Abstract

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae1. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg−1 GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

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Accessions

Data deposits

Viral genomic sequences have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and accession numbers are supplied in Extended Data Table 5. Small molecule X-ray crystallographic coordinates and structure factor files have been deposited in the Cambridge Structural Database (http://www.ccdc.cam.ac.uk/) and accession numbers are supplied in the Supplementary Information.

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Acknowledgements

T. Bocan, A. Duplantier, R. Panchal, and C. Kane provided scientific input. B. Norquist assisted with manuscript preparation. C. Cooper provided scientific input with human macrophage cultures for high-content image assessments. S. Tritsch and G. Gomba assisted with GS-5734 dose preparations for efficacy studies. C. Rice provided animal husbandry support services. X. Wei, W. Garner, and L. Zhong provided additional support for statistical analyses. K. Wang, K. Brendza, T. Alfredson, and L. Serafini assisted with analytical methods; S. Bondy and R. Seemayer procured key raw materials; L. Heumann, R. Polniaszeck, E. Rueden, A. Chtchemelinine, K. Brak, and B. Hoang contributed to synthesis; and Y. Zherebina helped with chiral separations. G. Lee supported the RSV antiviral assay, and G. Stepan, S. Ahmadyar, and H. Yu conducted part of the cytotoxicity testing. J. Knox contributed to polymerase modelling. A. L. Rheingold performed the X-ray crystallographic analysis (Supplementary Information). Studies at USAMRIID were in part supported by The Joint Science and Technology Office for Chemical and Biological Defense (JSTO-CBD) of the Defense Threat Reduction Agency (DTRA) under plan #CB10218. Work in the Fearns laboratory was supported by NIH R01AI113321. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army or the Centers for Disease Control and Prevention, US Department of Health and Human Services.

Author information

Author notes

    • Lisa S. Welch
    •  & Douglas L. Mayers

    Present addresses: LOKET Consulting, Clarksburg, Maryland 20871, USA (L.S.W.); Cocrystal Pharma, Tucker, Georgia 30084, USA (D.L.M).

Affiliations

  1. United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21702, USA

    • Travis K. Warren
    • , Veronica Soloveva
    • , Jay Wells
    • , Kelly S. Stuthman
    • , Sean A. Van Tongeren
    • , Nicole L. Garza
    • , Ginger Donnelly
    • , Amy C. Shurtleff
    • , Cary J. Retterer
    • , Dima Gharaibeh
    • , Rouzbeh Zamani
    • , Tara Kenny
    • , Brett P. Eaton
    • , Elizabeth Grimes
    • , Lisa S. Welch
    • , Laura Gomba
    • , Catherine L. Wilhelmsen
    • , Donald K. Nichols
    • , Jonathan E. Nuss
    • , Elyse R. Nagle
    • , Jeffrey R. Kugelman
    • , Gustavo Palacios
    • , Douglas L. Mayers
    •  & Sina Bavari
  2. United States Army Medical Research Institute of Infectious Diseases, Therapeutic Development Center, Frederick, Maryland 21702, USA

    • Travis K. Warren
    • , Veronica Soloveva
    • , Laura Gomba
    • , Jonathan E. Nuss
    •  & Sina Bavari
  3. Gilead Sciences, Foster City, California 94404, USA

    • Robert Jordan
    • , Adrian S. Ray
    • , Richard L. Mackman
    • , Dustin Siegel
    • , Michel Perron
    • , Roy Bannister
    • , Hon C. Hui
    • , Nate Larson
    • , Robert Strickley
    • , Edward Doerffler
    • , Sean Neville
    • , Ernest Carra
    • , Michael O. Clarke
    • , Lijun Zhang
    • , Willard Lew
    • , Bruce Ross
    • , Queenie Wang
    • , Kwon Chun
    • , Lydia Wolfe
    • , Darius Babusis
    • , Yeojin Park
    • , Kirsten M. Stray
    • , Iva Trancheva
    • , Joy Y. Feng
    • , Ona Barauskas
    • , Yili Xu
    • , Pamela Wong
    • , Shan-Shan Chen
    • , Swami Swaminathan
    • , William A. Lee
    •  & Tomas Cihlar
  4. Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA

    • Michael K. Lo
    • , Mike Flint
    • , Laura K. McMullan
    • , Christina F. Spiropoulou
    •  & Stuart T. Nichol
  5. Boston University School of Medicine, Boston, Massachusetts 02118, USA

    • Molly R. Braun
    •  & Rachel Fearns

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Contributions

R.L.M., D.S., H.C.H., E.D., S.N., E.C., M.O.C., L.Z., W.L., B.S., Q.W., K.C., and L.W. were responsible for the synthesis, characterization, and scale-up of small molecules. T.K.W. designed and supervised activities associated with efficacy evaluations, and interpreted study results. J.W., K.S.S., N.L.G., G.D., S.A.V.T., and J.E.N. conducted in vivo efficacy studies and performed associated sample analyses. A.C.S., L.S.W., and L.G. coordinated efficacy study activities. M.K.L., M.F., L.K.M., designed and executed the initial in vitro antiviral testing against EBOV and analysed data. V.S., R.Z., C.J.R., D.G, T.K., and B.P.E. designed and executed cell-based infection assays and analysed these data. E.G. conducted quantitative PCR analysis. D.K.N. and C.L.W. performed anatomic pathology examinations and analyses of all nonhuman primate subjects. E.R.N., J.R.K., and G.P. conducted viral genomic sequence analyses. N.L., I.T., and R.S. developed and tested drug formulations. A.S.R., D.B., Y.P., and K.M.S. designed and executed the pharmacokinetic and metabolism studies and summarized results. M.P., O.B., M.R.B., and R.F. designed and conducted biochemical enzymatic assays. K.M.S., J.Y.F., and Y.X. conducted cell-based assays for cytotoxicity. P.W. conducted statistical analysis and S.-S.C. oversaw the analysis. A.S.R., R.J., R.L.M., V.S., R.B., S.S., D.L.M., C.F.S., S.T.N., W.A.L., T.C., and S.B. designed experiments, evaluated results, and provided project oversight. T.K.W., A.S.R., R.J., D.S., M.P., and T.C. outlined and wrote the manuscript.

Competing interests

The authors affiliated with Gilead Sciences are employees of the company and may own company stock.

Corresponding authors

Correspondence to Tomas Cihlar or Sina Bavari.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Methods describing the synthesis of GS-5734 and the analytical data that support the synthetic steps.

  2. 2.

    Supplementary Information

    This file contains (1) Supplementary Figure, which shows the labeled uncropped gel depicted in Figure 1f and (2) Supplementary Table 1, which shows the list of primary and secondary antibodies used in immunostaining assay.

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DOI

https://doi.org/10.1038/nature17180

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