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.

Figure 1. Pharmacological characterization of BCX4430.

a, BCX4430 chemical structure and properties. LogP, log of partition coefficient. b, Conversion of ³H-BCX4430 or ³H-adenosine to diphosphate (DP) or triphosphate (TP) forms in Huh-7 cells (n = 1). c, Effect of BCX4430 on replication of an EBOV minigenome RNA replicon in cultured cells (n = 6). No L, l-polymerase encoding plasmid omitted from reaction. d, BCX4430-TP suppresses HCV NS5B (NS5B-1b-Δ21) RNA polymerase activity (n = 2). e, RNA products synthesized by purified HCV polymerase, in a template-directed primer (³²P-5′-GG) extension assay. nt, nucleotides. f, Expression of EBOV and MARV glycoprotein in infected HeLa cells (n = 5–6). g, MARV-infected HeLa cells. Green, α-MARV glycoprotein; blue, Hoechst dye. Scale bar, 100 µm. h, Production of intracellular MARV RNA (n = 4) and infectious virus in MARV-infected HeLa cells (n = 2 technical replicates). Data in c, d, and h are expressed as mean + standard deviation (s.d.). Data in f are expressed as mean + standard error of the mean (s.e.m.). PowerPoint slide Source data

Figure 2. BCX4430 in vivo efficacy and pharmacokinetics characterization.

a, Survival of RAVV-infected mice (n = 9–10 per group). BCX4430 treatments (Tx) were administered i.m. twice daily at 150 mg kg⁻¹ doses for 9 days beginning at the indicated time before infection (BI) or post-infection (PI). b, Pharmacokinetics of BCX4430 and BCX4430-TP in Sprague–Dawley rats (n = 3) after single-dose i.m. administration of BCX4430. *P < 0.05 treatment versus vehicle survival curves by log-rank (Mantel–Cox) test. Data in b are expressed as mean ± s.e.m. PowerPoint slide

Figure 3. Post-exposure protection of MARV-infected cynomolgus macaques by BCX4430.

a–f, Animals (n = 6) were challenged with MARV by subcutaneous injection, and BCX4430 (Tx) (15 mg kg⁻¹ twice daily) or vehicle was administered i.m. beginning at the indicated times after challenge. a, Kaplan–Meier survival curves. PI, post-infection. b, Serum viral RNA load. GE, genomic equivalents. c–f, Individual animal maximal values of serum aspartate aminotransferase concentrations (c), conjugated bilirubin concentrations (d), PT (e) and aPTT (f). Non-survivors are represented by open symbols. *P < 0.05 for comparison of treatment versus vehicle by log-rank (Mantel–Cox) test (a), two-tailed analyses using the Holm–Sidak method (b), or two-tailed Kruskal–Wallis test followed by Dunn’s post-test comparison (c–f). Data in b are expressed as geometric mean + s.d. Horizontal bars in c–f represent group means. PowerPoint slide

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.