Members of the family Filoviridae include Ebola virus (EBOV), Marburg virus (MARV), Ravn virus (RAVV), Sudan virus (SUDV) and Bundibugyo virus (BDBV), all of which cause severe viral haemorrhagic fevers in humans. In nature, filoviruses are transmitted by physical contact between infected individuals, presumably via infected bodily fluids2. Initial filovirus disease manifestations include fever, headache, vomiting and diarrhoea3. Fatal cases are characterized by viraemia, elevated liver-associated enzyme levels, coagulopathy and haemorrhage. Filovirus disease outbreaks occur sporadically, most frequently in sub-Saharan Africa, with reported case fatality rates exceeding 90% (ref. 1). In 2012, simultaneous outbreaks involved MARV, SUDV and BDBV, an emergent filovirus isolated in 20074. The historical occurrence of independent and simultaneous emergence of distinct filoviruses highlights the need for the identification and development of an efficacious broad-spectrum antiviral product.

Although no licensed antiviral preventative or therapeutic agents are currently available to combat filovirus disease in humans, a number of candidates are being developed and have been evaluated in non-human primate filovirus disease models. These models closely reproduce the known clinical and pathophysiological aspects of fatal human infection. Nucleic-acid-based products, antibody therapies and therapeutic vaccines have successfully protected non-human primates from filovirus disease5,6,7,8,9,10, but these approaches rely on virus-specific designs that inherently limit the spectrum of activity and potential utility of individual treatments. The development of a single therapeutic agent active against multiple filoviruses would provide a key, cost-effective component of public-health preparedness plans in outbreak-prone regions.

The broad-spectrum antiviral agent ribavirin, a trizole nucleoside effective against multiple pathogenic RNA viruses, is not active against filoviruses11. Other small molecules—including the adenosine analogue 3-deazaneplanocin A (c3-Npc A) and T-705 (favipiravir), a substituted pyrazine compound —have conferred a high-degree of protection against filoviruses in rodents but have not been reported to protect non-human primates12,13,14,15,16,17,18.

BCX4430, a novel nucleoside analogue (Fig. 1a), was synthesized (Supplementary Information) as part of a small-molecule library designed as inhibitors of viral RNA polymerase activity. BCX4430 is designed to inhibit viral RNA polymerase activity indirectly through non-obligate RNA chain termination, a mechanism requiring anabolism of the parent compound to BCX4430-triphosphate (BCX4430-TP). Then, after pyrophosphate cleavage, incorporation of BCX4430-monophosphate (BCX4430-MP) into nascent viral RNA strands would be expected to cause premature termination of transcription and replication of viral RNA. In support of this proposed mechanism, BCX4430 is rapidly phosphorylated to BCX4430-TP in cultured cell lines and primary hepatocytes, similar to the natural adenosine nucleoside (Fig. 1b and Extended Data Fig. 1a). Addition of BCX4430 reduces expression of green fluorescent protein (GFP) in an artificial EBOV minigenome replicon assay (Fig. 1c and Extended Data Fig. 1b), in which virion structural proteins comprising the viral ribonucleoprotein complex mediate transcription and replication of an RNA replicon template containing a GFP-reporter cassette. BCX4430-TP inhibits hepatitis C virus (HCV) RNA polymerase transcriptional activity in a cell-free, isolated enzyme transcription assay (Fig. 1d) (an isolated filovirus RNA polymerase enzyme assay has yet to be reported) and induces premature termination of RNA chain synthesis by HCV RNA polymerase during template-directed primer-extension assays (Fig. 1e and Extended Data Fig. 1c). In virus-infected cells, BCX4430 reduces surface-expressed MARV and EBOV glycoprotein and reduces the production of intracellular and extracellular MARV RNA (Fig. 1f–h and Extended Data Fig. 1d). Additionally, HeLa cells incubated with ≥25 µM BCX4430 produce no detectable infectious MARV virus (concentration providing 90% inhibition (IC90) = 5.4 µM) (Fig. 1h).

Figure 1: Pharmacological characterization of BCX4430.
figure 1

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.).

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Source data

Taken together, these assessments strongly support our hypothesis that BCX4430 inhibits viral RNA polymerase function by inducing RNA chain termination. Findings from primer-extension reactions suggest that termination occurs two bases after incorporation of BCX4430-MP, perhaps as a result of inhibitory stereochemical distortions of the nascent RNA chain. We observed no evidence of BCX4430-MP incorporation into human RNA or DNA on exposing human Huh-7 cells to concentrations of BCX4430 exceeding the MARV IC50 by more than tenfold (Extended Data Fig. 1f). The basis of the selectivity of BCX4430 for viral polymerases is not yet known.

To assess the spectrum of antiviral activity of BCX4430, we conducted additional high-content image (HCI)-based and neutral-red uptake antiviral assays using an array of other human viruses. BCX4430 exhibited antiviral activity against negative-sense RNA viruses representing the Filoviridae, Arenaviridae, Bunyaviridae, Orthomyxoviridae, Picornaviridae and Paramyxoviridae families, and positive-sense RNA viruses of the Flaviviridae and Coronaviridae families (Table 1 and Extended Data Fig. 2). Antiviral effects were specific and did not result from cellular toxicity or anti-proliferative effects, as assessed by enumeration of live cells during all HCI assays and by the lactate dehydrogenase (LDH) release assay. In HeLa cells, the 50% cytotoxic concentration (CC50) of BCX4430 exceeded 500 µM as determined by the LDH assay, and consistently exceeded 100 µM using the HCI-based assay (Table 1). An antiviral effect was confirmed in infected human macrophages cultured from normal peripheral blood monocytes, using EBOV (Extended Data Fig. 1e). It is important to note that immortalized cell lines exhibit a substantially attenuated capacity to convert BCX4430 to its active TP form, compared with primary hepatocytes (Extended Data Fig. 1a). Thus, the results obtained from antiviral assessments relying on immortalized cells may underrepresent the antiviral potency of BCX4430 achievable in vivo during experimental or clinical infections.

Table 1 BCX4430 antiviral activity

In preparation for in vivo pharmacokinetics and efficacy testing, we sought to further characterize potential adverse pharmacological features of BCX4430 that could limit its utility in vivo or clinically. BCX4430 exhibited no mutagenicity when tested using the Ames assay at 5 mg per plate, produced no detectable chromosomal aberrations in human lymphocytes at a concentration of 787 µM, and did not inhibit the hERG ion channel current at 30 µM (data not shown). The compound is metabolically stable (half-life (t1/2) > 54 min) when incubated with S9 liver fractions obtained from various animal species (Extended Data Table 1). In mouse, rat, guinea pig and cynomolgus macaque, BCX4430 pharmacokinetics were characterized by rapid clearance from the plasma with a half-life of <5 min (Fig. 2b, Extended Data Fig. 4c and Extended Data Table 2). By contrast, the half-life of the active BCX4430-TP form in the liver in rats was substantially longer at 6.2 h (Fig. 2b).

Figure 2: BCX4430 in vivo efficacy and pharmacokinetics characterization.
figure 2

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.

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Rodent models of filovirus disease do not always reliably reproduce the pathophysiology observed in humans and non-human primates. Nevertheless, rodent systems can provide proof-of-concept efficacy and inform dose and treatment-regimen selection for non-human primate efficacy trials. In RAVV-infected mice, intramuscular (i.m.) administration of BCX4430 conferred a high degree of protection (75–100%) in groups treated prophylactically at doses ≥3.3 mg kg−1 (Extended Data Fig. 3a, b). Notably, significant protection (P < 0.05) was also observed with doses of 150 mg kg−1 initiated at times as long as 96 h after infection (Fig. 2a). Additionally, we demonstrated that mice administered BCX4430 either i.m. or orally were protected against lethal EBOV challenge and that mice receiving i.m. treatments were protected against lethal Rift Valley fever virus challenge (Extended Data Fig. 3c, d).

The in vivo post-exposure efficacy of BCX4430 was further verified in two guinea pig models of MARV disease19 in which animals were challenged either by intraperitoneal (i.p.) injection, or, because filoviruses can be transmitted via aerosolized particles20, by exposure to aerosolized virus. In both the i.p. and aerosolized-virus challenge models, BCX4430 conferred significant post-exposure protection when treatment was initiated within 48 h of virus exposure (Extended Data Fig. 4a). In the respiratory exposure model, BCX4430 protected guinea pigs when treatment initiation was delayed to 72 h after infection, the longest delay tested (Extended Data Fig. 4b).

We further explored the post-exposure efficacy of BCX4430 using the cynomolgus macaque MARV disease model, which accurately reproduces filovirus disease manifestations observed in fatal human cases21. Cynomolgus macaques were experimentally infected with a lethal dose of wild-type MARV (Musoke variant), derived from a human clinical isolate22, and animals were administered with 15 mg kg−1 BCX4430 twice daily by i.m. injection beginning 1–48 h after infection and continuing for 14 days. The six infection-control subjects succumbed by day 12 (Fig. 3a), after having developed viraemia (Fig. 3b) and characteristic signs of filovirus disease, including behavioural inactivity, maculopapular rash, increases in liver injury markers such as aspartate aminotransferase and bilirubin (Fig. 3c, d), and prolongations of prothrombin time (PT) and activated partial thromboplastin time (aPTT) (Fig. 3e, f). All animals treated with BCX4430 beginning 24 or 48 h after infection survived. Five out of six (83%) animals treated with BCX4430 beginning 1 h after infection survived (Fig. 3a). Consistent with the proposed antiviral mechanism of action, BCX4430 significantly and substantially reduced serum MARV burden (Fig. 3b), without inducing type I interferon responses (Extended Data Fig. 4d), as has been observed for c3-Npc A in mice15. Additionally, BCX4430 ameliorated haemorrhagic disease manifestations, as evidenced by shorter PT and aPTT times and reduced laboratory indices of liver damage, such as serum AST and bilirubin concentrations, compared with vehicle treatment (Fig. 3c–f).

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

af, 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. cf, 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 (cf). Data in b are expressed as geometric mean + s.d. Horizontal bars in cf represent group means.

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Outbreaks in 2012 involving MARV, SUDV and BDBV reported in Uganda and the Democratic Republic of the Congo highlight the urgent need for development of an effective antiviral product to counter filovirus disease. For the first time, to our knowledge, we report the identification of a small molecule with efficacy against filovirus disease in non-human primates. Furthermore, we have provided evidence that BCX4430 exhibits broad-spectrum antiviral activity against other highly virulent RNA viruses (Table 1 and Extended Data Fig. 2). Additional evaluations are in progress to assess the in vivo efficacy of BCX4430 against EBOV and other highly virulent pathogens in non-human primates or other disease models that most closely recapitulate human disease.

BCX4430 was well tolerated, producing no overt signs of systemic toxicity or adverse local reactions in any of the efficacy studies. The substantial efficacy of BCX4430 observed with the i.m. route, which provides high bioavailability and rapid absorption, is conducive for use during outbreaks, potentially enabling administration by individuals lacking advanced medical training. In MARV-infected cynomolgus macaques, we did not observe any diminution of protective effects with delay of BCX4430 treatment initiation up to 48 h after infection, suggesting that even greater delays of treatment, yet to be tested, may also yield significant protective benefit. In preparation for advancing BCX4430 into human phase 1 clinical trials in the United States, additional studies are in progress to support filing of an Investigational New Drug Application.

Methods Summary

BCX4430 was synthesized by BioCryst Pharmaceuticals. Cell-based infection assays were conducted using HCI-based analyses as described previously23 for filoviruses and Rift Valley fever virus. For other viruses, inhibition of virus-induced cytopathic effect (CPE) was assessed in cell-based assays using neutral-red uptake, as described previously24. Replication of EBOV minigenome RNA constructs was assayed by using a plasmid-based reconstituted replication/transcription system25. Inhibition of RNA transcriptional activity was assessed in an isolated HCV polymerase assay26 and inhibition of HCV polymerase RNA synthesis activity was evaluated using a template-driven 32P-GG primer extension assay. Mouse models of EBOV, MARV and RAVV disease have been described previously27,28. Experiments were conducted using mouse-adapted strains of EBOV (Mayinga variant) and RAVV (Ravn variant). A guinea pig MARV-Musoke parenteral challenge model and virus aerosolization methods used for guinea pig exposures have been previously described19,29. BCX4430 was administered in a vehicle of sterile 0.9% saline for injection for all in vivo applications. Animal infection experiments were performed in biosafety level 4 containment facilities at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). All experimental treatment replication was conducted using biological replication, except for the virus-yield reduction assay (Fig. 1h), which relied on technical replication.

Research was conducted under an Institutional Animal Care and Use Committee approved protocol in compliance with the Animal Welfare Act, PHS Policy and other federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011.