ORIGINAL RESEARCH

Drug discovery against H1N1 virus (influenza A virus)via computational virtual screening approach

Ashwani Sharma • Ashish V. Tendulkar •

Pramod P. Wangikar

Received: 15 January 2010 / Accepted: 26 May 2010 / Published online: 15 June 2010

� Springer Science+Business Media, LLC 2010

Abstract The H1N1 virus is the causative agent of the

recent outbreak of Swine flu pandemic. Neuraminidase is

an enzyme that cleave glycosidic linkage of neuraminic

acid on viral cell surface and is known to occur as antigen

determinant to evoke immune response in host cell. It plays

an important role in life cycle of influenza virus. Inhibitors

of neuraminidase are, therefore, believed to have a poten-

tial in development of new drugs against swine flu. Using a

recently published model structure of neuraminidase, we

have carried out virtual screening of 70 compounds

obtained from Ligand databases. The ligands library also

included 57 natural plant metabolites from medicinal

plants. The virtual screening was performed via PatchDock

& GemDock softwares. Two of the plant metabolites,

Hesperidin & Narirutin showed significantly higher dock-

ing score than the currently marketed anti-influenza drug

Oseltamivir (Tamiflu).

Keywords H1N1 influenza virus � Swine flu �Antiviral compound � Docking � Oseltamivir (Tamiflu) �Hesperidin � Narirutin

Introduction

H1N1 (Influenza A) is a strain of influenza virus which is

responsible for world wide swine flu pandemic. The swine

flu outbreak has affected a large number of people and has

been instrumental in death of thousands across the world.

Till date, the source species of the virus has not been

clearly identified (Butler, 2009; Cohen and Enserink,

2009; Schnitzler and Schnitzler, 2009; Petrosillo et al.,

2009). It is believed that the deadly virus is formed by

reassortment of genes from three different flu viruses,

originated from human (Uyeki, 2009), bird (Kou et al.,

2009; Thomson et al., 2009), and pig (Smith et al., 2009).

Several drugs have been reported for treating infections

from conventional influenza viruses with varying degree

of specificity for different strains (Manzoni et al., 2007).

Among these, Oseltamivir (Tamiflu) is the most versatile

anti-influenza drug and is found to be effective against a

broad range of influenza strains including H1N1 (Schirmer

and Holodniy, 2009). Since the H1N1 virus is known to

develop rapid mutations in order to provide improved

resistance against several antiviral drugs (Baum, 2009;

Collins et al., 2009), we need to discover new anti-viral

compounds against it.

Here, we propose a computational virtual screening based

strategy for finding effective anti-viral compounds against

H1N1 virus. Recently, the genome of H1N1 is sequenced

(http://www.ncbi.nlm.nih.gov/genomes/FLU/SwineFlu.html)

and subsequently the three dimensional structure of viral

neuraminidase was obtained by homology modeling (Ooi

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00044-010-9375-5) contains supplementarymaterial, which is available to authorized users.

A. Sharma

Department of Bioscience and Bioengineering, Indian Institute

of Technology Bombay, Powai, Mumbai 400 076, India

e-mail: ashwani_iitb@iitb.ac.in

A. V. Tendulkar

Department of Computer Science and Engineering, Indian

Institute of Technology Madras, Chennai 600 036, India

P. P. Wangikar (&)

Department of Chemical Engineering, Indian Institute of

Technology Bombay, Powai, Mumbai 400 076, India

e-mail: pramodw@iitb.ac.in

123

Med Chem Res (2011) 20:1445–1449

DOI 10.1007/s00044-010-9375-5

MEDICINALCHEMISTRYRESEARCH

et al., 2009; Maurer-Stroh et al., 2009). The neuraminidase

enzyme is present on the viral surface (Dalakouras et al., 2006)

and performs a key role in attaching and detaching the newly

formed influenza viruses from the host cell. In addition, it

catalyzes cleavage of glycosidic linkages of neuraminic acids

hydrolysis of sialic acid in order to facilitate the mobility of

newly formed virions through respiratory tract mucus from the

infected host cell and hence also known as sialidase (Taube

et al., 2009). It is also used as antigenic determinant and is

responsible for initializing immune response. Due to its

importance in the virus life cycle, we plan to target neur-

aminidase for discovering new drugs against the H1N1 virus.

Material and method

The input to our scheme is three dimensional structure of

H1N1 neuraminidase and traditional drug compound

(Osetamivir) against the virus. The overall scheme is

depicted in Fig. 1. It has the following steps: (i) Obtain a

list of chemical compounds for virtual screening, (ii) Vir-

tual screening of compounds against the virus neuramini-

dase structure. Since the actual three dimensional structure

of viral neuraminidase is not available, we took the struc-

ture obtained from homology modeling (Maurer-Stroh

et al., 2009) as an input.

In order to perform virtual screening of chemical com-

pound on the three dimensional neuraminidase, we need to

first obtain a number of chemical compounds similar to

traditional drug molecule. We obtained such compounds

from ligand databases such as PubChem (Total 61), Drug

Bank (Total 8), and ZINC (Total 1) based on the similarity

with Oseltamivir and reported antiviral compounds from

literature (Supplementary Table 1). We searched Keyword

‘‘Oseltamivir’’ in PubChem database and obtained 4 iso-

mers of Oseltamivir compound. We selected some known

neuraminidase inhibitors and antiviral compounds from

Drug Bank and obtained their SMILE formats. Natural

antiviral plant metabolites were also obtained from litera-

ture study and their SMILE formats were downloaded from

Oseltamivir

Pubchem

chemDBZINC Plant metabolite

Drug bank

Inhibitor compounds

Docking

Docking score Docking pose Ligand interaction

Ranking

Best compound

1

6

5

4

3

2

H1N1 Neuraminidase

Similarity search& Literature

Residues at 6A0 radius 7

8

Fig. 1 Overall schematic of methodology. The method was divided

into following steps: (1) Oseltamivir has been taken as model anti-

influenza compound against neuraminidase of H1N1 virus and search

for similar compounds and reported antiviral compounds from ligands

databases, (2) Downloaded matched compounds from ligands

databases, (3) Screened the compounds against neuraminidase of

H1N1 virus (note that whole neuraminidase protein structure taken as

docking target), (4) Analyzed the results based on docking score,

docking pose and ligand interaction, (5) Ranking of compounds, (6)

Select the best compound, (7) Analysis the best hit compound for

binding at cavity of neuraminidase, (8) Prediction of function sites in

neuraminidase by determining the residues content at 6 A radius of

ligand (as center)

1446 Med Chem Res (2011) 20:1445–1449

123

Hesperidin

Patchdock score 6080

Hesperidin

Neuraminidase

R368

R118

R278

W179

R228

R152

Narirutin

Patchdock score 5832

Neuraminidase

R368

R152

W179

Narirutin

Oseltamivir

Patchdock score 4240

Neuraminidase

S247

D151

Oseltamivir

(a)

(b)

(c)

Fig. 2 Binding of (a)

Hesperidin, (b) Narirutin and (c)

Oseltamivir (Tamiflu) at cavity

of H1N1 neuraminidase protein.

The H-bonding (dash line)

residues are also shown

Med Chem Res (2011) 20:1445–1449 1447

123

PubChem Database. The three dimensional structures of

these compounds were obtained in PDB format using

CORINA server (http://www.molecular-networks.com/

online_demos/corina_demo).

The virtual screening of the compounds is performed

using PatchDock (Schneidman-Duhovny et al., 2005) and

GemDock (Yang and Chen, 2004) softwares. The whole

protein structure is taken as a target for docking the com-

pounds. Note that we did not select any particular active

site for docking analysis.

PatchDock employs geometry based approach that

combines geometric hashing with pose-clustering matching

(Schneidman-Duhovny et al., 2005). We subjected all the

70 compounds for docking against neuraminidase protein.

Here, the clustering RMSD was 4.0 and other parameters

were set to be default. After docking via Patchdock, the

compounds are ranked based on the geometric matching

score with protein and selected the top rank compound. On

the other hand, GemDock is a generic evolutionary method

for molecular docking. We used its drug screening platform

with population size of 200, number of generation 70, and

the number of solutions set to 3. Other parameters were set

to their default values. The compounds are screened against

neuraminidase in automated mood and ranked based on the

minimum interaction energy (Fitness value) with the pro-

tein. In addition the docking results were analyzed for

binding of the putative drug within the cavity of neur-

aminidase protein of H1N1 virus and prediction of putative

functional site residues surrounding the bound antiviral

compound at 6 A of radius as shown in Fig. 1.

Results and discussion

We constructed a library of 70 chemical and natural anti-

viral compounds. It included 57 potent natural anti-viral

plant metabolites. The compounds are listed in Supple-

mentary Table 1. PatchDock produced the highest docking

score for two plant metabolites namely Hesperidin (score:

6080) (Fig. 2a) and Narirutin (score: 5832) (Fig. 2b), while

the traditional anti-influenza drugs Oseltamivir (Tamiflu)

(score: 4240) (Fig. 2c) was ranked lower in our experi-

ments (Table 1, Fig. 3). In first run of GemDock program,

the Hesperidin (Fitness value -125.753) and Narirutin

(Fitness value -124.676) produced highest rank as com-

pared to Oseltamivir (Fitness value -71.224) among 70

compounds(Table 1, Fig. 3). Then, we selected top 10

compounds with fitness value cutoff [-100 and further

passed for second cycle of Drug screening through Gem-

dock. The Hesperidin (Fitness value -118.217) and Nari-

rutin (Fitness value -109.945) again achieved highest rank

in top 10 compounds with comparatively minimum energy

to other compounds (Supplementary Table 1). Thus, the

docking experiment predicted that Hesperidin and Nariru-

tin as more potent drugs against the H1N1 neuraminidase

enzyme. Both the plant metabolites were reported to be

found in medicinal plant Citrus junos v. Tanaka, Rutaceae

Table 1 Docking results of the top ranked compounds against

neuraminidase enzyme: (A) antiviral natural plant metabolites and (B)

known antiviral chemical compounds docking score with neuramin-

idase enzyme

Compound Patchdock

score

Gemdock fitness

value

(A) Plant metabolites

Hesperidin 6080 -125.752713

Narirutin 5832 -124.67651

Proanthocyanidin 5616 -111.15093

Ursolic 5402 -85.723006

Sitosterol 5096 -84.820896

Tangeretrin 5056 -88.479023

Abbyssinone 4970 -91.365788

Nobiletin 4956 -88.869808

Tannic_acid 4868 -97.647901

(B) Known antiviral chemical compounds

Amprenavir 5454 -104.339221

Peramivir 4410 -95.535586

Oseltamivir 4240 -71.223735

Abacavir 4130 -100.356469

Zanamivir 4026 -100.356469

Adefovir 3806 -108.036416

Cidofovir 3650 -89.747602

Carbovir 3306 -83.838397

Acyclovir 3158 -83.323266

PatchDock Score2000 3000 4000 5000 6000

Nu

mb

er o

f C

om

po

un

ds

0

2

4

6

8

10

12

14

16

18

20

Hes

per

idin

Oseltamivir

Nar

iru

tin

Oseltamivir 4240Hesperidin 6080Narirutin 5832

Fig. 3 The graph represents distribution of 70 antiviral compounds

based on their geometric affinity with modeled structure of neur-

aminidase protein. The plant metabolites Hesperidin (score: 6080)

and Narirutin (score: 5832) produced highest patchdock score as

compare to traditional anti-influenza drugs Oseltamivir (Tamiflu)

(score: 4240)

1448 Med Chem Res (2011) 20:1445–1449

123

(Wang et al., 2006). These plant metabolites are biofl-

avonoid compounds found in citrus fruits. Hesperidin is

found to inhibit the growth of Influenza A virus during the

experiments performed by Saha et al. (2009) and hence

confirms our finding. Hesperidin is also reported to boost

the immune system against the viral infections.

Further analysis reveals that the compounds were bound

in the cavity of the enzyme containing the following resi-

dues at 6 A (bound ligand as center): ARG118, GLU119,

LEU134, ILE149, ASP151, ARG152,TRP179, SER180,

ALA181, ASP199, ASN222, ILE223, ARG225, THR226,

GLU228, GLY245, PRO246, SER247, GLU277, GLU278,

ARG368,GLY401, TYR402, ARG430, PRO431, LYS432,

and THR438 (Fig. 2). The residues ARG118, ARG152,

TRP179, GLU228, GLU278, and ARG368 are involved in

making hydrogen bonds with Herperidin. On the other

hand, ARG152, TRP179, and ARG368 make H bond

interactions with Narirutin. Oseltamivir was also found to

bind at the same cavity with the following additional res-

idues: LYS150, ARG156, ASN248, and HIS275 and

shared hydrogen bond interactions with ASP151 and

SER247 (Fig. 2).

Conclusions

We have reported a couple of potential drug compounds

against H1N1 neuraminidase enzymes. These compounds

were predicted to be more potent than the existing drugs

such as Oseltamivir. Moreover, these compounds are based

on plant metabolites and are not expected to have unde-

sirable side effects as the traditional drugs. We further

believe that prediction of functional site will enable the

biologists to understand the specific functional mechanism

of H1N1 neuraminidase enzyme.

Acknowledgments Ashwani Sharma is grateful to Council of Sci-

entific and Industrial Research (CSIR) for his research fellowship.

Ashish V. Tendulkar gratefully acknowledges support from Depart-

ment of Biotechnology Govt. of India through Innovative Young

Biotechnologist Award 2008.

References

Baum SG (2009) Oseltamivir resistance: what does it mean

clinically? Clin Infect Dis 49:1836–1837

Butler D (2009) Swine flu goes global. Nature 458:1082–1083

Cohen J, Enserink M (2009) As swine flu circles globe, scientists

grapple with basic questions. Science 324:572–573

Collins PJ, Haire LF, Lin YP, Liu J, Russell RJ, Walker PA, Martin

SR, Daniels RS, Gregory V, Skehel JJ, Gamblin SJ, Hay AJ

(2009) Structural basis for oseltamivir resistance of influenza

viruses. Vaccine 27:6317–6323

Dalakouras T, Smith BJ, Platis D, Cox MMJ, Labrou NE (2006)

Development of recombinant protein-based influenza vaccine.

Expression and affinity purification of H1N1 influenza virus

neuraminidase. J Chromatogr A 1136:48–56

Kou Z, Li Y, Yin Z, Guo S, Wang M, Gao X, Li P, Tang L, Jiang P,

Luo Z, Xin Z, Ding C, He Y, Ren Z, Cui P, Zhao H, Zhang Z,

Tang S, Yan B, Lei F, Li T (2009) The survey of H5N1 flu virus

in wild birds in 14 provinces of China from 2004 to 2007. PLoS

ONE 4(9):1–8

Manzoni M, Colombi P, Papini N, Rubaga L, Tiso N, Preti A,

Venerando B, Tettamanti G, Bresciani R, Argenton F, Borsani

G, Monti E (2007) Molecular cloning and biochemical charac-

terization of sialidases from zebrafish (Danio rerio). Biochem J

408:395–406

Maurer-Stroh S, Ma J, Lee RTC, Sirota FL, Eisenhaber F (2009)

Mapping the sequence mutations of the 2009 H1N1 influenza A

virus neuraminidase relative to drug and antibody binding sites.

Biol Direct 4:1–9

Ooi HS, Kwo CY, Wildpaner M, Sirota FL, Eisenhaber B, Maurer-

Stroh S, Wong WC, Schleiffer A, Eisenhaber F, Schneider G

(2009) ANNIE: integrated de novo protein sequence annotation.

Nucl Acids Res 37:W435–W440

Petrosillo N, Di Bella S, Drapeau C, Grilli E (2009) The novel

influenza A (H1N1) virus pandemic: an update. Ann Thorac Med

4:163–172

Saha RK, Takahashi T, Suzuki T (2009) Glucosyl hesperidin prevents

influenza A virus replication in vitro by inhibition of viral

sialidase. Biol Pharm Bull 32:1188–1192

Schirmer P, Holodniy M (2009) Oseltamivir for treatment and prophy-

laxis of influenza infection. Expert Opin Drug Saf 8:357–371

Schneidman-Duhovny D, Inbar Y, Nussinov R, Wolfson HJ (2005)

PatchDock and SymmDock: servers for rigid and symmetric

docking. Nucl Acids Res 33:W363–W367

Schnitzler SU, Schnitzler P (2009) An update on swine-origin

influenza virus A/H1N1: a review. Virus Genes 39:279–292

Smith GJD, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus

OG, Ma SK, Cheung CL, Raghwani J, Bhatt S, Peiris JSM, Guan

Y, Rambaut A (2009) Origins and evolutionary genomics of the

2009 swine-origin H1N1 influenza A epidemic. Nature

459:1122–1125

Taube S, Perry JW, Yetming K, Patel SP, Auble H, Shu L, Nawar HF,

Chang HL, Connell TD, Shayman JA, Wobus CE (2009)

Ganglioside-linked terminal sialic acid moieties on murine

macrophages function as attachment receptors for murine

noroviruses. J Virol 83:4092–4101

Thomson RJ, Haselhorst T, Dyason JC, Von Itzstein M (2009)

Cracking the code for H5N1-bird flu and beyond. Curr Drug

Deliv 6:343–346

Uyeki TM (2009) Human infection with highly pathogenic avian

influenza A (H5N1) virus: review of clinical issues. Clin Infect

Dis 49:279–290

Wang X, Jia W, Zhao A, Wang X (2006) Anti-influenza agents from

plants and traditional Chinese medicine. Phytother Res 20:335–341

Yang JM, Chen CC (2004) GEMDOCK: a generic evolutionary

method for molecular docking. Proteins Struct Funct Genet

55:288–304

Med Chem Res (2011) 20:1445–1449 1449

123