An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus Challenge

Abstract

ST-246 is a low-molecular-weight compound (molecular weight = 376), that is potent (concentration that inhibited virus replication by 50% = 0.010 microM), selective (concentration of compound that inhibited cell viability by 50% = >40 microM), and active against multiple orthopoxviruses, including vaccinia, monkeypox, camelpox, cowpox, ectromelia (mousepox), and variola viruses. Cowpox virus variants selected in cell culture for resistance to ST-246 were found to have a single amino acid change in the V061 gene. Reengineering this change back into the wild-type cowpox virus genome conferred resistance to ST-246, suggesting that V061 is the target of ST-246 antiviral activity. The cowpox virus V061 gene is homologous to vaccinia virus F13L, which encodes a major envelope protein (p37) required for production of extracellular virus. In cell culture, ST-246 inhibited plaque formation and virus-induced cytopathic effects. In single-cycle growth assays, ST-246 reduced extracellular virus formation by 10 fold relative to untreated controls, while having little effect on the production of intracellular virus. In vivo oral administration of ST-246 protected BALB/c mice from lethal infection, following intranasal inoculation with 10x 50% lethal dose (LD(50)) of vaccinia virus strain IHD-J. ST-246-treated mice that survived infection acquired protective immunity and were resistant to subsequent challenge with a lethal dose (10x LD(50)) of vaccinia virus. Orally administered ST-246 also protected A/NCr mice from lethal infection, following intranasal inoculation with 40,000x LD(50) of ectromelia virus. Infectious virus titers at day 8 postinfection in liver, spleen, and lung from ST-246-treated animals were below the limits of detection (<10 PFU/ml). In contrast, mean virus titers in liver, spleen, and lung tissues from placebo-treated mice were 6.2 x 10(7), 5.2 x 10(7), and 1.8 x 10(5) PFU/ml, respectively. Finally, oral administration of ST-246 inhibited vaccinia virus-induced tail lesions in Naval Medical Research Institute mice inoculated via the tail vein. Taken together, these results validate F13L as an antiviral target and demonstrate that an inhibitor of extracellular virus formation can protect mice from orthopoxvirus-induced disease.

Figures

FIG. 1.

Chemical structure of ST-246.

FIG. 2.

A comparison of antiviral potency of ST-246 and CDV in Vero cell culture. Vero cells were incubated with increasing concentrations of ST-246 or CDV and infected with vaccinia virus strain NYCBH at 0.05 PFU/cell. The cultures were fixed and stained with crystal violet at 3 days postinfection. The extent of virus-induced CPE was quantified by measuring absorbance at 470 nm. The percent inhibition was calculated relative to untreated infected and mock-infected controls.

FIG. 3.

Plaque formation of wild-type cowpox virus in the presence and absence of ST-246. Vero cell monolayers (106 cells/well) were infected with cowpox virus at 0.0001, 0.001, and 0.01 PFU/cell in the presence and absence of 5 μM ST-246. At 3 days postinfection, the cultures were fixed in 5% glutaraldehyde and stained with crystal violet.

FIG. 4.

The effects of ST-246 on production of intracellular and extracellular virus. BSC-40 cells were infected with 2 PFU/cell of vaccinia virus strain IHD-J in the presence and absence of 5 μM ST-246. At 3-h intervals for 24 h postinfection, the cultures were harvested and separated into extracellular and intracellular virus fractions. The virus titers in each fraction were measured by plaque assay of BSC-40 cell monolayers. The error bars represent the standard deviation of the means of triplicate virus yield determinations.

FIG. 5.

Plaque formation of an ST-246-resistant cowpox virus variant in the presence and absence of ST-246. Vero cell monolayers (106 cells/well) were infected with a cowpox virus variant that exhibited reduced susceptibility to ST-246 at 0.0001, 0.001, and 0.01 PFU/cell in the presence and absence of 5 μM ST-246. At 3 days postinfection, the cultures were fixed in 5% glutaraldehyde and stained with crystal violet.

FIG. 6.

The effects of ST-246 in a lethal intranasal vaccinia virus challenge model. Mice (six animals per dose group) were inoculated on day 0 with 4 × 105 PFU (10× LD50) of vaccinia virus strain IHD-J with the exception of naïve and scarified groups, which remained uninfected and were used as controls to assess the immune status of animals that survived initial viral challenge. Mice were treated with ST-246 at 50 mg/kg b.i.d. or with vehicle administered by gavage at 8-h intervals for 14 days. A control group of mice were treated with CDV at 100 mg/kg administered by i.p. injection. The percent survival (A) or percent starting body weight for each treatment condition (B) was plotted as a function of days postinfection.

FIG. 7.

The effects of ST-246 and CDV on virus titers in liver, spleen, and lung at day 8 postinfection. A/NCr mice were inoculated with 50 PFU (100× LD50) of ectromelia virus via intranasal administration. CDV-treated mice were injected on day 0 with a single dose by the intraperitoneal route. ST-246- or placebo-dosed mice were treated by gavage on day 0 through day 14 at 8-h intervals and twice daily. At day 8 postinfection, five mice in each group were sacrificed, and tissue infectivity was measured in target organs by plaque assay.

FIG. 8.

The effects of ST-246 on vaccinia virus-induced tail lesion formation. NMRI mice (eight mice per placebo-treated group and six mice per compound-treated groups) were inoculated with 4,000 PFU of vaccinia virus administered by tail vein injection. Mice received placebo, ST-246 (50 mg/kg), or ST-246 (15 mg/kg) b.i.d. by gavage starting at 2 h postinoculation and continuing for 5 days. CDV was administered at 25 mg/kg as a single i.p. injection. Photographs of the tail lesions in placebo and ST-246 50-mg/kg dose groups at day 8 postinfection are shown. Lesions for all dose groups were quantified at day 8 postinfection (graph). The error bars represent the standard deviation of the mean lesion number.

FIG. 9.

A sequence comparison of F13L orthologs. (A) Graphic representation of F13L, illustrating the location of known sites of palmitoylation, resistance to the antiviral compounds ST-246 (G277C) and IMCBH (D280Y), and an HKD phospholipase motif implicated in F13L function. (B) Amino acid sequence comparison of F13L orthologs from amino acid positions 245 to 305 showing the sequence conservation surrounding the sites of resistance to ST-246 (amino acid position 277) and IMCBH (amino acid position 280), identified by asterisks. The shaded amino acids at positions 250 and 291 indicate deviations from the consensus sequence.