Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. Protein Expression and Purification
2.3. Data Acquisition, Quantification, and Analysis
3. Results
3.1. Competition between Remdesivir-TP and ATP
3.2. Selectivity Measurements
3.3. Chain Termination
3.4. Delayed Chain Termination
3.5. Susceptibility to Other Nucleotide Analogues
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- WHO. List of Blueprint Priority Diseases. Available online: http://www.who.int/blueprint/priority-diseases/en/ (accessed on 12 March 2019).
- CDC. 2014–2016 Ebola Outbreak in West Africa. Available online: https://www.cdc.gov/vhf/ebola/history/2014-2016-outbreak/index.html (accessed on 12 March 2019).
- Henao-Restrepo, A.M.; Longini, I.M.; Egger, M.; Dean, N.E.; Edmunds, W.J.; Camacho, A.; Carroll, M.W.; Doumbia, M.; Draguez, B.; Duraffour, S.; et al. Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial. Lancet 2015, 386, 857–866. [Google Scholar] [CrossRef]
- Davidson, E.; Bryan, C.; Fong, R.H.; Barnes, T.; Pfaff, J.M.; Mabila, M.; Rucker, J.B.; Doranz, B.J. Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies. J. Virol. 2015, 89, 10982–10992. [Google Scholar] [CrossRef]
- Qiu, X.; Wong, G.; Audet, J.; Bello, A.; Fernando, L.; Alimonti, J.B.; Fausther-Bovendo, H.; Wei, H.; Aviles, J.; Hiatt, E.; et al. Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp. Nature 2014, 514, 47–53. [Google Scholar] [CrossRef]
- Group, P.I.W.; Multi-National, P.I.I.S.T.; Davey, R.T., Jr.; Dodd, L.; Proschan, M.A.; Neaton, J.; Neuhaus Nordwall, J.; Koopmeiners, J.S.; Beigel, J.; Tierney, J.; et al. A Randomized, Controlled Trial of ZMapp for Ebola Virus Infection. N. Engl. J. Med. 2016, 375, 1448–1456. [Google Scholar] [CrossRef]
- Saphire, E.O.; Schendel, S.L.; Gunn, B.M.; Milligan, J.C.; Alter, G. Antibody-mediated protection against Ebola virus. Nat. Immunol. 2018, 19, 1169–1178. [Google Scholar] [CrossRef] [PubMed]
- Kimble, J.B.; Malherbe, D.C.; Meyer, M.; Gunn, B.M.; Karim, M.M.; Ilinykh, P.A.; Iampietro, M.; Mohamed, K.S.; Negi, S.; Gilchuk, P.; et al. Antibody-Mediated Protective Mechanisms Induced by a Trivalent Parainfluenza-Vectored Ebolavirus Vaccine. J. Virol. 2018. [Google Scholar] [CrossRef]
- Siegel, D.; Hui, H.C.; Doerffler, E.; Clarke, M.O.; Chun, K.; Zhang, L.; Neville, S.; Carra, E.; Lew, W.; Ross, B.; et al. Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses. J. Med. Chem. 2017, 60, 1648–1661. [Google Scholar] [CrossRef]
- Warren, T.K.; Jordan, R.; Lo, M.K.; Ray, A.S.; Mackman, R.L.; Soloveva, V.; Siegel, D.; Perron, M.; Bannister, R.; Hui, H.C.; et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature 2016, 531, 381–385. [Google Scholar] [CrossRef] [PubMed]
- Warren, T.K.; Wells, J.; Panchal, R.G.; Stuthman, K.S.; Garza, N.L.; Van Tongeren, S.A.; Dong, L.; Retterer, C.J.; Eaton, B.P.; Pegoraro, G.; et al. Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430. Nature 2014, 508, 402–405. [Google Scholar] [CrossRef]
- Bixler, S.L.; Bocan, T.M.; Wells, J.; Wetzel, K.S.; Van Tongeren, S.A.; Dong, L.; Garza, N.L.; Donnelly, G.; Cazares, L.H.; Nuss, J.; et al. Efficacy of favipiravir (T-705) in nonhuman primates infected with Ebola virus or Marburg virus. Antiviral Res. 2018, 151, 97–104. [Google Scholar] [CrossRef]
- Furuta, Y.; Komeno, T.; Nakamura, T. Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 2017, 93, 449–463. [Google Scholar] [CrossRef]
- Furuta, Y.; Takahashi, K.; Fukuda, Y.; Kuno, M.; Kamiyama, T.; Kozaki, K.; Nomura, N.; Egawa, H.; Minami, S.; Watanabe, Y.; et al. In vitro and in vivo activities of anti-influenza virus compound T-705. Antimicrob. Agents Chemother. 2002, 46, 977–981. [Google Scholar] [CrossRef]
- Agostini, M.L.; Andres, E.L.; Sims, A.C.; Graham, R.L.; Sheahan, T.P.; Lu, X.; Smith, E.C.; Case, J.B.; Feng, J.Y.; Jordan, R.; et al. Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. MBio 2018, 9. [Google Scholar] [CrossRef]
- Lo, M.K.; Jordan, R.; Arvey, A.; Sudhamsu, J.; Shrivastava-Ranjan, P.; Hotard, A.L.; Flint, M.; McMullan, L.K.; Siegel, D.; Clarke, M.O.; et al. GS-5734 and its parent nucleoside analog inhibit Filo-, Pneumo-, and Paramyxoviruses. Sci. Rep. 2017, 7, 43395. [Google Scholar] [CrossRef]
- Julander, J.G.; Siddharthan, V.; Evans, J.; Taylor, R.; Tolbert, K.; Apuli, C.; Stewart, J.; Collins, P.; Gebre, M.; Neilson, S.; et al. Efficacy of the broad-spectrum antiviral compound BCX4430 against Zika virus in cell culture and in a mouse model. Antiviral Res. 2017, 137, 14–22. [Google Scholar] [CrossRef]
- Furuta, Y.; Takahashi, K.; Shiraki, K.; Sakamoto, K.; Smee, D.F.; Barnard, D.L.; Gowen, B.B.; Julander, J.G.; Morrey, J.D. T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections. Antiviral Res. 2009, 82, 95–102. [Google Scholar] [CrossRef]
- Zaraket, H.; Saito, R. Japanese Surveillance Systems and Treatment for Influenza. Curr. Treat. Options Infect. Dis. 2016, 8, 311–328. [Google Scholar] [CrossRef]
- Furuta, Y.; Takahashi, K.; Kuno-Maekawa, M.; Sangawa, H.; Uehara, S.; Kozaki, K.; Nomura, N.; Egawa, H.; Shiraki, K. Mechanism of action of T-705 against influenza virus. Antimicrob. Agents Chemother. 2005, 49, 981–986. [Google Scholar] [CrossRef]
- Jin, Z.; Smith, L.K.; Rajwanshi, V.K.; Kim, B.; Deval, J. The ambiguous base-pairing and high substrate efficiency of T-705 (Favipiravir) Ribofuranosyl 5′-triphosphate towards influenza A virus polymerase. PLoS ONE 2013, 8, e68347. [Google Scholar] [CrossRef]
- Delang, L.; Abdelnabi, R.; Neyts, J. Favipiravir as a potential countermeasure against neglected and emerging RNA viruses. Antiviral Res. 2018, 153, 85–94. [Google Scholar] [CrossRef] [PubMed]
- Guedj, J.; Piorkowski, G.; Jacquot, F.; Madelain, V.; Nguyen, T.H.T.; Rodallec, A.; Gunther, S.; Carbonnelle, C.; Mentré, F.; Raoul, H.; et al. Antiviral efficacy of favipiravir against Ebola virus: A translational study in cynomolgus macaques. PLoS Med. 2018, 15, e1002535. [Google Scholar] [CrossRef] [PubMed]
- Sissoko, D.; Laouenan, C.; Folkesson, E.; M’Lebing, A.B.; Beavogui, A.H.; Baize, S.; Camara, A.M.; Maes, P.; Shepherd, S.; Danel, C.; et al. Experimental Treatment with Favipiravir for Ebola Virus Disease (the JIKI Trial): A Historically Controlled, Single-Arm Proof-of-Concept Trial in Guinea. PLoS Med. 2016, 13, e1001967. [Google Scholar] [CrossRef]
- Feld, J.J.; Jacobson, I.M.; Sulkowski, M.S.; Poordad, F.; Tatsch, F.; Pawlotsky, J.M. Ribavirin revisited in the era of direct-acting antiviral therapy for hepatitis C virus infection. Liver Int. 2017, 37, 5–18. [Google Scholar] [CrossRef]
- Witkowski, J.T.; Robins, R.K.; Sidwell, R.W.; Simon, L.N. Design, synthesis, and broad spectrum antiviral activity of 1- -D-ribofuranosyl-1,2,4-triazole-3-carboxamide and related nucleosides. J. Med. Chem. 1972, 15, 1150–1154. [Google Scholar] [CrossRef] [PubMed]
- Sidwell, R.W.; Huffman, J.H.; Khare, G.P.; Allen, L.B.; Witkowski, J.T.; Robins, R.K. Broad-spectrum antiviral activity of Virazole: 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide. Science 1972, 177, 705–706. [Google Scholar] [CrossRef] [PubMed]
- McHutchison, J.G.; Gordon, S.C.; Schiff, E.R.; Shiffman, M.L.; Lee, W.M.; Rustgi, V.K.; Goodman, Z.D.; Ling, M.H.; Cort, S.; Albrecht, J.K. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N. Engl. J. Med. 1998, 339, 1485–1492. [Google Scholar] [CrossRef]
- Smither, S.J.; Eastaugh, L.S.; Steward, J.A.; Nelson, M.; Lenk, R.P.; Lever, M.S. Post-exposure efficacy of oral T-705 (Favipiravir) against inhalational Ebola virus infection in a mouse model. Antiviral Res. 2014, 104, 153–155. [Google Scholar] [CrossRef]
- Oestereich, L.; Ludtke, A.; Wurr, S.; Rieger, T.; Munoz-Fontela, C.; Gunther, S. Successful treatment of advanced Ebola virus infection with T-705 (favipiravir) in a small animal model. Antiviral Res. 2014, 105, 17–21. [Google Scholar] [CrossRef]
- Jordan, P.C.; Liu, C.; Raynaud, P.; Lo, M.K.; Spiropoulou, C.F.; Symons, J.A.; Beigelman, L.; Deval, J. Initiation, extension, and termination of RNA synthesis by a paramyxovirus polymerase. PLoS Pathog. 2018, 14, e1006889. [Google Scholar] [CrossRef]
- Fearns, R.; Plemper, R.K. Polymerases of paramyxoviruses and pneumoviruses. Virus Res. 2017, 234, 87–102. [Google Scholar] [CrossRef]
- Tchesnokov, E.P.; Raeisimakiani, P.; Ngure, M.; Marchant, D.; Gotte, M. Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus. Sci. Rep. 2018, 8, 3970. [Google Scholar] [CrossRef] [PubMed]
- Noton, S.L.; Deflube, L.R.; Tremaglio, C.Z.; Fearns, R. The respiratory syncytial virus polymerase has multiple RNA synthesis activities at the promoter. PLoS Pathog. 2012, 8, e1002980. [Google Scholar] [CrossRef] [PubMed]
- Deval, J.; Hong, J.; Wang, G.; Taylor, J.; Smith, L.K.; Fung, A.; Stevens, S.K.; Liu, H.; Jin, Z.; Dyatkina, N.; et al. Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2′-Fluoro-4′-Chloromethyl-Cytidine Triphosphate. PLoS Pathog. 2015, 11, e1004995. [Google Scholar] [CrossRef]
- Rahmeh, A.A.; Morin, B.; Schenk, A.D.; Liang, B.; Heinrich, B.S.; Brusic, V.; Walz, T.; Whelan, S.P. Critical phosphoprotein elements that regulate polymerase architecture and function in vesicular stomatitis virus. Proc. Natl. Acad. Sci. USA 2012, 109, 14628–14633. [Google Scholar] [CrossRef] [PubMed]
- Muhlberger, E.; Weik, M.; Volchkov, V.E.; Klenk, H.D.; Becker, S. Comparison of the transcription and replication strategies of marburg virus and Ebola virus by using artificial replication systems. J. Virol. 1999, 73, 2333–2342. [Google Scholar] [PubMed]
- Smidansky, E.D.; Arnold, J.J.; Reynolds, S.L.; Cameron, C.E. Human mitochondrial RNA polymerase: evaluation of the single-nucleotide-addition cycle on synthetic RNA/DNA scaffolds. Biochemistry 2011, 50, 5016–5032. [Google Scholar] [CrossRef] [PubMed]
- Berger, I.; Fitzgerald, D.J.; Richmond, T.J. Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 2004, 22, 1583–1587. [Google Scholar] [CrossRef]
- Bieniossek, C.; Richmond, T.J.; Berger, I. MultiBac: Multigene baculovirus-based eukaryotic protein complex production. Curr. Protoc. Protein Sci. 2008, 51, 5–20. [Google Scholar]
- Carroll, S.S.; Tomassini, J.E.; Bosserman, M.; Getty, K.; Stahlhut, M.W.; Eldrup, A.B.; Bhat, B.; Hall, D.; Simcoe, A.L.; LaFemina, R.; et al. Inhibition of hepatitis C virus RNA replication by 2′-modified nucleoside analogs. J. Biol. Chem. 2003, 278, 11979–11984. [Google Scholar] [CrossRef]
- Fung, A.; Jin, Z.; Dyatkina, N.; Wang, G.; Beigelman, L.; Deval, J. Efficiency of incorporation and chain termination determines the inhibition potency of 2′-modified nucleotide analogs against hepatitis C virus polymerase. Antimicrob. Agents Chemother. 2014, 58, 3636–3645. [Google Scholar] [CrossRef]
- Deval, J.; Symons, J.A.; Beigelman, L. Inhibition of viral RNA polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand RNA viruses beyond hepatitis C virus. Curr. Opin. Virol. 2014, 9, 1–7. [Google Scholar] [CrossRef] [PubMed]
- Uebelhoer, L.S.; Albarino, C.G.; McMullan, L.K.; Chakrabarti, A.K.; Vincent, J.P.; Nichol, S.T.; Towner, J.S. High-throughput, luciferase-based reverse genetics systems for identifying inhibitors of Marburg and Ebola viruses. Antiviral Res. 2014, 106, 86–94. [Google Scholar] [CrossRef]
- Bixler, S.L.; Duplantier, A.J.; Bavari, S. Discovering Drugs for the Treatment of Ebola Virus. Curr Treat. Options Infect. Dis. 2017, 9, 299–317. [Google Scholar] [CrossRef] [PubMed]
- Jin, Z.; Kinkade, A.; Behera, I.; Chaudhuri, S.; Tucker, K.; Dyatkina, N.; Rajwanshi, V.K.; Wang, G.; Jekle, A.; Smith, D.B.; et al. Structure-activity relationship analysis of mitochondrial toxicity caused by antiviral ribonucleoside analogs. Antiviral Res. 2017, 143, 151–161. [Google Scholar] [CrossRef] [PubMed]
- Jin, Z.; Tucker, K.; Lin, X.; Kao, C.C.; Shaw, K.; Tan, H.; Symons, J.; Behera, I.; Rajwanshi, V.K.; Dyatkina, N.; et al. Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2′-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases. Antimicrob. Agents Chemother. 2015, 59, 7504–7516. [Google Scholar] [CrossRef] [PubMed]
- Furuta, Y.; Gowen, B.B.; Takahashi, K.; Shiraki, K.; Smee, D.F.; Barnard, D.L. Favipiravir (T-705), a novel viral RNA polymerase inhibitor. Antiviral Res. 2013, 100, 446–454. [Google Scholar] [CrossRef]
- Tchesnokov, E.P.; Obikhod, A.; Schinazi, R.F.; Gotte, M. Delayed chain termination protects the anti-hepatitis B virus drug entecavir from excision by HIV-1 reverse transcriptase. J. Biol. Chem. 2008, 283, 34218–34228. [Google Scholar] [CrossRef] [PubMed]
- Domaoal, R.A.; McMahon, M.; Thio, C.L.; Bailey, C.M.; Tirado-Rives, J.; Obikhod, A.; Detorio, M.; Rapp, K.L.; Siliciano, R.F.; Schinazi, R.F.; et al. Pre-steady-state kinetic studies establish entecavir 5′-triphosphate as a substrate for HIV-1 reverse transcriptase. J. Biol. Chem. 2008, 283, 5452–5459. [Google Scholar] [CrossRef]
- Seifer, M.; Hamatake, R.K.; Colonno, R.J.; Standring, D.N. In vitro inhibition of hepadnavirus polymerases by the triphosphates of BMS-200475 and lobucavir. Antimicrob. Agents Chemother. 1998, 42, 3200–3208. [Google Scholar] [CrossRef]
- Innaimo, S.F.; Seifer, M.; Bisacchi, G.S.; Standring, D.N.; Zahler, R.; Colonno, R.J. Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus. Antimicrob. Agents Chemother. 1997, 41, 1444–1448. [Google Scholar] [CrossRef]
- McMahon, M.A.; Jilek, B.L.; Brennan, T.P.; Shen, L.; Zhou, Y.; Wind-Rotolo, M.; Xing, S.; Bhat, S.; Hale, B.; Hegarty, R.; et al. The HBV drug entecavir—effects on HIV-1 replication and resistance. N Engl. J. Med. 2007, 356, 2614–2621. [Google Scholar] [CrossRef] [PubMed]
RNA Polymerase | EBOV | RSV | h-mtRNAP | ||||
---|---|---|---|---|---|---|---|
Substrate | ATP | Remdesivir-TP | ATP | Remdesivir-TP | ATP | Remdesivir-TP | |
Vmaxa (product fraction) | 0.84 d ± 0.027 e (3%) f | 0.75 ± 0.039 (5%) | 0.76 ± 0.022 (3%) | 0.82 ± 0.027 (3%) | 0.98 ± 0.018 (2%) | 0.81 ± 0.013 (2%) | |
Kmb (μM) | 1.5 ± 0.23 (15%) | 5.7 ± 1.1 (19%) | 0.17 ± 0.023 (14%) | 0.50 ± 0.089 (18%) | 0.050 ± 0.0037 (7%) | 21 ± 0.096 (5%) | |
Vmax/Km | 0.56 | 0.15 | 4.5 | 1.6 | 19.6 | 0.039 | |
Selectivity c (fold) | Ref. g | 3.8 | Ref. | 2.7 | Ref. | 508 |
RNA Polymerase | EBOV | RSV | |||
---|---|---|---|---|---|
Primer 3′-end (base) | A | Remdesivir | A | Remdesivir | |
Substrate | UTP | ||||
Vmaxa (product fraction) | 0.64 d ± 0.020 e (3%) f | 0.65 ± 0.016 (3%) | 0.69 ± 0.017 (3%) | 0.61 ± 0.014 (2%) | |
Kmb (μM) | 1.2 ± 0.18 (15%) | 0.51 ± 0.07 (15%) | 1.0 ± 0.12 (12%) | 5.5 ± 0.49 (9%) | |
Vmax/Km | 0.53 | 1.3 | 0.69 | 0.11 | |
Inhibition c (fold) | Ref. g | 0.42 | Ref. | 6.2 |
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Tchesnokov, E.P.; Feng, J.Y.; Porter, D.P.; Götte, M. Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir. Viruses 2019, 11, 326. https://doi.org/10.3390/v11040326
Tchesnokov EP, Feng JY, Porter DP, Götte M. Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir. Viruses. 2019; 11(4):326. https://doi.org/10.3390/v11040326
Chicago/Turabian StyleTchesnokov, Egor P., Joy Y. Feng, Danielle P. Porter, and Matthias Götte. 2019. "Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir" Viruses 11, no. 4: 326. https://doi.org/10.3390/v11040326
APA StyleTchesnokov, E. P., Feng, J. Y., Porter, D. P., & Götte, M. (2019). Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir. Viruses, 11(4), 326. https://doi.org/10.3390/v11040326