexpression of the two key enzymes of this pathway, S-adenosylmethionine decarboxylase (SAMDC) and ornithine decarboxylase,is upregulated [102, 103]. This argues for a role of polyamines inthe HSV-1 replication cycle. Indeed, HSV-1 particles contain twotypes of polyamine, spermidine in the envelope and spermine in the

nucleocapsid. Furthermore, polyamines are involved in thereplication of HSV-1 DNA [104, 105]. The use of methylglyoxal

bis(guanylhydrazone), a specific SAMDC inhibitor, reveals thatSAMDC activity is required for HSV-1 and HSV-2 replication incell culture. Furthermore, HSV-1 infection is severely repressedwhen cells are pretreated with methylglyoxal bis(guanylhydrazone)and when treatment is delayed until the viral adsorption or theimmediate-early and early steps of infection. Moreover, inhibitionof SAMDC activity prevents the in vitro replication of HSV-1 andHSV-2 laboratory strains and of clinical mutant isolates that areresistant to conventional antiviral drugs such as ACV and foscarnet[103]. Hence, SAMDC and other key enzymes in the polyamine

pathway represent potentially interesting anti-HSV targets.

A few reports indicate that molecules that target cellularreceptors or extracellular proteoglycans can, under some circum-stances, efficiently block viral penetration into a specific tissue or

organ. Infection of primary cultures of human T lymphocytes byHSV-1 can be inhibited by an HVEM-specific antiserum [106].Moreover, it has been demonstrated that an anti-HVEM antibodyinhibits the entry of HSV-1 into primary cultures of human oculartrabecular meshwork cells, suggesting that molecules inhibiting theinteractions between HVEM and viral glycopro-teins could be avalid strategy to prevent the eye infections, at least with HSV-1,whose complication contributes to the development of blindingdisease [107].

Recently, it was shown that the apoEdp fragment of theapolipoprotein E is able to inhibit viral infection via a mechanismthat has not yet been defined but that is partially mediated by directinhibition of viral attachment, perhaps with the blockage ofextracellular proteoglycan sites or via binding to low-densitylipoprotein receptor-related proteins [108].

Cellular topoisomerases are essential enzymes in the unwin-ding of the double-stranded DNA and have also been consideredvery recently as possible anti-herpes targets. Herpes virus infec-tions are known to require topoisomerases I and II; inhibitors oftopoisomerase II derived from substituted acridines are able toinhibit herpes infections, most probably by blocking topoiso-merase

binding to DNA [109, 110].

CONCLUSIONS

Despite intensive research, no prophylactic HSV vaccine hasproven to be effective. An alternative approach to the control ofHSV infections is to develop chemotherapeutic drugs with anti-


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HSV activity. The emergence of virus strains which are resistant tothe available anti-herpes drugs has accelerated the antiviral research

process. Until the last decade, viral functions have been the targetsof choice in the anti-herpes drug discovery field, essentially

because drugs directed against v iral targets were expected to havehigh selectivity and low toxicity and because almost all theessential viral proteins have been identified. The resultingmolecules have very specific antiviral activity, since they are

restricted to herpes infections. In fact, these drugs inhibit infectionof just one or a few members of the herpes family and rarely have

broad-spectrum activity. Moreover, the occurrence of resistance tothese new potential antiviral drugs, or at least those targeting thehelicase primase complex, appears to be less frequent than forACV.

More recently, a range of studies have highlighted theimportance of host cellular factors in regulating HSV infection.Some of these cellular proteins have been used as novel targets forantiviral therapeutics. Drugs targeting cellular functions areexpected to have different advantages, including lower selectivity(i.e. inhibition of a broader range of viruses) and the diminishedappearance of resistant viral strains. Indeed, cdk2 inhibitors preventinfection by HSV-1, HSV-2, other herpes viruses, drug-resistantviruses and even unrelated viruses that require active cdk2 forreplication [111-113]. One can speculate that inhibitors of cellular

proteins necessary for viral replication would be active against abroad range of viruses, and that targeting cellular proteins neededfor viral infection may also help to prevent or slow the emergenceof drug-resistant viral strains. One drawback of this strategy is thatmolecules acting on cellular proteins could display cytotoxiceffects. This point should be taken into account for the futuredevelopments of this type of antiviral molecules.

It is interesting to note that some drugs targeting HSV-2ribonucleotide reductase or the cellular cdk2 seem also to inhibitHSV-2 and HSV-1 reactivation, respectively.

Significant progress has been made in understanding thedynamics of viral infection and reactivation from latency. This willhelp in the identification of new, relevant drug targets and thereforein the development of new antiviral drugs with greater efficacy andsafety. Management of viral infections will be facilitated by using

unrelated target proteins to develop drugs with non-overlappingmechanisms of action.

In theory at least, every protein whether or cellular or viralorigin involved in each step of the virus cycle still represents a

potential target for the development of anti-herpes drug. How-ever,not enough data are yet available to clearly demonstrate that

proteins other than those described in this review have beenidentified as good potential targets.

The alternate use of drugs in development, the combination ofthese drugs with conventional ones or the combination of drugsdirected respectively against viral and cellular targets all constitute

promising strategies for reducing the appearance and disseminationof drug-resistant viruses and preventing reacti-vation from latency.

ACKNOWLEDGEMENTS

We are thankful to Dr Henri Agut for his comments andsuggestions about this review. A. Greco and J-J Diaz are membersof the Institut National de la Sant et de la Recherche Mdicale(INSERM).

ABBREVIATIONS

ACV = Acyclovir

cdk = Cyclin-dependent kinase

HSV = Herpes simplex virus

HSV-1 = Herpes simplex virus type 1

HSV-2 = Herpes simplex virus type 2

HVEM = Herpes virus entry mediator

PCI = Pharmacological cdk inhibitor

Pol = Polymerase

RR = Ribonucleotide reductase

SAMDC = S-adenosyl methionine decarboxylase

TK = Thymidine kinase

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Received: July 11, 2006 Accepted: October 13, 2006

Novel Targets for the Development of Anti-Herpes Compounds

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