Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430

Abstract

Filoviruses are emerging pathogens and causative agents of viral haemorrhagic fever. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, exceeding 90% (ref. 1). Licensed therapeutic or vaccine products are not available to treat filovirus diseases. Candidate therapeutics previously shown to be efficacious in non-human primate disease models are based on virus-specific designs and have limited broad-spectrum antiviral potential. Here we show that BCX4430, a novel synthetic adenosine analogue, inhibits infection of distinct filoviruses in human cells. Biochemical, reporter-based and primer-extension assays indicate that BCX4430 inhibits viral RNA polymerase function, acting as a non-obligate RNA chain terminator. Post-exposure intramuscular administration of BCX4430 protects against Ebola virus and Marburg virus disease in rodent models. Most importantly, BCX4430 completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 hours after infection. In addition, BCX4430 exhibits broad-spectrum antiviral activity against numerous viruses, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. This is the first report, to our knowledge, of non-human primate protection from filovirus disease by a synthetic drug-like small molecule. We provide additional pharmacological characterizations supporting the potential development of BCX4430 as a countermeasure against human filovirus diseases and other viral diseases representing major public health threats.

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Figure 1: Pharmacological characterization of BCX4430.
Figure 2: BCX4430 in vivo efficacy and pharmacokinetics characterization.
Figure 3: Post-exposure protection of MARV-infected cynomolgus macaques by BCX4430.

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Acknowledgements

J. Kuhn and J. Huggins provided insightful discussions and critically reviewed the manuscript. R. Kincaid and G. Feuerstein provided advice and guidance for BCX4430 development efforts. These studies were in part supported by The Joint Science and Technology Office for Chemical and Biological Defense of the Defense Threat Reduction Agency (proposal #TMTI0048_09_RD_T and CB3675) to S. Bavari. S. Radoshitzky assisted with the EBOV minigenome replicon assay. J. Reifman was essential to algorithm development of HCI image assessments. C. Basler provided the BHK-21-derived cell line constitutively expressing the T7 RNA polymerase. Plasmids encoding viral products and the EBOV minigenome replicon were provided by P. Kranzusch and S. Whelan. Neutral-red uptake antiviral assays were conducted by: D. L. Barnard, G. W. Day, B. Gowan, J. G. Julander, B. Tarbet, D. F. Smee and J. D. Morrey of Utah State University under National Institute of Allergy and Infectious Diseases (NIAID) contract HHSN272201100019I, BioQual Inc. under NIAID contract HHSN27220110005I, and at the University of Alabama Birmingham under NIAID contract HHSN272201100016I. Cell-based metabolism studies were conducted by C. Parker, X. Cheng, R. Upshaw and Y. Luo. A. Nalca, E. E. Zumbrun, H. Bloomfield, D. Dyer and J. Yeager assisted with virus aerosolization. C. Cooper provided assistance with the culture of human macrophage cell culture and R. Zamani provided assistance with high-content image assessments. S. Tritsch assisted with Nipah virus antiviral assays. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the US Army.

Author information

Y.S.B. and P.K. were responsible for the synthesis of BCX4430 and other small molecules. T.K.W. designed and supervised activities associated with rodent and non-human primate efficacy evaluations, evaluated study results, and wrote the manuscript. J.W., K.S.D., N.L.G. and S.A.V.T. conducted the rodent and non-human primate efficacy studies and performed sample analyses. R.G.P., G.P., C.J.R. and B.P.E. designed and executed cell-based filovirus assays and analysed these data. S. Bantia, Y.S.B., D.M.M., W.P.S., B.R.T. and others designed and analysed data from cell-based antiviral assays. L.D. conducted quantitative PCR analysis. B.R.T. conducted statistical evaluations of in vivo study results. S.H. performed post-mortem analysis of all non-human primate subjects. Y.S.B. supervised the pharmacokinetics studies of BCX4430 and W.P.S. conducted pharmacokinetics data analysis. S. Bantia conducted assessments of BCX4430 metabolite analysis and incorporation into host nucleic acids. X.C. conducted chain termination experiments. T.K.W., D.M.M., L.S.W., B.R.T., Y.S.B., W.P.S. and S. Bavari designed experiments, evaluated results and provided project oversight.

Correspondence to Sina Bavari.

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Competing interests

S. Bantia, P.K., Y.S.B. and BioCryst Pharmaceuticals, Inc. claim intellectual property regarding BCX4430 for treatment of viral infections. S. Bantia, X.C., P.K., Y.S.B. and W.P.S. are employees of BioCryst Pharmaceuticals, Inc.

Extended data figures and tables

Extended Data Figure 1 Phosphorylation and antiviral mechanism of action of BCX4430.

a, Conversion of BCX4430 to the active triphosphate (TP) form in cultured cell lines and fresh primary hepatocytes (n = 3–6). Right axis, values normalized to mean 24 h value for human hepatocytes. b, Expression of EBOV NP and VP35 in an EBOV minigenome RNA replicon assay, in BHK-21-derived cells (n = 6). Right three lanes, plasmids expressing the indicated viral protein were omitted from the transfection mix. Gel image cropped for clarity. c, RNA products synthesized by purified HCV polymerase, in a template-directed primer (32P-5′-GG) extension assay. d, Production of intra- and extracellular MARV RNA and cell-surface expression of viral glycoprotein in MARV-infected HeLa cells (n = 4) treated with BCX4430 either 18 h before infection, or 1, 12 or 24 h after infection. e, Expression of EBOV glycoprotein in monocyte-derived primary human macrophages (n = 4). f, Incorporation of 3H-BCX4430 (3H-4430) or 3H-adenosine (3H-AD) in human Huh-7 cells after 24 h incubation (n = 1). CPM, counts per min. Percentage inhibition assessed against the average of medium-only infection-control wells. Data in a are expressed as mean ± s.d. Data in d are expressed as mean + s.e.m. Data in e are expressed as mean ± s.e.m. Source data

Extended Data Figure 2 Broad-spectrum antiviral activity of BCX4430.

Antiviral activity was assessed in cell-based assays (n = 3–5; n = 2, MERS-CoV), either using high-content image-based analysis or neutral-red uptake, using cell lines described in Methods. Cells were pre-treated with BCX4430 for 18 h before infection. Definitions of virus abbreviations are provided in Methods. Except for the top row, viruses are arranged in rows by taxonomic family. Percentage inhibition of BCX4430-treatment wells was assessed against the average of medium-only infection-control wells. Negative inhibition values were transformed to zero for curve fit analysis and display. Data are expressed as mean + s.e.m. Source data

Extended Data Figure 3 Efficacy of BCX4430 in mouse disease models.

a, b, BCX4430 dose versus survival of RAVV-infected mice (a, n = 9–10). BCX4430 treatments (Tx) were administered for 9 days beginning 4 h before infection. Numbers in panel a indicate mg kg−1 doses. c, Survival of mice (n = 10) infected with EBOV. BCX4430 was administered twice daily i.m. or orally at a dose of 150 mg kg−1. d, Survival of mice (n = 10) infected with RVFV. BCX4430 was administered twice daily at doses of 5–150 mg kg−1 by i.m. injection. *P < 0.05 for comparison of treatment versus vehicle survival curves by log-rank (Mantel–Cox) test.

Extended Data Figure 4 In vivo activity of BCX4430 in guinea pigs and cynomolgus macaques.

a, b, Survival of guinea pigs (n = 8 per group) infected by i.p. injection with MARV-Musoke (a) or by exposure to aerosolized MARV-Angola (b). BCX4430 (i.m., 50 mg kg−1 twice daily) treatments (Tx) began at the indicated times before infection (BI) or post-infection (PI). c, Pharmacokinetics of BCX4430 in guinea pigs and cynomolgus macaques (n = 3) after single-dose i.m. administration. d, Individual animal maximal values of interferon-α2a in MARV-infected cynomolgus macaques. *P < 0.05 for comparison of treatment versus vehicle survival curves by log-rank (Mantel–Cox) test. Data in c are expressed as mean ± s.e.m. Horizontal bars in d represent group means.

Extended Data Table 1 In vitro metabolic stability of BCX4430 in liver S9 fractions
Extended Data Table 2 BCX4430 and BCX4430-TP pharmacokinetics

Supplementary information

Supplementary Information

This file contains Supplementary Methods describing the synthesis of BCX4430 and the analytical data that support the synthetic steps, Supplementary Figures 1-29 and additional references. (PDF 6744 kb)

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Warren, T., Wells, J., Panchal, R. et al. Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430. Nature 508, 402–405 (2014) doi:10.1038/nature13027

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