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Kumari S, Chakraborty S, Ahmad M, Kumar V, Tailor PB, Biswal BK. Identification of probable inhibitors for the DNA polymerase of the Monkeypox virus through the virtual screening approach. Int J Biol Macromol 2023; 229:515-528. [PMID: 36584781 PMCID: PMC9794403 DOI: 10.1016/j.ijbiomac.2022.12.252] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2022] [Revised: 12/09/2022] [Accepted: 12/22/2022] [Indexed: 12/29/2022]
Abstract
Given the paucity of antiviral treatments for monkeypox disease, caused by the Monkeypox virus (MPXV), there is a pressing need for the development/identification of new drugs to treat the infection. MPXV possesses a linear dsDNA genome that is replicated by a DNA replication complex of which DNA polymerase (DPol) forms an important component. Owing to the importance of DPol in the viral life cycle, identifying/designing small molecules abolishing its function could yield new antivirals. In this study, we first used the AlphaFold artificial intelligence program to model the 3D structure of the MPXV DPol; like the fold of DPol from other organisms, the MPXV DPol structure has the characteristic exonuclease, thumb, palm, and fingers sub-domains arrangement. Subsequently, we have identified several inhibitors through virtual screening of ZINC and antiviral libraries. Molecules with phenyl scaffold along with alanine-based and tetrazole-based molecules showed the best docking score of -8 to -10 kcal/mol. These molecules bind in the palm and fingers sub-domains interface region, which partially overlaps with the DNA binding path. The delineation of DPol/inhibitor interactions showed that majorly active site residues ASP549, ASP753, TYR550, ASN551, SER552, and ASN665 interact with the inhibitors. These compounds exhibit good Absorption, Distribution, Metabolism and Excretion properties.
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Affiliation(s)
- Swati Kumari
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
| | - Sayan Chakraborty
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
| | - Mohammed Ahmad
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
| | - Varun Kumar
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
| | | | - Bichitra K Biswal
- National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
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2
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Nasr T, Aboshanab AM, Mpekoulis G, Drakopoulos A, Vassilaki N, Zoidis G, Abouzid KAM, Zaghary W. Novel 6-Aminoquinazolinone Derivatives as Potential Cross GT1-4 HCV NS5B Inhibitors. Viruses 2022; 14:v14122767. [PMID: 36560772 PMCID: PMC9782603 DOI: 10.3390/v14122767] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2022] [Revised: 12/04/2022] [Accepted: 12/07/2022] [Indexed: 12/15/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infections are a worldwide medical problem responsible for diverse types of liver diseases. The NS5B polymerase enzyme has become a very interesting target for the development of anti-HCV drugs owing to its fundamental role in viral replication. Here we report the synthesis of a novel series of 1-substituted phenyl-4(1H)-quinazolinone and 2-methyl-1-substituted phenyl-4(1H)-quinazolinone derivatives and evaluate their activity against HCV in HCV subgenomic replicon assays. The biological data revealed that compound 11a showed the highest activity against HCV GT1b at a micromolar concentration (EC50 = 0.984 µM) followed by compound 11b (EC50 = 1.38 µM). Both compounds 11a and 11b had high selectivity indices (SI = CC50/EC50), 160.71 and 71.75, respectively, which make them very interesting candidates for further development of more potent and selective anti-HCV agents.
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Affiliation(s)
- Tamer Nasr
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Ain-Helwan, Cairo 11795, Egypt
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, MTI University, Cairo 12055, Egypt
- Correspondence: (T.N.); (G.Z.)
| | - Ahmed M. Aboshanab
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Ain-Helwan, Cairo 11795, Egypt
| | - George Mpekoulis
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 11521 Athens, Greece
| | - Antonios Drakopoulos
- Department of Chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden
| | - Niki Vassilaki
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 11521 Athens, Greece
| | - Grigoris Zoidis
- Department of Pharmacy, Division of Pharmaceutical Chemistry, School of Health Sciences, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, 15771 Athens, Greece
- Correspondence: (T.N.); (G.Z.)
| | - Khaled A. M. Abouzid
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt
| | - Wafaa Zaghary
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, Ain-Helwan, Cairo 11795, Egypt
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3
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Zenchenko AA, Drenichev MS, Il’icheva IA, Mikhailov SN. Antiviral and Antimicrobial Nucleoside Derivatives: Structural Features and Mechanisms of Action. Mol Biol 2021; 55:786-812. [PMID: 34955556 PMCID: PMC8682041 DOI: 10.1134/s0026893321040105] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Revised: 04/03/2021] [Accepted: 04/09/2021] [Indexed: 11/23/2022]
Abstract
The emergence of new viruses and resistant strains of pathogenic microorganisms has become a powerful stimulus in the search for new drugs. Nucleosides are a promising class of natural compounds, and more than a hundred drugs have already been created based on them, including antiviral, antibacterial and antitumor agents. The review considers the structural and functional features and mechanisms of action of known nucleoside analogs with antiviral, antibacterial or antiprotozoal activity. Particular attention is paid to the mechanisms that determine the antiviral effect of nucleoside analogs containing hydrophobic fragments. Depending on the structure and position of the hydrophobic substituent, such nucleosides can either block the process of penetration of viruses into cells or inhibit the stage of genome replication. The mechanisms of inhibition of viral enzymes by compounds of nucleoside and non-nucleoside nature have been compared. The stages of creation of antiparasitic drugs, which are based on the peculiarities of metabolic transformations of nucleosides in humans body and parasites, have been considered. A new approach to the creation of drugs is described, based on the use of prodrugs of modified nucleosides, which, as a result of metabolic processes, are converted into an effective drug directly in the target organ or tissue. This strategy makes it possible to reduce the general toxicity of the drug to humans and to increase the effectiveness of its action on cells infected by the virus.
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Affiliation(s)
- A. A. Zenchenko
- Engelhardt Institute of Molecular Biology, 119991 Moscow, Russia
| | - M. S. Drenichev
- Engelhardt Institute of Molecular Biology, 119991 Moscow, Russia
| | - I. A. Il’icheva
- Engelhardt Institute of Molecular Biology, 119991 Moscow, Russia
| | - S. N. Mikhailov
- Engelhardt Institute of Molecular Biology, 119991 Moscow, Russia
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4
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Ahmad M, Dwivedy A, Mariadasse R, Tiwari S, Kar D, Jeyakanthan J, Biswal BK. Prediction of Small Molecule Inhibitors Targeting the Severe Acute Respiratory Syndrome Coronavirus-2 RNA-dependent RNA Polymerase. ACS OMEGA 2020; 5:18356-18366. [PMID: 32743211 PMCID: PMC7391942 DOI: 10.1021/acsomega.0c02096] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/06/2020] [Accepted: 06/30/2020] [Indexed: 05/08/2023]
Abstract
The current COVID-19 outbreak warrants the design and development of novel anti-COVID therapeutics. Using a combination of bioinformatics and computational tools, we modelled the 3D structure of the RdRp (RNA-dependent RNA polymerase) of SARS-CoV2 (severe acute respiratory syndrome coronavirus-2) and predicted its probable GTP binding pocket in the active site. GTP is crucial for the formation of the initiation complex during RNA replication. This site was computationally targeted using a number of small molecule inhibitors of the hepatitis C RNA polymerase reported previously. Further optimizations suggested a lead molecule that may prove fruitful in the development of potent inhibitors against the RdRp of SARS-CoV2.
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Affiliation(s)
- Mohammed Ahmad
- National
Institute of Immunology, New Delhi 110067, India
| | | | - Richard Mariadasse
- Department
of Bioinformatics, Alagappa University, karaikudi 630004, Tamil Nadu, India
| | - Satish Tiwari
- National
Institute of Immunology, New Delhi 110067, India
| | - Deepsikha Kar
- National
Institute of Immunology, New Delhi 110067, India
| | - Jeyaraman Jeyakanthan
- Department
of Bioinformatics, Alagappa University, karaikudi 630004, Tamil Nadu, India
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Mittal L, Kumari A, Suri C, Bhattacharya S, Asthana S. Insights into structural dynamics of allosteric binding sites in HCV RNA-dependent RNA polymerase. J Biomol Struct Dyn 2019; 38:1612-1625. [PMID: 31057089 DOI: 10.1080/07391102.2019.1614480] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Inhibition of the viral RNA-dependent RNA polymerase (RdRp) to resolve chronic infection is a useful therapeutic strategy against Hepatitis C virus (HCV). Non-nucleoside inhibitors (NNIs) of RdRp are small molecules that bind tightly with allosteric sites on the enzyme, thereby inhibiting polymerase activity. A large number of crystal structures (176) were studied to establish the structure-activity relationship along with the mechanism of inhibition and resistance between HCV RdRp and NNIs at different allosteric sites. The structure and the associated dynamics are the blueprint to understand the function of the protein. We have implemented the ligand-based pharmacophore and molecular dynamic simulations to extract the possible local and global characteristics of RdRp upon NNI binding and the structural-dynamical features possessed by the known actives. Our results suggest that the NNI binding induces significant fluctuations at the atomic level which are critical for enzymatic activity, with minimal global structural alterations. Residue-wise mapping of interactions of NNIs at different sites exhibited some conserved interaction patterns of key amino acids and water molecules. Here, the structural insights are explored to understand the correlation between the dynamics of protein's subdomains and function at the molecular level, useful for genotype-specific rational designing of NNIs.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Lovika Mittal
- Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Faridabad, India
| | - Anita Kumari
- Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Faridabad, India
| | - Charu Suri
- Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Faridabad, India
| | - Sankar Bhattacharya
- Vaccine and Infectious Disease Research Center (VIDRC), Translational Health Science and Technology Institute (THSTI), Faridabad, India
| | - Shailendra Asthana
- Drug Discovery Research Center (DDRC), Translational Health Science and Technology Institute (THSTI), Faridabad, India
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6
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Polamreddy P, Vishwakarma V, Arumugam P, Bheemanati R, Esram P, Mahto MK, Kacker P. Discovery of hit molecules targeting allosteric site of hepatitis C virus NS5B polymerase. J Biomol Struct Dyn 2019; 38:1448-1466. [PMID: 31007134 DOI: 10.1080/07391102.2019.1608864] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Nonstructural protein 5B (NS5B), the RNA-dependent RNA polymerase of Hepatitis C Virus (HCV), plays a key role in viral amplification and is an attractive and most explored target for discovery of new therapeutic agents for Hepatitis C. Though safe and effective, NS5B inhibitors were launched in 2013 (Sovaldi) and 2014 (Harvoni, Viekira Pak), the high price tags of these medications limit their use among poor people in developing countries. Hence, still there exists a need for cost-effective and short duration anti-HCV agents especially those targeting niche patient population who were non-respondent to earlier therapies or with comorbid conditions. The present study describes the discovery of novel non-nucleoside (NNI) inhibitors of NS5B using a series of rational drug design techniques such as virtual screening, scaffold matching and molecular docking. 2D and 3D structure based virtual screening technique identified 300 hit compounds. Top 20 hits were screened out from identified hits using molecular docking technique. Four molecules, that are representative of 20 hits were evaluated for binding affinity under in vitro conditions using surface plasmon resonance-based assay and the results emphasized that compound with CoCoCo ID: 412075 could exhibit good binding response toward NS5B and could be a potential candidate as NS5B inhibitor.Communicated by Ramaswamy H. Sarma.
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Affiliation(s)
- Prasanthi Polamreddy
- Centre for Nanoscience and Nanotechnology, Sathyabama Institute of Science and Technology, Chennai, India.,Excelra Knowledge Solutions Pvt Ltd, Hyderabad, India
| | - Vinita Vishwakarma
- Centre for Nanoscience and Nanotechnology, Sathyabama Institute of Science and Technology, Chennai, India
| | | | | | | | | | - Puneet Kacker
- Excelra Knowledge Solutions Pvt Ltd, Hyderabad, India
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7
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Bessa LM, Launay H, Dujardin M, Cantrelle FX, Lippens G, Landrieu I, Schneider R, Hanoulle X. NMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5B. J Biol Chem 2017; 292:18024-18043. [PMID: 28912275 DOI: 10.1074/jbc.m117.813766] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2017] [Revised: 09/06/2017] [Indexed: 12/13/2022] Open
Abstract
Non-structural protein 5B (NS5B) is the RNA-dependent RNA polymerase that catalyzes replication of the hepatitis C virus (HCV) RNA genome and therefore is central for its life cycle. NS5B interacts with the intrinsically disordered domain 2 of NS5A (NS5A-D2), another essential multifunctional HCV protein that is required for RNA replication. As a result, these two proteins represent important targets for anti-HCV chemotherapies. Despite this importance and the existence of NS5B crystal structures, our understanding of the conformational and dynamic behavior of NS5B in solution and its relationship with NS5A-D2 remains incomplete. To address these points, we report the first detailed NMR spectroscopic study of HCV NS5B lacking its membrane anchor (NS5BΔ21). Analysis of constructs with selective isotope labeling of the δ1 methyl groups of isoleucine side chains demonstrates that, in solution, NS5BΔ21 is highly dynamic but predominantly adopts a closed conformation. The addition of NS5A-D2 leads to spectral changes indicative of binding to both allosteric thumb sites I and II of NS5BΔ21 and induces long-range perturbations that affect the RNA-binding properties of the polymerase. We compared these modifications with the short- and long-range effects triggered in NS5BΔ21 upon binding of filibuvir, an allosteric inhibitor. We demonstrate that filibuvir-bound NS5BΔ21 is strongly impaired in the binding of both NS5A-D2 and RNA. NS5A-D2 induces conformational and functional perturbations in NS5B similar to those triggered by filibuvir. Thus, our work highlights NS5A-D2 as an allosteric regulator of the HCV polymerase and provides new insight into the dynamics of NS5B in solution.
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Affiliation(s)
- Luiza M Bessa
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Hélène Launay
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Marie Dujardin
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - François-Xavier Cantrelle
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Guy Lippens
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Isabelle Landrieu
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Robert Schneider
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
| | - Xavier Hanoulle
- From the University of Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000 Lille, France
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8
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Ismail NSM, Elzahabi HSA, Sabry P, Baselious FN, AbdelMalak AS, Hanna F. A study of the allosteric inhibition of HCV RNA-dependent RNA polymerase and implementing virtual screening for the selection of promising dual-site inhibitors with low resistance potential. J Recept Signal Transduct Res 2016; 37:341-354. [PMID: 27829320 DOI: 10.1080/10799893.2016.1248293] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Structure-based pharmacophores were generated and validated using the bioactive conformations of different co-crystallized enzyme-inhibitor complexes for allosteric palm-1 and thumb-2 inhibitors of NS5B. Two pharmacophore models were obtained, one for palm-1 inhibitors with sensitivity = 0.929 and specificity = 0.983, and the other for thumb-2 inhibitors with sensitivity = 1 and specificity = 0.979. In addition, a quantitative structure activity relationship (QSAR) models were developed based on using the values of different scoring functions as descriptors predicting the activity on both allosteric binding sites (palm-1 and thumb-2). QSAR studies revealed good predictive and statistically significant two descriptor models (r2 = .837, r2adjusted = .792 and r2prediction = .688 for palm-1 model and r2 = .927, r2adjusted = .908 and r2prediction = .779 for thumb-2 model). External validation for the QSAR models assured their prediction power with r2ext = .72 and .89 for palm-1 and thumb-2, respectively. Different docking protocols were examined for their validity to predict the correct binding poses of inhibitors inside their respective binding sites. Virtual screening was carried out on ZINC database using the generated pharmacophores, the selected valid docking algorithms and QSAR models to find compounds that could theoretically bind to both sites simultaneously.
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Affiliation(s)
- Nasser S M Ismail
- a Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries , Future University , Cairo , Egypt
| | - Heba S A Elzahabi
- b Department of Pharmaceutical Chemistry, Faculty of Pharmacy , Al-Azhar University , Cairo , Egypt
| | - Peter Sabry
- c National Organization for Drug Control and Research , Dokki , Cairo , Egypt
| | - Fady N Baselious
- d Department of Research and Development , Global Napi Pharmaceuticals , 6th October City , Giza , Egypt
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9
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Speranzini V, Rotili D, Ciossani G, Pilotto S, Marrocco B, Forgione M, Lucidi A, Forneris F, Mehdipour P, Velankar S, Mai A, Mattevi A. Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features. SCIENCE ADVANCES 2016; 2:e1601017. [PMID: 27626075 PMCID: PMC5017823 DOI: 10.1126/sciadv.1601017] [Citation(s) in RCA: 57] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/06/2016] [Accepted: 08/11/2016] [Indexed: 06/06/2023]
Abstract
Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors.
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Affiliation(s)
- Valentina Speranzini
- Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy
| | - Dante Rotili
- Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
| | - Giuseppe Ciossani
- Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy
| | - Simona Pilotto
- Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy
| | - Biagina Marrocco
- Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy
| | - Mariantonietta Forgione
- Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
- Center for Life Nano Science@Sapienza, Italian Institute of Technology, Viale Regina Elena 291, 00161 Rome, Italy
| | - Alessia Lucidi
- Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
| | - Federico Forneris
- Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy
| | - Parinaz Mehdipour
- Department of Experimental Oncology, European Institute of Oncology, via Adamello 16, 20139 Milan, Italy
| | - Sameer Velankar
- European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Cambridge, Cambridgeshire CB10 1SD, U.K
| | - Antonello Mai
- Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
- Pasteur Institute–Cenci Bolognetti Foundation, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome, Italy
| | - Andrea Mattevi
- Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy
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Abstract
The treatment of HCV infection has evolved at an extremely rapid pace over the past few years. The development of direct-acting antiviral agents, which potently inhibit different stages in the viral life cycle, has led to the replacement of interferon with well-tolerated oral therapies with cure rates of >90% in most patient populations. Understanding the mechanisms of action of the various agents as well as related issues, including the molecular basis for resistance, helps to guide drug development and clinical use. In this Review, we provide a mechanistic description of NS3/4A protease inhibitors, nucleotide and non-nucleotide inhibitors of the NS5B viral polymerase and inhibitors of the NS5A protein, followed by a summary of clinical data from studies of each drug class alone and in combination. Remaining challenges in drug development efforts are also discussed.
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11
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Davis BC, Brown JA, Thorpe IF. Allosteric inhibitors have distinct effects, but also common modes of action, in the HCV polymerase. Biophys J 2016; 108:1785-1795. [PMID: 25863069 DOI: 10.1016/j.bpj.2015.03.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2014] [Revised: 02/27/2015] [Accepted: 03/04/2015] [Indexed: 12/28/2022] Open
Abstract
The RNA-dependent RNA polymerase from the Hepatitis C Virus (gene product NS5B) is a validated drug target because of its critical role in genome replication. There are at least four distinct allosteric sites on the polymerase to which several small molecule inhibitors bind. In addition, numerous crystal structures have been solved with different allosteric inhibitors bound to the polymerase. However, the molecular mechanisms by which these small molecules inhibit the enzyme have not been fully elucidated. There is evidence that allosteric inhibitors alter the intrinsic motions and distribution of conformations sampled by the enzyme. In this study we use molecular dynamics simulations to understand the structural and dynamic changes that result when inhibitors are bound at three different allosteric binding sites on the enzyme. We observe that ligand binding at each site alters the structure and dynamics of NS5B in a distinct manner. Nonetheless, our studies also highlight commonalities in the mechanisms of action of the different inhibitors. Each inhibitor alters the conformational states sampled by the enzyme, either by rigidifying the enzyme and preventing transitions between functional conformational states or by destabilizing the enzyme and preventing functionally relevant conformations from being adequately sampled. By illuminating the molecular mechanisms of allosteric inhibition, these studies delineate the intrinsic functional properties of the enzyme and pave the way for designing novel and more effective polymerase inhibitors. This information may also be important to understand how allosteric regulation occurs in related viral polymerases and other enzymes.
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Affiliation(s)
- Brittny C Davis
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland
| | - Jodian A Brown
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland
| | - Ian F Thorpe
- Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland.
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12
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Inhibitors of the Hepatitis C Virus Polymerase; Mode of Action and Resistance. Viruses 2015; 7:5206-24. [PMID: 26426038 PMCID: PMC4632376 DOI: 10.3390/v7102868] [Citation(s) in RCA: 86] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2015] [Revised: 09/17/2015] [Accepted: 09/17/2015] [Indexed: 12/19/2022] Open
Abstract
The hepatitis C virus (HCV) is a pandemic human pathogen posing a substantial health and economic burden in both developing and developed countries. Controlling the spread of HCV through behavioural prevention strategies has met with limited success and vaccine development remains slow. The development of antiviral therapeutic agents has also been challenging, primarily due to the lack of efficient cell culture and animal models for all HCV genotypes, as well as the large genetic diversity between HCV strains. On the other hand, the use of interferon-α-based treatments in combination with the guanosine analogue, ribavirin, achieved limited success, and widespread use of these therapies has been hampered by prevalent side effects. For more than a decade, the HCV RNA-dependent RNA polymerase (RdRp) has been targeted for antiviral development. Direct acting antivirals (DAA) have been identified which bind to one of at least six RdRp inhibitor-binding sites, and are now becoming a mainstay of highly effective and well tolerated antiviral treatment for HCV infection. Here we review the different classes of RdRp inhibitors and their mode of action against HCV. Furthermore, the mechanism of antiviral resistance to each class is described, including naturally occurring resistance-associated variants (RAVs) in different viral strains and genotypes. Finally, we review the impact of these RAVs on treatment outcomes with the newly developed regimens.
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13
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RNA-Dependent RNA Polymerases of Picornaviruses: From the Structure to Regulatory Mechanisms. Viruses 2015; 7:4438-60. [PMID: 26258787 PMCID: PMC4576190 DOI: 10.3390/v7082829] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2015] [Revised: 07/24/2015] [Accepted: 07/29/2015] [Indexed: 12/25/2022] Open
Abstract
RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication within the infected cells. RdRP function is critical not only for the virus life cycle but also for its adaptive potential. The combination of low fidelity of replication and the absence of proofreading and excision activities within the RdRPs result in high mutation frequencies that allow these viruses a rapid adaptation to changing environments. In this review, we summarize the current knowledge about structural and functional aspects on RdRP catalytic complexes, focused mainly in the Picornaviridae family. The structural data currently available from these viruses provided high-resolution snapshots for a range of conformational states associated to RNA template-primer binding, rNTP recognition, catalysis and chain translocation. As these enzymes are major targets for the development of antiviral compounds, such structural information is essential for the design of new therapies.
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14
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Harikishore A, Li E, Lee JJ, Cho NJ, Yoon HS. Combination of pharmacophore hypothesis and molecular docking to identify novel inhibitors of HCV NS5B polymerase. Mol Divers 2015; 19:529-39. [PMID: 25862642 DOI: 10.1007/s11030-015-9591-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2014] [Accepted: 03/25/2015] [Indexed: 01/03/2023]
Abstract
Hepatitis C virus (HCV) infection or HCV-related liver diseases are now shown to cause more than 350,000 deaths every year. Adaptability of HCV genome to vary its composition and the existence of multiple strains makes it more difficult to combat the emergence of drug-resistant HCV infections. Among the HCV polyprotein which has both the structural and non-structural regions, the non-structural protein NS5B RNA-dependent RNA polymerase (RdRP) mainly mediates the catalytic role of RNA replication in conjunction with its viral protein machinery as well as host chaperone proteins. Lack of such RNA-dependent RNA polymerase enzyme in host had made it an attractive and hotly pursued target for drug discovery efforts. Recent drug discovery efforts targeting HCV RdRP have seen success with FDA approval for sofosbuvir as a direct-acting antiviral against HCV infection. However, variations in drug-binding sites induce drug resistance, and therefore targeting allosteric sites could delay the emergence of drug resistance. In this study, we focussed on allosteric thumb site II of the non-structural protein NS5B RNA-dependent RNA polymerase and developed a five-feature pharmacophore hypothesis/model which estimated the experimental activity with a strong correlation of 0.971 & 0.944 for training and test sets, respectively. Further, the Güner-Henry score of 0.6 suggests that the model was able to discern the active and inactive compounds and enrich the true positives during a database search. In this study, database search and molecular docking results supported by experimental HCV viral replication inhibition assays suggested ligands with best fitness to the pharmacophore model dock to the key residues involved in thumbs site II, which inhibited the HCV 1b viral replication in sub-micro-molar range.
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Affiliation(s)
- Amaravadhi Harikishore
- School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore, 637551, Singapore,
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15
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Reich S, Kovermann M, Lilie H, Knick P, Geissler R, Golbik RP, Balbach J, Behrens SE. Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template. Biochemistry 2014; 53:7002-12. [PMID: 25310724 PMCID: PMC4230328 DOI: 10.1021/bi5006656] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
![]()
The
hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is
a central enzyme of the intracellular replication of the viral (+)RNA
genome. Here, we studied the individual steps of NS5B-catalyzed RNA
synthesis by a combination of biophysical methods, including real-time
1D 1H NMR spectroscopy. NS5B was found to bind to a nonstructured
and a structured RNA template in different modes. Following NTP binding
and conversion to the catalysis-competent ternary complex, the polymerase
revealed an improved affinity for the template. By monitoring the
folding/unfolding of 3′(−)SL by 1H NMR, the
base pair at the stem’s edge was identified as the most stable
component of the structure. 1H NMR real-time analysis of
NS5B-catalyzed RNA synthesis on 3′(−)SL showed that
a pronounced lag phase preceded the processive polymerization reaction.
The presence of the double-stranded stem with the edge base pair acting
as the main energy barrier impaired RNA synthesis catalyzed by NS5B.
Our observations suggest a crucial role of RNA-modulating factors
in the HCV replication process.
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Affiliation(s)
- Stefan Reich
- Institute of Biochemistry and Biotechnology, Section of Microbial Biotechnology, ‡Institute of Physics, Section of Biophysics, §Institute of Biochemistry and Biotechnology, Section of Technical Biochemistry, Martin Luther University Halle-Wittenberg , D-06120 Halle/Saale, Germany
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16
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Cross-genotypic examination of hepatitis C virus polymerase inhibitors reveals a novel mechanism of action for thumb binders. Antimicrob Agents Chemother 2014; 58:7215-24. [PMID: 25246395 DOI: 10.1128/aac.03699-14] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Direct-acting antivirals (DAAs) targeting proteins encoded by the hepatitis C virus (HCV) genome have great potential for the treatment of HCV infections. However, the efficacy of DAAs designed to target genotype 1 (G1) HCV against non-G1 viruses has not been characterized fully. In this study, we investigated the inhibitory activities of nonnucleoside inhibitors (NNIs) against the HCV RNA-dependent RNA polymerase (RdRp). We examined the ability of six NNIs to inhibit G1b, G2a, and G3a subgenomic replicons in cell culture, as well as in vitro transcription by G1b and G3a recombinant RdRps. Of the six G1 NNIs, only the palm II binder nesbuvir demonstrated activity against G1, G2, and G3 HCV, in both replicon and recombinant enzyme models. The thumb I binder JTK-109 also inhibited G1b and G3a replicons and recombinant enzymes but was 41-fold less active against the G2a replicon. The four other NNIs, which included a palm I binder (setrobuvir), two thumb II binders (lomibuvir and filibuvir), and a palm β-hairpin binder (tegobuvir), all showed at least 40-fold decreases in potency against G2a and G3a replicons and the G3a enzyme. This antiviral resistance was largely conferred by naturally occurring amino acid residues in the G2a and G3a RdRps that are associated with G1 resistance. Lomibuvir and filibuvir (thumb II binders) inhibited primer-dependent but not de novo activity of the G1b polymerase. Surprisingly, these compounds instead specifically enhanced the de novo activity at concentrations of ≥ 100 nM. These findings highlight a potential differential mode of RdRp inhibition for HCV NNIs, depending on their prospective binding pockets, and also demonstrate a surprising enhancement of de novo activity for thumb RdRp binders. These results also provide a better understanding of the antiviral coverage for these polymerase inhibitors, which will likely be used in future combinational interferon-free therapies.
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17
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Pourbasheer E, Aalizadeh R, Shokouhi Tabar S, Ganjali MR, Norouzi P, Shadmanesh J. 2D and 3D Quantitative Structure–Activity Relationship Study of Hepatitis C Virus NS5B Polymerase Inhibitors by Comparative Molecular Field Analysis and Comparative Molecular Similarity Indices Analysis Methods. J Chem Inf Model 2014; 54:2902-14. [DOI: 10.1021/ci500216c] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Affiliation(s)
- Eslam Pourbasheer
- Department
of Chemistry, Payame Noor University (PNU), P. O. Box 19395-3697, Tehran, Iran
| | | | - Samira Shokouhi Tabar
- Department
of Chemistry, Payame Noor University (PNU), P. O. Box 19395-3697, Tehran, Iran
| | - Mohammad Reza Ganjali
- Center
of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, P.O. Box 143981-7435, Tehran, Iran
- Biosensor
Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences
Institute, Tehran University of Medical Sciences, P. O. Box,
14114-13137, Tehran, Iran
| | - Parviz Norouzi
- Center
of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, P.O. Box 143981-7435, Tehran, Iran
- Biosensor
Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences
Institute, Tehran University of Medical Sciences, P. O. Box,
14114-13137, Tehran, Iran
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18
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Genotypic and phenotypic analyses of hepatitis C virus variants observed in clinical studies of VX-222, a nonnucleoside NS5B polymerase inhibitor. Antimicrob Agents Chemother 2014; 58:5456-65. [PMID: 24982088 DOI: 10.1128/aac.03052-14] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
VX-222, a thiophene-2-carboxylic acid derivative, is a selective nonnucleoside inhibitor of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In phase 1 and 2 clinical studies, VX-222 demonstrated effective antiviral efficacy, with substantial reductions in plasma HCV RNA in patients chronically infected with genotype 1 HCV. To characterize the potential for selection of VX-222-resistant variants in HCV-infected patients, the HCV NS5B gene was sequenced at baseline and during and after 3 days of VX-222 dosing (monotherapy) in a phase 1 study. Variants with the substitutions L419C/I/M/P/S/V, R422K, M423I/T/V, I482L/N/T, A486S/T/V, and V494A were selected during VX-222 dosing, and their levels declined over time after the end of dosing. Phenotypic analysis of these variants was conducted using HCV replicons carrying site-directed mutations. Of the 17 variants, 14 showed reduced susceptibility to VX-222 compared with the wild type, with the L419C/S and R422K variants having higher levels of resistance (>200-fold) than the rest of the variants (6.8- to 76-fold). The M423I and A486S variants remained susceptible to VX-222. The 50% effective concentration (EC50) for the L419P variant could not be obtained due to the poor replication of this replicon. The majority of the variants (15/17) were less fit than the wild type. A subset of the variants, predominately the L419S and R422K variants, were observed when the efficacy and safety of VX-222- and telaprevir-based regimens given for 12 weeks were investigated in genotype 1 HCV-infected patients in a phase 2 study. The NS3 and NS5B variants selected during the dual combination therapy showed reduced susceptibility to both telaprevir and VX-222 and had a lower replication capacity than the wild type. The phase 1b study has the ClinicalTrials.gov identifier NCT00911963, and the phase 2a study has ClinicalTrials.gov identifier NCT01080222.
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López-Jiménez AJ, Clemente-Casares P, Sabariegos R, Llanos-Valero M, Bellón-Echeverría I, Encinar JA, Kaushik-Basu N, Froeyen M, Mas A. Hepatitis C virus polymerase-polymerase contact interface: significance for virus replication and antiviral design. Antiviral Res 2014; 108:14-24. [PMID: 24815023 DOI: 10.1016/j.antiviral.2014.04.009] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2014] [Revised: 04/13/2014] [Accepted: 04/21/2014] [Indexed: 11/25/2022]
Abstract
The hepatitis C virus (HCV) replicates its genome in replication complexes located in micro-vesicles derived from endoplasmic reticulum. The composition of these replication complexes indicates that proteins, both viral and cellular in origin, are at high concentrations. Under these conditions, protein-protein interactions must occur although their role in the replication pathways is unknown. HCV RNA-dependent RNA-polymerase (NS5B) initiates RNA synthesis in these vesicles by a de novo (DN) mechanism. After initiation, newly synthesized dsRNA could induce conformational changes that direct the transition from an initiating complex into a processive elongation complex. In this report, we analyze the role played by NS5B-NS5B intermolecular interactions controlling these conformational rearrangements. Based on NS5B protein-protein docking and molecular dynamics simulations, we constructed mutants of residues predicted to be involved in protein-protein interactions. Changes at these positions induced severe defects in both the activity of the enzyme and the replication of a subgenomic replicon. Thus, mutations at the interaction surface decreased both DN synthesis initiation and processive elongation activities. Based on this analysis, we define at an atomic level an NS5B homomeric interaction model that connects the T-helix in the thumb subdomain of one monomer, with the F-helix of the fingers subdomain in other monomer. Knowing the molecular determinants involved in viral replication could be helpful to delineate new and powerful antiviral strategies.
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Affiliation(s)
- Alberto José López-Jiménez
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain
| | - Pilar Clemente-Casares
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain; School of Pharmacy, Universidad de Castilla-La Mancha, Albacete 02008, Spain; Viral Hepatitis Study Group, Spanish Society of Virology, Madrid, Spain
| | - Rosario Sabariegos
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain; School of Medicine, Universidad de Castilla-La Mancha, Albacete 02008, Spain; Viral Hepatitis Study Group, Spanish Society of Virology, Madrid, Spain
| | - María Llanos-Valero
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain
| | - Itxaso Bellón-Echeverría
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain
| | - José Antonio Encinar
- Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche 03202, Spain
| | - Neerja Kaushik-Basu
- Department of Biochemistry and Molecular Biology, Rutgers, The State University of New Jersey, New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, United States
| | - Mathy Froeyen
- Laboratory for Medicinal Chemistry, Rega Institute for Medical Research, K.U. Leuven, Belgium
| | - Antonio Mas
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla-La Mancha, Albacete 02008, Spain; School of Pharmacy, Universidad de Castilla-La Mancha, Albacete 02008, Spain; Viral Hepatitis Study Group, Spanish Society of Virology, Madrid, Spain; Unidad de Biomedicina, CSIC-UCLM, Spain.
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20
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Gaetano JN. Benefit-risk assessment of new and emerging treatments for hepatitis C: focus on simeprevir and sofosbuvir. DRUG HEALTHCARE AND PATIENT SAFETY 2014; 6:37-45. [PMID: 24729731 PMCID: PMC3976205 DOI: 10.2147/dhps.s43304] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Greater understanding of the hepatitis C virus (HCV) genome and life cycle of the HCV virion allows for new targets for therapy that directly act on the viral machinery to inhibit replication. Numerous direct-acting antivirals are in development, and four have been brought to market. Simeprevir, a second-generation protease inhibitor, has been approved for HCV genotype 1 patients in combination with pegylated interferon-α and ribavirin. Sofosbuvir, a novel nucleotide analog, has pangenotypic coverage and has been approved for HCV genotype 1 patients with ribavirin and pegylated interferon-α. For HCV genotypes 2 and 3, an all-oral regimen of sofosbuvir with ribavirin has become the new gold standard for treatment. The efficacy and safety for these two novel therapies among various subpopulations of those infected with chronic hepatitis C are discussed in the following review. In addition, off-label and future therapeutic regimens are addressed, as well as the concerns about cost of current and future therapies.
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Affiliation(s)
- John N Gaetano
- Section of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Chicago Hospitals, Chicago, IL, USA
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21
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Therese PJ, Manvar D, Kondepudi S, Battu MB, Sriram D, Basu A, Yogeeswari P, Kaushik-Basu N. Multiple e-pharmacophore modeling, 3D-QSAR, and high-throughput virtual screening of hepatitis C virus NS5B polymerase inhibitors. J Chem Inf Model 2014; 54:539-52. [PMID: 24460140 DOI: 10.1021/ci400644r] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRP) is a crucial and unique component of the HCV RNA replication machinery and a validated target for drug discovery. Multiple crystal structures of NS5B inhibitor complexes have facilitated the identification of novel compound scaffolds through in silico analysis. With the goal of discovering new NS5B inhibitor leads, HCV NS5B crystal structures bound with inhibitors in the palm and thumb allosteric pockets in combination with ligands with known inhibitory potential were explored for a comparative pharmacophore analyses. The energy-based and 3D-QSAR-based pharmacophore models were validated using enrichment analysis, and the six models thus developed were employed for high-throughput virtual screening and docking to identify nonpeptidic leads. The hits derived at each stage were analyzed for diversity based on the six pharmacophore models, followed by molecular docking and filtering based on their interaction with amino acids in the NS5B allosteric pocket and 3D-QSAR predictions. The resulting 10 hits displaying diverse scaffold were then screened employing biochemical and cell-based NS5B and anti-HCV inhibition assays. Of these, two molecules H-5 and H-6 were the most promising, exhibiting IC50 values of 28.8 and 47.3 μM against NS5B polymerase and anti-HCV inhibition of 96% and 86% at 50 μM, respectively. The identified leads comprised of benzimidazole (H-5) and pyridine (H-6) scaffolds thus constitute prototypical molecules for further optimization and development as NS5B inhibitors.
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Affiliation(s)
- Patrisha Joseph Therese
- Computer-Aided Drug Design Lab, Department of Pharmacy, Birla Institute of Technology & Science-Pilani , Hyderabad campus, Jawahar Nagar, Hyderabad-500078, Andhra Pradesh, India
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22
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Beaulieu PL. Design and Development of NS5B Polymerase Non‐nucleoside Inhibitors for the Treatment of Hepatitis C Virus Infection. SUCCESSFUL STRATEGIES FOR THE DISCOVERY OF ANTIVIRAL DRUGS 2013. [DOI: 10.1039/9781849737814-00248] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
The hepatitis C virus (HCV) infects an estimated 130–170 million people worldwide and is associated with life‐threatening liver diseases. The recent introduction of the first two HCV direct‐acting antivirals (DAAs) as a complement to the interferon/ribavirin standard of care has provided patients with improved outcomes. Still, 25–30% of subjects infected with genotype 1 HCV do not respond adequately to treatment owing to the emergence of resistant virus and many suffer from severe side effects. A paradigm shift towards the development of interferon‐free combinations of DAAs with complementary modes of action is currently taking place. Virally encoded proteins and enzymes have become the target of HCV drug discovery efforts and several promising new agents are currently being evaluated in the clinic for treatment of chronic HCV infection. The NS5B RNA‐dependent RNA polymerase is responsible for replication of viral RNA and plays a pivotal role in the virus life cycle. NS5B is undoubtedly the most druggable HCV target and is susceptible to several classes of allosteric inhibitors that bind to four distinct sites on the enzyme. This chapter describes successful strategies that have led to the discovery of HCV NS5B antivirals. It is divided according to allosteric sites and describes how each of the known families of inhibitors was discovered, characterized and optimized to provide clinical candidates. When available, the strategies adopted by medicinal chemists to optimize initial leads and address challenges and liabilities encountered on the path to candidate selection are described, along with reported clinical outcomes.
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Affiliation(s)
- Pierre L. Beaulieu
- Boehringer Ingelheim (Canada) Ltd. 2100 Cunard Street, Laval, Québec Canada, H7S 2G5 resgeneral.lav@boehringer‐ingelheim.com
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23
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Weidlich IE, Filippov IV, Brown J, Kaushik-Basu N, Krishnan R, Nicklaus MC, Thorpe IF. Inhibitors for the hepatitis C virus RNA polymerase explored by SAR with advanced machine learning methods. Bioorg Med Chem 2013; 21:3127-37. [PMID: 23608107 PMCID: PMC3653294 DOI: 10.1016/j.bmc.2013.03.032] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2013] [Revised: 03/10/2013] [Accepted: 03/18/2013] [Indexed: 12/30/2022]
Abstract
Hepatitis C virus (HCV) is a global health challenge, affecting approximately 200 million people worldwide. In this study we developed SAR models with advanced machine learning classifiers Random Forest and k Nearest Neighbor Simulated Annealing for 679 small molecules with measured inhibition activity for NS5B genotype 1b. The activity was expressed as a binary value (active/inactive), where actives were considered molecules with IC50 ≤0.95 μM. We applied our SAR models to various drug-like databases and identified novel chemical scaffolds for NS5B inhibitors. Subsequent in vitro antiviral assays suggested a new activity for an existing prodrug, Candesartan cilexetil, which is currently used to treat hypertension and heart failure but has not been previously tested for anti-HCV activity. We also identified NS5B inhibitors with two novel non-nucleoside chemical motifs.
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Affiliation(s)
- Iwona E. Weidlich
- Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
- Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, NIH, DHHS, Frederick National Laboratory for Cancer Research, 376 Boyles Street, Frederick, MD 21702
- Computational Drug Design Systems (CODDES) LLC, Rockville, MD
| | - Igor V. Filippov
- Chemical Biology Laboratory, Center for Cancer Research, SAIC-Frederick, Inc., 376 Boyles Street, Frederick, MD 21702
| | - Jodian Brown
- Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
| | - Neerja Kaushik-Basu
- Department of Biochemistry and Molecular Biology, UMDNJ New Jersey Medical School, 185 South Orange Ave, Newark, NJ 07103
| | - Ramalingam Krishnan
- Department of Biochemistry and Molecular Biology, UMDNJ New Jersey Medical School, 185 South Orange Ave, Newark, NJ 07103
| | - Marc C. Nicklaus
- Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, NIH, DHHS, Frederick National Laboratory for Cancer Research, 376 Boyles Street, Frederick, MD 21702
| | - Ian F. Thorpe
- Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250
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Garriga D, Ferrer-Orta C, Querol-Audí J, Oliva B, Verdaguer N. Role of motif B loop in allosteric regulation of RNA-dependent RNA polymerization activity. J Mol Biol 2013; 425:2279-87. [PMID: 23542342 DOI: 10.1016/j.jmb.2013.03.034] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2013] [Revised: 03/15/2013] [Accepted: 03/21/2013] [Indexed: 01/22/2023]
Abstract
Increasing amounts of data show that conformational dynamics are essential for protein function. Unveiling the mechanisms by which this flexibility affects the activity of a given enzyme and how it is controlled by other effectors opens the door to the design of a new generation of highly specific drugs. Viral RNA-dependent RNA polymerases (RdRPs) are not an exception. These enzymes, essential for the multiplication of all RNA viruses, catalyze the formation of phosphodiester bonds between ribonucleotides in an RNA-template-dependent fashion. Inhibition of RdRP activity will prevent genome replication and virus multiplication. Thus, RdRPs, like the reverse transcriptase of retroviruses, are validated targets for the development of antiviral therapeutics. X-ray crystallography of RdRPs trapped in multiple steps throughout the catalytic process, together with NMR data and molecular dynamics simulations, have shown that all polymerase regions contributing to conserved motifs required for substrate binding, catalysis and product release are highly flexible and some of them are predicted to display correlated motions. All these dynamic elements can be modulated by external effectors, which appear as useful tools for the development of effective allosteric inhibitors that block or disturb the flexibility of these enzymes, ultimately impeding their function. Among all movements observed, motif B, and the B-loop at its N-terminus in particular, appears as a new potential druggable site.
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Affiliation(s)
- Damià Garriga
- Institut de Biología Molecular de Barcelona (CSIC), Parc Científic de Barcelona, Baldiri i Reixac 10-12, 08028 Barcelona, Spain
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25
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Rosnoblet C, Fritzinger B, Legrand D, Launay H, Wieruszeski JM, Lippens G, Hanoulle X. Hepatitis C virus NS5B and host cyclophilin A share a common binding site on NS5A. J Biol Chem 2012; 287:44249-60. [PMID: 23152499 DOI: 10.1074/jbc.m112.392209] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Nonstructural protein 5B (NS5B) is essential for hepatitis C virus (HCV) replication as it carries the viral RNA-dependent RNA polymerase enzymatic activity. HCV replication occurs in a membrane-associated multiprotein complex in which HCV NS5A and host cyclophilin A (CypA) have been shown to be present together with the viral polymerase. We used NMR spectroscopy to perform a per residue level characterization of the molecular interactions between the unfolded domains 2 and 3 of NS5A (NS5A-D2 and NS5A-D3), CypA, and NS5B(Δ21). We show that three regions of NS5A-D2 (residues 250-262 (region A), 274-287 (region B), and 306-333 (region C)) interact with NS5B(Δ21), whereas NS5A-D3 does not. We show that both NS5B(Δ21) and CypA share a common binding site on NS5A that contains residues Pro-306 to Glu-323. No direct molecular interaction has been detected by NMR spectroscopy between HCV NS5B(Δ21) and host CypA. We show that cyclosporine A added to a sample containing NS5B(Δ21), NS5A-D2, and CypA specifically inhibits the interaction between CypA and NS5A-D2 without altering the one between NS5A-D2 and NS5B(Δ21). A high quality heteronuclear NMR spectrum of HCV NS5B(Δ21) has been obtained and was used to characterize the binding site on the polymerase of NS5A-D2. Moreover these data highlight the potential of using NMR of NS5B(Δ21) as a powerful tool to characterize in solution the interactions of the HCV polymerase with all kinds of molecules (proteins, inhibitors, RNA). This work brings new insights into the comprehension of the molecular interplay between NS5B, NS5A, and CypA, three essentials proteins for HCV replication.
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Golub AG, Gurukumar KR, Basu A, Bdzhola VG, Bilokin Y, Yarmoluk SM, Lee JC, Talele TT, Nichols DB, Kaushik-Basu N. Discovery of new scaffolds for rational design of HCV NS5B polymerase inhibitors. Eur J Med Chem 2012; 58:258-64. [PMID: 23127989 DOI: 10.1016/j.ejmech.2012.09.010] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2012] [Revised: 09/05/2012] [Accepted: 09/07/2012] [Indexed: 02/02/2023]
Abstract
Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of anti-HCV drugs. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening and in vitro NS5B inhibition assays. One hundred and sixty thousand compounds from the Otava database were virtually screened against the thiazolone inhibitor binding site on NS5B (thumb pocket-2, TP-2), resulting in a sequential down-sizing of the library by 2.7 orders of magnitude to yield 59 NS5B non-nucleoside inhibitor (NNI) candidates. In vitro evaluation of the NS5B inhibitory activity of the 59 selected compounds resulted in a 14% hit rate, yielding 8 novel structural scaffolds. Of these, compound 1 bearing a 4-hydrazinoquinazoline scaffold was the most active (IC(50) = 16.0 μM). The binding site of all 8 NNIs was mapped to TP-2 of NS5B as inferred by a decrease in their inhibition potency against the M423T NS5B mutant, employed as a screen for TP-2 site binders. At 100 μM concentration, none of the eight compounds exhibited any cytotoxicity, and all except compound 8 exhibited between 40 and 60% inhibition of intracellular NS5B polymerase activity in BHK-NS5B-FRLuc reporter cells. These inhibitor scaffolds will form the basis for future optimization and development of more potent NS5B inhibitors.
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Affiliation(s)
- Andriy G Golub
- Department of Combinatorial Chemistry, Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine, 150 Zabolotnogo Street, 03143 Kyiv, Ukraine
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27
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Davis BC, Thorpe IF. Thumb inhibitor binding eliminates functionally important dynamics in the hepatitis C virus RNA polymerase. Proteins 2012; 81:40-52. [PMID: 22855387 DOI: 10.1002/prot.24154] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2012] [Revised: 07/12/2012] [Accepted: 07/23/2012] [Indexed: 11/09/2022]
Abstract
Hepatitis C virus (HCV) has infected almost 200 million people worldwide, typically causing chronic liver damage and severe complications such as liver failure. Currently, there are few approved treatments for viral infection. Thus, the HCV RNA-dependent RNA polymerase (gene product NS5B) has emerged as an important target for small molecule therapeutics. Potential therapeutic agents include allosteric inhibitors that bind distal to the enzyme active site. While their mechanism of action is not conclusively known, it has been suggested that certain inhibitors prevent a conformational change in NS5B that is crucial for RNA replication. To gain insight into the molecular origin of long-range allosteric inhibition of NS5B, we employed molecular dynamics simulations of the enzyme with and without an inhibitor bound to the thumb domain. These studies indicate that the presence of an inhibitor in the thumb domain alters both the structure and internal motions of NS5B. Principal components analysis identified motions that are severely attenuated by inhibitor binding. These motions may have functional relevance by facilitating interactions between NS5B and RNA template or nascent RNA duplex, with presence of the ligand leading to enzyme conformations with narrower and thus less accessible RNA binding channels. This study provides the first evidence for a mechanistic basis of allosteric inhibition in NS5B. Moreover, we present evidence that allosteric inhibition of NS5B results from intrinsic features of the enzyme free energy landscape, suggesting a common mechanism for the action of diverse allosteric ligands.
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Affiliation(s)
- Brittny C Davis
- Department of Chemistry and Biochemistry, University of Maryland-Baltimore County, Baltimore, Maryland 21250, USA
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28
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Discovery of substituted N-phenylbenzenesulphonamides as a novel class of non-nucleoside hepatitis C virus polymerase inhibitors. Antiviral Res 2012; 95:182-91. [DOI: 10.1016/j.antiviral.2012.04.008] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2012] [Revised: 04/23/2012] [Accepted: 04/28/2012] [Indexed: 01/10/2023]
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29
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Canales E, Carlson JS, Appleby T, Fenaux M, Lee J, Tian Y, Tirunagari N, Wong M, Watkins WJ. Tri-substituted acylhydrazines as tertiary amide bioisosteres: HCV NS5B polymerase inhibitors. Bioorg Med Chem Lett 2012; 22:4288-92. [DOI: 10.1016/j.bmcl.2012.05.025] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2012] [Revised: 05/05/2012] [Accepted: 05/08/2012] [Indexed: 11/25/2022]
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Nurisso A, Bravo J, Carrupt PA, Daina A. Molecular docking using the molecular lipophilicity potential as hydrophobic descriptor: impact on GOLD docking performance. J Chem Inf Model 2012; 52:1319-27. [PMID: 22462609 DOI: 10.1021/ci200515g] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
GOLD is a molecular docking software widely used in drug design. In the initial steps of docking, it creates a list of hydrophobic fitting points inside protein cavities that steer the positioning of ligand hydrophobic moieties. These points are generated based on the Lennard-Jones potential between a carbon probe and each atom of the residues delimitating the binding site. To thoroughly describe hydrophobic regions in protein pockets and properly guide ligand hydrophobic moieties toward favorable areas, an in-house tool, the MLP filter, was developed and herein applied. This strategy only retains GOLD hydrophobic fitting points that match the rigorous definition of hydrophobicity given by the molecular lipophilicity potential (MLP), a molecular interaction field that relies on an atomic fragmental system based on 1-octanol/water experimental partition coefficients (log P(oct)). MLP computations in the binding sites of crystallographic protein structures revealed that a significant number of points considered hydrophobic by GOLD were actually polar according to the MLP definition of hydrophobicity. To examine the impact of this new tool, ligand-protein complexes from the Astex Diverse Set and the PDB bind core database were redocked with and without the use of the MLP filter. Reliable docking results were obtained by using the MLP filter that increased the quality of docking in nonpolar cavities and outperformed the standard GOLD docking approach.
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Affiliation(s)
- Alessandra Nurisso
- School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest-Ansermet 30, CH-1211, Geneva 4, Switzerland
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31
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Biochemical study of the comparative inhibition of hepatitis C virus RNA polymerase by VX-222 and filibuvir. Antimicrob Agents Chemother 2011; 56:830-7. [PMID: 22143520 DOI: 10.1128/aac.05438-11] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Filibuvir and VX-222 are nonnucleoside inhibitors (NNIs) that bind to the thumb II allosteric pocket of the hepatitis C virus (HCV) RNA-dependent RNA polymerase. Both compounds have shown significant promise in clinical trials and, therefore, it is relevant to better understand their mechanisms of inhibition. In our study, filibuvir and VX-222 inhibited the 1b/Con1 HCV subgenomic replicon, with 50% effective concentrations (EC(50)s) of 70 nM and 5 nM, respectively. Using several RNA templates in biochemical assays, we found that both compounds preferentially inhibited primer-dependent RNA synthesis but had either no or only modest effects on de novo-initiated RNA synthesis. Filibuvir and VX-222 bind to the HCV polymerase with dissociation constants of 29 and 17 nM, respectively. Three potential resistance mutations in the thumb II pocket were analyzed for effects on inhibition by the two compounds. The M423T substitution in the RNA polymerase was at least 100-fold more resistant to filibuvir in the subgenomic replicon and in the enzymatic assays. This resistance was the result of a 250-fold loss in the binding affinity (K(d)) of the mutated enzyme to filibuvir. In contrast, the inhibitory activity of VX-222 was only modestly affected by the M423T substitution but more significantly affected by an I482L substitution.
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32
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An objective assessment of conformational variability in complexes of hepatitis C virus polymerase with non-nucleoside inhibitors. J Mol Biol 2011; 414:370-84. [PMID: 22008450 DOI: 10.1016/j.jmb.2011.10.001] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2011] [Revised: 08/24/2011] [Accepted: 10/01/2011] [Indexed: 12/14/2022]
Abstract
A major target for antiviral therapy against hepatitis C virus (HCV) is the HCV polymerase nonstructural protein 5B (NS5B). Huge efforts have been devoted to the development of nucleoside and non-nucleoside inhibitors (NNIs) of NS5B. An offshoot of these efforts has been the structural characterization of the interaction of NS5B with NNIs by X-ray crystallography. These works have shown that the conformation of recombinant NS5B is very similar across strains, constructs and complexes, making evaluation of the long-range conformational effects of NNIs nontrivial. Using procedures appropriate to the evaluation of such minor but potentially important differences, we objectively assessed the conformational diversity in the 78 available genotype 1b NS5B structures in the Protein Data Bank. We find that there are 20 significantly different NS5B conformations available, but all are geometrically close to a closed, RNA synthesis initiation-competent one. Within this fairly restricted range, differences can be mapped to movements of NS5B domains and subregions. Most of this information is actually defined by small but significant changes in complexes with NNIs. We thus establish rigorously the moving parts of the NS5B molecular machine and the previously unrecognized hinge points that come into play upon NNI binding. We propose that NNIs binding at three of the four distinct sites specifically inhibit the initiation step by the same mechanism: they prevent NS5B's "thumb" from quite reaching the proper initiation-competent position. Furthermore, we suggest that a small number of critical hinges in the NS5B structure may emerge as sites of resistance mutations during future antiviral treatment.
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Abstract
Given its essential role in the process of hepatitis C virus (HCV) replication, the viral NS3/4A serine protease is arguably the most thoroughly characterized HCV enzyme and the most intensively pursued anti-HCV target for drug development thus far. Recent data have demonstrated promise for the NS3 protease inhibitor boceprevir, which, when added to the standard of care peginterferon and ribavirin, improves sustained virological response while shortening duration of therapy in genotype-1-infected individuals. This review discusses the mechanism of action of boceprevir, its effects on HCV, and its viral resistance.
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Affiliation(s)
- Paul Y Kwo
- Liver Transplantation, Gastroenterology/Hepatology Division, Indiana University School of Medicine, Indianapolis, IN 46202-5121, USA.
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34
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Nucleoside analog inhibitors of hepatitis C viral replication: recent advances, challenges and trends. Future Med Chem 2011; 1:1429-52. [PMID: 21426058 DOI: 10.4155/fmc.09.88] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infection is a global health problem, with over 170 million people infected worldwide. The current therapy, pegylated interferon (PEG-IFN) plus ribavirin (RBV), provides only approximately a 40% sustained virological response (undetectable HCV RNA for greater than 24 weeks after cessation of therapy), in genotype 1-infected individuals. In addition to the limited sustained virological response, PEG-IFN/RBV treatment is associated with serious adverse effects. Nucleosides have long been the cornerstone of antiviral therapy because of their proven efficacy and high barrier to resistance. Through the use of surrogate viruses or the HCV subgenomic replicon, several classes of nucleoside analogs or their monophosphate prodrugs have been identified that inhibit HCV RNA replication. Nucleoside analogs that possess the 2´-C-methyl modification vary in their ability to be phosphorylated and to act as alternative substrate inhibitors of the HCV RNA polymerase. Herein, we discuss various classes of nucleoside inhibitors, with a focus on available structure-activity relationships, their mode of action and resistance profile.
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Clemente-Casares P, López-Jiménez AJ, Bellón-Echeverría I, Encinar JA, Martínez-Alfaro E, Pérez-Flores R, Mas A. De novo polymerase activity and oligomerization of hepatitis C virus RNA-dependent RNA-polymerases from genotypes 1 to 5. PLoS One 2011; 6:e18515. [PMID: 21490973 PMCID: PMC3072391 DOI: 10.1371/journal.pone.0018515] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2010] [Accepted: 03/04/2011] [Indexed: 01/20/2023] Open
Abstract
Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The Vmax/Km ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in Vmax values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions.
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Affiliation(s)
- Pilar Clemente-Casares
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla La Mancha, Albacete, Spain
| | - Alberto J. López-Jiménez
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla La Mancha, Albacete, Spain
- Infectious Disease Unit, Complejo Hospitalario Universitario de Albacete, Albacete, Spain
| | - Itxaso Bellón-Echeverría
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla La Mancha, Albacete, Spain
| | - José Antonio Encinar
- Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain
| | - Elisa Martínez-Alfaro
- Infectious Disease Unit, Complejo Hospitalario Universitario de Albacete, Albacete, Spain
| | - Ricardo Pérez-Flores
- Digestive Department, Complejo Hospitalario Universitario de Albacete, Albacete, Spain
| | - Antonio Mas
- Centro Regional de Investigaciones Biomédicas (CRIB), Universidad de Castilla La Mancha, Albacete, Spain
- * E-mail:
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36
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Structure-based library design in efficient discovery of novel inhibitors. Methods Mol Biol 2011; 685:175-90. [PMID: 20981524 DOI: 10.1007/978-1-60761-931-4_9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Structure-based library design employs both structure-based drug design (SBDD) and combinatorial library design. Combinatorial library design concepts have evolved over the past decade, and this chapter covers several novel aspects of structure-based library design together with successful case studies in the anti-viral drug design HCV target area. Discussions include reagent selections, diversity library designs, virtual screening, scoring/ranking, and post-docking pose filtering, in addition to the considerations of chemistry synthesis. Validation criteria for a successful design include an X-ray co-crystal complex structure, in vitro biological data, and the number of compounds to be made, and these are addressed in this chapter as well.
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Rigat K, Wang Y, Hudyma TW, Ding M, Zheng X, Gentles RG, Beno BR, Gao M, Roberts SB. Ligand-induced changes in hepatitis C virus NS5B polymerase structure. Antiviral Res 2010; 88:197-206. [PMID: 20813137 DOI: 10.1016/j.antiviral.2010.08.014] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2010] [Revised: 08/20/2010] [Accepted: 08/26/2010] [Indexed: 12/28/2022]
Abstract
Hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B) is required for viral replication. Crystal structures of the NS5B apoprotein show that the finger and thumb domains interact to encircle the active site, and that inhibitors defined by P495 resistance that bind to the thumb-finger interface displace the Δ1 finger loop and disrupt this structure. Since crystal structures may not reveal all of the conformations of a protein in solution we have developed an alternative method, using limited trypsin protease digestion, to investigate the impact of inhibitors as well as substrates on the movement of the Δ1 loop. This assay can be used to study NS5B under conditions that support enzymatic activity. In the absence of inhibitors, no specific region of NS5B was hypersensitive to trypsin, and no specific intermediate cleavage products were formed. Binding of P495-site inhibitors to NS5B induced specific trypsin hypersensitivity at lysine residues 50 and 51. Previously characterized inhibitors and mutant polymerases were used to link this specific trypsin hypersensitivity to movement of the Δ1 loop. Trypsin hypersensitivity identical to the inhibitor pattern was also induced by the binding of the RNA template. The addition of primer to the NS5B-template complex eliminated the hypersensitivity. The data are consistent with displacement of the Δ1 finger loop from the thumb by the binding of template, and reversal by the addition of primer or NTP. Our results complement inhibitor-enzyme co-crystal studies, and the assay provides a rapid and sensitive method to study dynamic changes in HCV NS5B polymerase conformation under conditions that support functional activity.
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Affiliation(s)
- Karen Rigat
- Department of Virology, Bristol-Myers Squibb Co., Research & Development, Wallingford, CT 06492, USA.
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Musmuca I, Caroli A, Mai A, Kaushik-Basu N, Arora P, Ragno R. Combining 3-D quantitative structure-activity relationship with ligand based and structure based alignment procedures for in silico screening of new hepatitis C virus NS5B polymerase inhibitors. J Chem Inf Model 2010; 50:662-76. [PMID: 20225870 DOI: 10.1021/ci9004749] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The viral NS5B RNA-dependent RNA-polymerase (RdRp) is one of the best-studied and promising targets for the development of novel therapeutics against hepatitis C virus (HCV). Allosteric inhibition of this enzyme has emerged as a viable strategy toward blocking replication of viral RNA in cell based systems. Herein, we describe how the combination of a complete computational procedure together with biological studies led to the identification of novel molecular scaffolds, hitherto untested toward NS5B polymerase. Structure based 3-D quantitative structure-activity relationship (QSAR) models were generated employing NS5B non-nucleoside inhibitors (NNIs), whose bound conformations were readily available from the protein database (PDB). These were grouped into two training sets of structurally diverse NS5B NNIs, based on their binding to the enzyme thumb (15 NNIs) or palm (10 NNIs) domains. Ligand based (LB) and structure based (SB) alignments were rigorously investigated to assess the reliability on the correct molecular alignment for unknown binding mode modeled compounds. Both Surflex and Autodock programs were able to reproduce with minimal errors the experimental binding conformations of 24 experimental NS5B allosteric inhibitors. Eighty-one (thumb) and 223 (palm) modeled compounds taken from literature were LB and SB aligned and used as external validation sets for the development of 3-D QSAR models. Low error of prediction proved the 3-D QSARs to be useful scoring functions for the in silico screening procedure. Finally, the virtual screening of the NCI Diversity Set led to the selection for enzymatic assays of 20 top-scoring molecules for each final model. Among the 40 selected molecules, preliminary data yielded four derivatives exhibiting IC(50) values ranging between 45 and 75 microM. Binding mode analysis of hit compounds within the NS5B polymerase thumb domain showed that one of them, NSC 123526, exhibited a docked conformation which was in good agreement with the thumb training set most active compound (6).
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Affiliation(s)
- Ira Musmuca
- Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Universita di Roma, P le A Moro 5, 00185 Rome, Italy
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Abstract
IMPORTANCE OF THE FIELD Currently, 170 million people worldwide are affected by the HCV. Chronic HCV infection is amongst the leading causes of chronic liver disease and its complications such as cirrhosis and hepatocellular carcinoma, making it the most common reason for liver transplantation. The current standard of treatment for HCV is pegylated IFN-α plus ribavirin. This treatment, when administered for the standard duration, allows sustained virological response (SVR) in ∼ 50% of patients infected with HCV and about 40% for HCV genotype 1, the most prevalent form of HCV in the US. SVR rates for populations with co-morbidities (patients with chronic renal disease) and certain ethnic backgrounds (African Americans and Hispanics) are lower. Given the high prevalence and relatively low cure rates of current antiviral therapy, the burden of HCV is enormous. AREAS COVERED IN THIS REVIEW Faced with this urgent and growing medical need, research into novel therapeutic compounds for the treatment of HCV is a rapidly growing industry. Several novel compounds are in advanced stages of clinical development, such as HCV protease inhibitors (particularly those against NS3-4A protease), HCV polymerase inhibitors (including both nucleoside and non-nucleoside analogs) and cyclophilin inhibitors. WHAT THE READER WILL GAIN HCV treatment has seen many advances in the last decade and the discovery process has been fraught with both successes and disappointments. Through a process of rigorous research, the current late stage novel HCV therapeutics seem to have overcome some of the obstacles met by their early predecessors and offer the promise of meeting the shortfalls of the current standard of treatment. TAKE HOME MESSAGE Data from clinical trials are encouraging and suggest that combination therapies of these novel agents may have the potential to shorten treatment duration and increase viral clearance when used in conjunction with pegylated IFN-α and ribavirin.
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Affiliation(s)
- Aybike Birerdinc
- Betty and Guy Beatty Center for Integrated Research, Inova Health System, Falls Church, VA, USA
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40
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Bellón-Echeverría I, López-Jiménez AJ, Clemente-Casares P, Mas A. Monitoring hepatitis C virus (HCV) RNA-dependent RNA polymerase oligomerization by a FRET-based in vitro system. Antiviral Res 2010; 87:57-66. [DOI: 10.1016/j.antiviral.2010.04.009] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2010] [Revised: 04/15/2010] [Accepted: 04/19/2010] [Indexed: 12/23/2022]
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Ontoria JM, Rydberg EH, Di Marco S, Tomei L, Attenni B, Malancona S, Martin Hernando JI, Gennari N, Koch U, Narjes F, Rowley M, Summa V, Carroll SS, Olsen DB, De Francesco R, Altamura S, Migliaccio G, Carfì A. Identification and biological evaluation of a series of 1H-benzo[de]isoquinoline-1,3(2H)-diones as hepatitis C virus NS5B polymerase inhibitors. J Med Chem 2010; 52:5217-27. [PMID: 19877603 DOI: 10.1021/jm900517t] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for inhibition. Herein, we present 1H-benzo[de]isoquinoline-1,3(2H)-diones as a new series of selective inhibitors of HCV NS5B polymerase. The HTS hit 1 shows submicromolar potency in two different HCV replicons (1b and 2b) and displays no activity on other polymerases (HIV-RT, Polio-pol, GBV-b-pol). These inhibitors act during the pre-elongation phase by binding to NS5B non-nucleoside binding site Thumb Site II as demonstrated by crystal structure of compound 1 with the DeltaC55-1b and DeltaC21-2b enzymes and by mutagenesis studies. SAR in this new series reveals inhibitors, such as 20, with low micromolar activity in the HCV replicon and with good activity/toxicity window in cells.
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Affiliation(s)
- Jesus M Ontoria
- Istituto Di Ricerche Di Biologia Molecolare, P. Angeletti, S.p.A. (IRBM-MRL Rome), Via Pontina Km 30,600, I-00040 Pomezia, Italy.
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Delang L, Coelmont L, Neyts J. Antiviral therapy for hepatitis C virus: beyond the standard of care. Viruses 2010; 2:826-866. [PMID: 21994657 PMCID: PMC3185663 DOI: 10.3390/v2040826] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2009] [Revised: 03/09/2010] [Accepted: 03/17/2010] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) represents a major health burden, with an estimated 180 million chronically infected individuals worldwide. These patients are at increased risk of developing liver cirrhosis and hepatocellular carcinoma. Infection with HCV is the leading cause of liver transplantation in the Western world. Currently, the standard of care (SoC) consists of pegylated interferon alpha (pegIFN-α) and ribavirin (RBV). However this therapy has a limited efficacy and is associated with serious side effects. Therefore more tolerable, highly potent inhibitors of HCV replication are urgently needed. Both Specifically Targeted Antiviral Therapy for HCV (STAT-C) and inhibitors that are believed to interfere with the host-viral interaction are discussed.
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Affiliation(s)
| | | | - Johan Neyts
- Rega Institute for Medical Research, KULeuven, Minderbroedersstraat 10, 3000 Leuven, Belgium; E-Mails: (L.D.); (L.C.)
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Abstract
Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)(20)]. Chemical modification of the initial "hit" improved the compound potency to an IC(50) (that is, a concentration that inhibits 50% RdRp activity) of 0.7 microM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC(50) of 5 microM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC(50)s of >100 microM against hepatitis C virus RdRp and human DNA polymerase alpha and beta. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.
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44
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1a/1b subtype profiling of nonnucleoside polymerase inhibitors of hepatitis C virus. J Virol 2010; 84:2923-34. [PMID: 20071590 DOI: 10.1128/jvi.01980-09] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.
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45
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Bikadi Z, Hazai E. Application of the PM6 semi-empirical method to modeling proteins enhances docking accuracy of AutoDock. J Cheminform 2009; 1:15. [PMID: 20150996 PMCID: PMC2820493 DOI: 10.1186/1758-2946-1-15] [Citation(s) in RCA: 371] [Impact Index Per Article: 23.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2009] [Accepted: 09/11/2009] [Indexed: 11/18/2022] Open
Abstract
BACKGROUND Molecular docking methods are commonly used for predicting binding modes and energies of ligands to proteins. For accurate complex geometry and binding energy estimation, an appropriate method for calculating partial charges is essential. AutoDockTools software, the interface for preparing input files for one of the most widely used docking programs AutoDock 4, utilizes the Gasteiger partial charge calculation method for both protein and ligand charge calculation. However, it has already been shown that more accurate partial charge calculation - and as a consequence, more accurate docking- can be achieved by using quantum chemical methods. For docking calculations quantum chemical partial charge calculation as a routine was only used for ligands so far. The newly developed Mozyme function of MOPAC2009 allows fast partial charge calculation of proteins by quantum mechanical semi-empirical methods. Thus, in the current study, the effect of semi-empirical quantum-mechanical partial charge calculation on docking accuracy could be investigated. RESULTS The docking accuracy of AutoDock 4 using the original AutoDock scoring function was investigated on a set of 53 protein ligand complexes using Gasteiger and PM6 partial charge calculation methods. This has enabled us to compare the effect of the partial charge calculation method on docking accuracy utilizing AutoDock 4 software. Our results showed that the docking accuracy in regard to complex geometry (docking result defined as accurate when the RMSD of the first rank docking result complex is within 2 A of the experimentally determined X-ray structure) significantly increased when partial charges of the ligands and proteins were calculated with the semi-empirical PM6 method. Out of the 53 complexes analyzed in the course of our study, the geometry of 42 complexes were accurately calculated using PM6 partial charges, while the use of Gasteiger charges resulted in only 28 accurate geometries. The binding affinity estimation was not influenced by the partial charge calculation method - for more accurate binding affinity prediction development of a new scoring function for AutoDock is needed. CONCLUSION Our results demonstrate that the accuracy of determination of complex geometry using AutoDock 4 for docking calculation greatly increases with the use of quantum chemical partial charge calculation on both the ligands and proteins.
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Affiliation(s)
- Zsolt Bikadi
- Virtua Drug Ltd, Csalogany Street 4C Budapest, Hungary
| | - Eszter Hazai
- Virtua Drug Ltd, Csalogany Street 4C Budapest, Hungary
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Beaulieu PL. Recent advances in the development of NS5B polymerase inhibitors for the treatment of hepatitis C virus infection. Expert Opin Ther Pat 2009; 19:145-64. [PMID: 19441916 DOI: 10.1517/13543770802672598] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
BACKGROUND 170 to 200 million people worldwide are believed to suffer from chronic hepatitis C virus (HCV) infection, a blood-born disease that targets the liver and progresses to organ cirrhosis and primary cancer in a significant proportion of patients. The currently available treatment has limited efficacy and suffers from restricting side effects. HCV infection is the principal cause of liver transplant in industrialized nations and between 8000 and 10,000 deaths result annually from the disease in the United States alone. Virus-specific, more efficacious, and better-tolerated anti-HCV therapies are thus required to address the unmet medical need. OBJECTIVE To review progress achieved since 2005 in the development of HCV NS5B polymerase inhibitors as potential therapy for the treatment of HCV infection with a primary focus on available patent and medical literature. RESULTS/CONCLUSION Several classes of small-molecule inhibitors of HCV NS5B have progressed into clinical development and demonstrated efficacy in reducing viral load in infected patients. The results so far provide an encouraging foundation for the development of novel, more tolerable therapies and addressing emergence of resistance through combination of antiviral agents with complementary mechanisms of action.
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Structural Basis for Resistance of the Genotype 2b Hepatitis C Virus NS5B Polymerase to Site A Non-Nucleoside Inhibitors. J Mol Biol 2009; 390:1048-59. [DOI: 10.1016/j.jmb.2009.06.012] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2009] [Revised: 05/29/2009] [Accepted: 06/03/2009] [Indexed: 12/21/2022]
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Belon CA, Frick DN. Helicase inhibitors as specifically targeted antiviral therapy for hepatitis C. Future Virol 2009; 4:277-293. [PMID: 20161209 PMCID: PMC2714653 DOI: 10.2217/fvl.09.7] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The hepatitis C virus (HCV) leads to chronic liver disease and affects more than 2% of the world's population. Complications of the disease include fibrosis, cirrhosis and hepatocellular carcinoma. Current therapy for chronic HCV infection, a combination of ribavirin and pegylated IFN-alpha, is expensive, causes profound side effects and is only moderately effective against several common HCV strains. Specifically targeted antiviral therapy for hepatitis C (STAT-C) will probably supplement or replace present therapies. Leading compounds for STAT-C target the HCV nonstructural (NS)5B polymerase and NS3 protease, however, owing to the constant threat of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase domain of the HCV NS3 protein. The HCV helicase uses energy derived from ATP hydrolysis to separate based-paired RNA or DNA. This article discusses unique features of the HCV helicase, recently discovered compounds that inhibit HCV helicase catalyzed reactions and HCV cellular replication, and new methods to monitor helicase action in a high-throughput format.
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Affiliation(s)
- Craig A Belon
- New York Medical College, Department of Biochemistry & Molecular Biology, Valhalla, NY 10595, USA, Tel.: +1 914 594 3537; Fax: +1 914 594 4058;
| | - David N Frick
- New York Medical College, Department of Biochemistry & Molecular Biology, Valhalla, NY 10595, USA, Tel.: +1 914 594 4190; Fax: +1 914 594 4058;
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Schinazi RF, Coats SJ, Bassit LC, Lennerstrand J, Nettles JH, Hurwitz SJ. Approaches for the development of antiviral compounds: the case of hepatitis C virus. Handb Exp Pharmacol 2009:25-51. [PMID: 19048196 DOI: 10.1007/978-3-540-79086-0_2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Traditional methods for general drug discovery typically include evaluating random compound libraries for activity in relevant cell-free or cell-based assays. Success in antiviral development has emerged from the discovery of more focused libraries that provide clues about structure activity relationships. Combining these with more recent approaches including structural biology and computational modeling can work efficiently to hasten discovery of active molecules, but that is not enough. There are issues related to biology, toxicology, pharmacology, and metabolism that have to be addressed before a hit compound becomes nominated for clinical development. The objective of gaining early preclinical knowledge is to reduce the risk of failure in Phases 1, 2, and 3, leading to the goal of approved drugs that benefit the infected individual. This review uses hepatitis C virus (HCV), for which we still do not have an ideal therapeutic modality, as an example of the multidisciplinary efforts needed to discover new antiviral drugs for the benefit of humanity.
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Affiliation(s)
- Raymond F Schinazi
- Laboratory of Biochemical Pharmacology, VA Medical Center, Emory University School of Medicine, 1670 Clairmont Road, Decatur, GA 30033, USA.
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1,5-benzodiazepines, a novel class of hepatitis C virus polymerase nonnucleoside inhibitors. Antimicrob Agents Chemother 2008; 52:4420-31. [PMID: 18852280 DOI: 10.1128/aac.00669-08] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors.
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