Non-nucleosidic inhibition of Herpes simplex virus DNA polymerase: mechanistic insights into the anti-herpetic mode of action of herbal drug withaferin A
© Grover et al; licensee BioMed Central Ltd. 2011
Published: 30 November 2011
Herpes Simplex Virus 1 and 2 causes several infections in humans including cold sores and encephalitis. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that can inhibit viral polymerase and terminate the replicating viral DNA. However, these drugs bear an intrinsic non-specificity as they can also inhibit cellular polymerase apart from the viral one. The present study is an attempt to elucidate the action mechanism of naturally occurring withaferin A in inhibiting viral DNA polymerase, thus providing an evidence for its development as a novel anti-herpetic drug.
Withaferin A was found to bind very similarly to that of the previously reported 4-oxo-DHQ inhibitor. Withaferin A was observed binding to the residues Gln 617, Gln 618, Asn 815 and Tyr 818, all of which are crucial to the proper functioning of the polymerase. A comparison of the conformation obtained from docking and the molecular dynamics simulations shows that substantial changes in the binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured.
We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that withaferin A is a potential ligand to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus.
Herpes Simplex Virus type 1 and type 2 (HSV-1 and HSV-2) are two members of Herpesviridae family, which infect almost 85% of the world population . They are the causative agents of a gamut of diseases ranging from mild ones like cold sores in mouth, eye cornea and genitals to more severe life threatening ones like the fatal herpes encephalitis . People with suppressed immune system like those suffering from AIDS are more prone to get infections from HSV than others .
Drugs for HSV
There is no permanent cure for these infections till date. Present day treatment involves the use of antiviral drugs to reduce the physical severity of outbreak-associated lesions and viral shedding, though this helps decreasing the chances of transmission to others only by maximum 50% . There are two types of drugs that are clinically useful against HSV infections. The first category consists of nucleoside analogs like acyclovir and its prodrug valacyclovir, ganciclovir, penciclovir and its prodrug famciclovir, sorivudine and brivudine. These require phosphorylation by viral thymidine kinase to form triphosphates that are active inhibitors of viral DNA polymerase. The second category consists of direct viral DNA polymerase inhibitors like vidarbine, foscarnet and cidofovir . Thus, both types of drugs target in dysfunctioning the replication centre i.e. DNA polymerase of the viral genome .
Development of alternative treatments for HSV
However, in past few years, a number of acyclovir drug resistant viral strains have been isolated especially from immuno-compromised patients [7–9]. In this era, where the number of immuno-suppressed patients like those suffering from HIV is continuously increasing, there is an immediate need to find new drugs to treat HSV infections which have a higher efficacy or have an alternative mode of action . Resistance to acyclovir is mainly due to mutations in the viral thymidine kinase (TK) gene which impair the initial drug phosphorylation . These drug resistant strains have been of significant clinical attention [12, 13], indicating the need for alternative anti- HSV drugs. Previous antiviral studies on herpes viruses have focussed on developing nucleoside analogues that will inhibit viral polymerase and terminate the replicating viral DNA. A number of new anti-viral drugs against HSV DNA polymerase are currently under research and development; these focus other domains of the polymerase than those targeted by the commercially available drugs . One such novel class of compounds is that of 4-oxo-DHQs belonging to the non-nucleoside anti-herpetic drugs family .
Medicinal plants products have been used over centuries as traditional remedies for different kinds of diseases including viral diseases. Recently, there have been studies which report anti-viral activities of extracts from plants like Swertia chirata, Aloe forex and Withania somnifera against HSV [16, 17]. These plant extracts inhibit the formation of HSV-1 plaque above a certain minimum concentration and their activities can be compared to the commercial drugs like acyclovir.
Withania somnifera or Winter Cherry or Indian ginseng is a proud herb of Ayurveda, classified as Rasayan (the most esteemed of Ayurveda herbs) . It is held in high repute in traditional Indian medicine mainly because of its constituents called withanolides . They are built on an ergostane framework, which is oxidized at C-22 and C-26 to form a six-member lactone ring. Withaferin A (WA), the first withanolide to be isolated and the major withanolide present in Indian variety of plants has been widely researched for its pharamacological activities including anti-inflammatory, anti-cancer, anti-stress and immunomodulatory, adaptogenic, central nervous system, endocrine and cardiovascular activities [20–25]. Leaves of Withania somnifera have been reported to have the highest content of WA (around 0.001 to 0.5% dry weight of leaves) .
In this study, we report a possible mode of action of withaferin A against HSV by inhibition of its DNA polymerase. Molecular docking studies have been used to identify the binding modes. Dynamic structural patterns were studied using Molecular dynamics simulations.
Ligand and receptors
Semi-flexible molecular docking of the HSV DNA polymerase along with ligand WA was implemented using AutoDock 4.0 . The general procedure for performing docking is described elsewhere . The outputs from AutoDock were rendered with PyMOL  and Accelerys ViewerLite 5.0. Confirmation of the results were achieved using ParDOCK .
MD simulations in water
The energy minimization and MD simulations of HSV POL and its complex with WA were carried out using AMBER package as fully described elsewhere by the authors .
Results and discussion
Docking of withaferin A into HSV POL
Properties of the docked conformation
Total internal energy
MD simulations in water
To probe the dynamic flexibility changes in the protein, due to the inhibitor binding in its critical regions, B-factors (Bn) for the Cα atoms were calculated using the following relation:
Bn = 8/3 π rn2
Analysis of pre- and post-MD simulated structures
Comparison of different parameters of docking of withaferin A onto DNA POL in pre- and post- MD simulated structures
Non-nucleosidic inhibition of HSV POL by WA
It has been earlier reported that 4-oxo-dihydroquinolines (4-oxo-DHQs) have shown broad anti-HSV activity [35, 36]. This class of compounds inhibit most human herpes viruses, which is associated with DNA polymerase inhibition. From the binding assays of PNU-183792 , a radiolabelled DHQ on HSV POL, it was observed that this inhibitor binds only to HSV POL in complex with the DNA duplex; while no binding was observed either with HSV POL or with DNA duplex alone . However, in our case we observed that WA is able to bind to the segregated HSV POL itself. From visual inspection of the structures of WA and the 4-oxo-DHQ inhibitor, it was observed that both these ligands contain 2 ketonic groups, an oxygen containing heterocyclic ring and a tail containing an electronegative atom. In the present study, the binding mode of WA was found similar to that of the radiolabelled 4-oxo-DHQ in which the residues Gln 617, Gln 618, Asn 815 and Tyr 818 play critical role in the stabilization of the ligand. It can be deduced from our studies that WA can be a potent non-nucleosidic inhibitor of HSV POL whose binding would result in a conformational change in the polymerase that distorts the positioning of the residues that bind DNA, inhibiting polymerization. 4-oxo-DHQs have shown high specificity index in inhibiting DNA polymerases belonging to the herpesviridae family because unrelated DNA and RNA viruses were not susceptible to their inhibitory effect, and they also proved to have broad spectrum antiviral effects [35, 36]. The same can be expected for WA owing to its analogous mode of action. The inhibition constant of WA found in our study (0.6 µm) was also quite comparable to the currently established nucleosidic drugs (0.1-0.6 µm) .
The emergence of HSV resistance to antiviral drugs is also a major concern. Three basic mechanisms have been identified: altered thymidine kinase substrate specificity, absent or partial production of viral thymidine kinase and altered viral DNA polymerase . The most common mechanism found in clinical isolates is deficient TK activity. For the foremost approved drug- Acyclovir, resistant isolates of HSV have been observed in immuno-compromised individuals, especially AIDS patients . Since WA is observed exerting its inhibitory effect via interaction with a viral DNA polymerase site that is less important for the binding of deoxynucleoside triphosphates, it holds potential to exert its influence even on these resistant isolates. The non-nucleosidic mode of action of WA holds promise for prevention of infection, as it can selectively target only the viral enzymes. Moreover being a naturally occurring herbal drug candidate, WA will also be able to address the issue of safety and bioavailability.
Based on the results from WA-HSV POL complex, it appears that interactions with the residues Gln 617, Gln 618, Tyr 722, Asn 815 and Asp 888 of HSV POL are important for inhibitory activity of WA. A comparison between the conformation obtained from docking and that from molecular dynamics simulations show that substantial changes in binding conformations have occurred. These results indicate that the initial receptor-ligand interaction observed after docking can be limited due to the receptor rigid docking algorithm and that the conformations and interactions observed after simulation runs are more energetically favoured and should be better representations of derivative poses in receptor.
We have performed docking and molecular dynamics simulation studies to elucidate the binding mechanism of prospective herbal drug withaferin A onto the structure of DNA polymerase of Herpes simplex virus. Our docking simulations results give high binding affinity of the ligand to the receptor. Long de novo MD simulations for 10 ns performed allowed us to evaluate the dynamic behaviour of the system studied and corroborate the docking results, as well as identify key residues in the enzyme-inhibitor interactions. The present MD simulations support the hypothesis that WA is a prospective ligand that has potential to target/inhibit DNA polymerase of the Herpes simplex virus. Results of these studies will also guide the design of selective inhibitors of DNA POL with high specificity and potent activity in order to strengthen the therapeutic arsenal available today against the dangerous biological warfare agent represented by Herpes Simplex Virus.
Research in the laboratory of DS is supported by grants from Lady Tata Memorial Trust and Department of Biotechnology (DBT) Government of India. The authors would like to thankfully acknowledge the Supercomputing Facility for Bioinformatics and Computational Biology (SCFBio) at IIT Delhi for the use of its facilities.
This article has been published as part of BMC Bioinformatics Volume 12 Supplement 13, 2011: Tenth International Conference on Bioinformatics – First ISCB Asia Joint Conference 2011 (InCoB/ISCB-Asia 2011): Bioinformatics. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/12?issue=S13.
- Prevention and control of Herpes virus diseases In Clinical and laboratory diagnosis and chemotherapy. Volume 63. Bulletin of the WHO; 1985:182–185.Google Scholar
- Whitley RJ, Gnann JWJ: The Human Herpesviruses. In The Human Herpesviruses Edited by: Roizmann B, Whitley RJ, Lopez C. 1993, 69–105.Google Scholar
- Wild K, Bohner T, Folkers G, Schulz GE: The structures of thymidine kinase from Herpes simplex virus type 1 in complex with substrates and a substrate analogue. Protein Science 1997, 6: 2097–2106.PubMed CentralView ArticlePubMedGoogle Scholar
- Corey L, Wald A, Patel R, Sacks SL, Tyring SK, Warren T, Douglas JM, Paavonen J, Morrow RA, Beutner KR, et al.: Once-daily valacyclovir to reduce the risk of transmission of genital herpes. New England Journal of Medicine 2004, 350: 11–20. 10.1056/NEJMoa035144View ArticlePubMedGoogle Scholar
- Siakallis G, Spandidos DA, Sourvinos G: Herpesviridae and novel inhibitors. Antiviral Therapy 2009, 14: 1051–1064.View ArticlePubMedGoogle Scholar
- Coen DM, Schaffer PA: Antiherpesvirus drugs: a promising spectrum of new drugs and drug targets. Nat Rev Drug Discov 2003, 2: 278–288. 10.1038/nrd1065View ArticlePubMedGoogle Scholar
- Chatis PA, Miller CH, Schrager LE, Crumpacker CS: Successful treatment with foscarnet of an acyclovir-resistant mucocutaneous infection with Herpes-simplex virus in a patient with Acquired Immunodeficiency Syndrome. New England Journal of Medicine 1989, 320: 297–300. 10.1056/NEJM198902023200507View ArticlePubMedGoogle Scholar
- Erice A, Chou S, Biron KK, Stanat SC, Balfour HH, Jordan MC: Progressive disease due to ganciclovir-resistant cytomegalo-virus in immunocompromised patients. New England Journal of Medicine 1989, 320: 289–293. 10.1056/NEJM198902023200505View ArticlePubMedGoogle Scholar
- Erlich KS, Mills J, Chatis P, Mertz GJ, Busch DF, Follansbee SE, Grant RM, Crumpacker CS: Acyclovir-Resistant Herpes-Simplex Virus-Infections in Patients with the Acquired Immunodeficiency Syndrome. New England Journal of Medicine 1989, 320: 293–296. 10.1056/NEJM198902023200506View ArticlePubMedGoogle Scholar
- Field AK, Biron KK: The end of innocence revisited - resistance of Herpesviruses to antiviral drugs. Clinical Microbiology Reviews 1994, 7: 1–13.PubMed CentralPubMedGoogle Scholar
- Gilbert C, Bestman-Smith J, Boivin G: Resistance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms. Drug Resist Updat 2002, 5: 88–114. 10.1016/S1368-7646(02)00021-3View ArticlePubMedGoogle Scholar
- Hirsch MS, Schooley RT: Resistance to antiviral drugs: the end of innocence. N Engl J Med 1989, 320: 313–314. 10.1056/NEJM198902023200510View ArticlePubMedGoogle Scholar
- Kost RG, Hill EL, Tigges M, Straus SE: Brief report: recurrent acyclovir-resistant genital herpes in an immunocompetent patient. N Engl J Med 1993, 329: 1777–1782. 10.1056/NEJM199312093292405View ArticlePubMedGoogle Scholar
- Greco A, Diaz JJ, Thouvenot D, Morfin F: Novel targets for the development of anti-herpes compounds. Infect Disord Drug Targets 2007, 7: 11–18. 10.2174/187152607780090766View ArticlePubMedGoogle Scholar
- Liu S, Knafels JD, Chang JS, Waszak GA, Baldwin ET, Deibel MR Jr., Thomsen DR, Homa FL, Wells PA, Tory MC, et al.: Crystal structure of the herpes simplex virus 1 DNA polymerase. Journal of Biological Chemistry 2006, 281: 18193–18200. 10.1074/jbc.M602414200View ArticlePubMedGoogle Scholar
- Kambizi L, Goosen BM, Taylor MB, Afolayan AJ: Anti-viral effects of aqueous extracts of Aloe ferox and Withania somnifera on herpes simplex virus type 1 in cell culture. South African Journal of Science 2007, 103: 359–360.Google Scholar
- Verma H, Patil PR, Kolhapure RM, Gopalkrishna V: Antiviral activity of the Indian medicinal plant extract, Swertia chirata against herpes simplex viruses: A study by in-vitro and molecular approach. Indian Journal of Medical Microbiology 2008, 26: 322–326. 10.4103/0255-0857.43561View ArticlePubMedGoogle Scholar
- Widodo N, Takagi Y, Shrestha BG, Ishii T, Kaul SC, Wadhwa R: Selective killing of cancer cells by leaf extract of Ashwagandha: Components, activity and pathway analyses. Cancer Letters 2008, 262: 37–47. 10.1016/j.canlet.2007.11.037View ArticlePubMedGoogle Scholar
- Mishra L, Singh B, Dagenias S: Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Alternative Medicine Review 2000, 5: 334–336.PubMedGoogle Scholar
- Panda S, Kar A: Changes in thyroid hormone concentrations after administration of Ashwagandha root extract to adult male mice. Journal of Pharmacy and Pharmacology 1998, 50: 1065–1068. 10.1111/j.2042-7158.1998.tb06923.xView ArticlePubMedGoogle Scholar
- Budhiraja RD, Sudhir S: Review of Biological-Activity of Withanolides. Journal of Scientific & Industrial Research 1987, 46: 488–491.Google Scholar
- Kulkarni S, George B, Mathur R: Protective effect of Withania somnifera root extract on electrographic activity in a lithium pilocarpine model of status epilepticus. Phytotherapy Research 1998, 12: 451–453. 10.1002/(SICI)1099-1573(199809)12:6<451::AID-PTR328>3.0.CO;2-CView ArticleGoogle Scholar
- Bhattacharya A, Ghosal S, Bhattacharya SK: Anti-oxidant effect of Withania somnifera glycowithanolides in chronic footshock stress-induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum. Journal of Ethnopharmacology 2001, 74: 1–6. 10.1016/S0378-8741(00)00309-3View ArticlePubMedGoogle Scholar
- Bhattacharya SK, Muruganandam AV: Adaptogenic activity of Withania somnifera : an experimental study using a rat model of chronic stress. Pharmacology Biochemistry and Behavior 2003, 75: 547–555. 10.1016/S0091-3057(03)00110-2View ArticleGoogle Scholar
- Chaudhary G, Sharma U, Jagannathan NR, Gupta YK: Evaluation of Withania somnifera in a middle cerebral artery occlusion model of stroke in rats. Clinical and Experimental Pharmacology and Physiology 2003, 30: 399–404. 10.1046/j.1440-1681.2003.03849.xView ArticlePubMedGoogle Scholar
- Mirjalili MH, Moyano E, Bonfill M, Cusido RM, Palazon J: Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 2009, 14: 2373–2393. 10.3390/molecules14072373View ArticlePubMedGoogle Scholar
- Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE: The protein data bank. Nucleic Acids Research 2000, 28: 235–242. 10.1093/nar/28.1.235PubMed CentralView ArticlePubMedGoogle Scholar
- Grover A, Shandilya A, Agrawal V, Pratik P, Bhasme D, Bisaria VS, Sundar D: Hsp90/Cdc37 Chaperone/co-chaperone complex, a novel junction anticancer target elucidated by the mode of action of herbal drug Withaferin A. BMC Bioinformatics 2011, 12(Suppl 1):S30. 10.1186/1471-2105-12-S1-S30PubMed CentralView ArticlePubMedGoogle Scholar
- NCBI-PubChem Compound database[http://pubchem.ncbi.nlm.nih.gov/]
- Case DA DT, Cheatham TE, Simmerling CL, Wang J, Duke RE, Luo R, Walker RC, Zhang W, Merz KM, Roberts B, Wang B, Hayik S, Roitberg A, Seabra G, Kolossváry I, Wong IF, Paesani F, Vanicek J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Cai Q, Ye X, Wang J, Hsieh MJ, Cui G, Roe DR, Mathews DH, Seetin MG, Sagui C, Babin V, Luchko T, Gusarov S, Kovalenko A, Kollman PA: AMBER 11. San Francisco: University of California; 2010.Google Scholar
- Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ: Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. Journal of Computational Chemistry 1998, 19: 1639–1662. 10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-BView ArticleGoogle Scholar
- DeLano W: The PyMOL Molecular Graphics System 2002. San Carlos, CA: DeLano Scientific; 2002.Google Scholar
- Jain T, Jayaram B: An all atom energy based computational protocol for predicting binding affinities of protein-ligand complexes. Febs Letters 2005, 579: 6659–6666. 10.1016/j.febslet.2005.10.031View ArticlePubMedGoogle Scholar
- Wallace AC, Laskowski RA, Thornton JM: LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng 1995, 8: 127–134. 10.1093/protein/8.2.127View ArticlePubMedGoogle Scholar
- Brideau RJ, Knechtel ML, Huang A, Vaillancourt VA, Vera EE, Oien NL, Hopkins TA, Wieber JL, Wilkinson KF, Rush BD, et al.: Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses. Antiviral Res 2002, 54: 19–28. 10.1016/S0166-3542(01)00208-XView ArticlePubMedGoogle Scholar
- Oien NL, Brideau RJ, Hopkins TA, Wieber JL, Knechtel ML, Shelly JA, Anstadt RA, Wells PA, Poorman RA, Huang A, et al.: Broad-spectrum antiherpes activities of 4-hydroxyquinoline carboxamides, a novel class of herpesvirus polymerase inhibitors. Antimicrob Agents Chemother 2002, 46: 724–730. 10.1128/AAC.46.3.724-730.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Vajpayee M, Malhotra N: Antiviral drugs against herpes infection. Indian Journal of Pharmacology 2000, 32: 330–338.Google Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.