Identification of a broad-spectrum arenavirus entry inhibitor

Abstract

Several arenaviruses, including Lassa virus (LASV), are causative agents of hemorrhagic fever, for which effective therapeutic options are lacking. The LASV envelope glycoprotein (GP) gene was used to generate lentiviral pseudotypes to identify small-molecule inhibitors of viral entry. A benzimidazole derivative with potent antiviral activity was identified from a high-throughput screen utilizing this strategy. Subsequent lead optimization for antiviral activity identified a modified structure, ST-193, with a 50% inhibitory concentration (IC(50)) of 1.6 nM against LASV pseudotypes. ST-193 inhibited pseudotypes generated with other arenavirus envelopes as well, including the remaining four commonly associated with hemorrhagic fever (IC(50)s for Junín, Machupo, Guanarito, and Sabiá were in the 0.2 to 12 nM range) but exhibited no antiviral activity against pseudotypes incorporating either the GP from the LASV-related arenavirus lymphocytic choriomeningitis virus (LCMV) or the unrelated G protein from vesicular stomatitis virus, at concentrations of up to 10 microM. Determinants of ST-193 sensitivity were mapped through a combination of LASV-LCMV domain-swapping experiments, genetic selection of viral variants, and site-directed mutagenesis. Taken together, these studies demonstrate that sensitivity to ST-193 is dictated by a segment of about 30 amino acids within the GP2 subunit. This region includes the carboxy-terminal region of the ectodomain and the predicted transmembrane domain of the envelope protein, revealing a novel antiviral target within the arenavirus envelope GP.

FIG. 1.

Structure and antiviral activity of ST-37 and ST-193. (A) Benzimidazole derivative ST-37 (left) was modified to generate ST-193 (right). (B) Inhibition of LASV GP- or VSVg-pseudotyped HIV infection with ST-37 or ST-193. Infectivity was measured with a luciferase reporter relative to controls with no compound. Each point is an average of three replicates, with error bars designating standard deviations.

FIG. 2.

ST-193 inhibition of TCRV infection. TCRV was diluted and used to infect Vero cell monolayers in 35-mm wells in the presence of 0.1% DMSO with or without ST-193. Bars represent averages of six replicates (error bars designate standard errors of the means).

FIG. 3.

Domain swapping demonstrates that the C-terminal third of GP2 determines sensitivity to ST-193. (A) Schematic representation of LASV-LCMV chimeric GPs. Predicted TMD and heptad repeat domains (HR1 and HR2) are indicated. The two GPs are spliced 12 aa N terminal of the predicted TMD. (B) Pseudotype infectivity as in Fig. 1, using the arenavirus GPs shown (from a representative experiment). Average IC₅₀s ± standard errors of the means for these constructs (across at least four experiments) were as follows: LASV, 0.0016 ± 0.0003 μM; LCMV, 31 ± 4 μM; LASV_N-LCMV_C, 13.6 ± 2.2 μM; and LCMV_N-LASV_C, 0.0005 ± 0.0003 μM. WT, wild type.

FIG. 4.

ST-193 sensitivity determinants. (A) Schematic representation of an arenavirus GP2 subunit and the sites of 193R variations. The TCRV amino acid sequence is shown for the region indicated, with sites identified as determinants of ST-193 sensitivity indicated with raised letters and numbering (TCRV GP amino acid numbering). Predicted TMD and heptad repeat domains (HR1 and HR2) are indicated (the TMD is underlined in the sequence). (B) Single-amino-acid variations that reduce ST-193 sensitivity in TCRV GP. Resistance is the fold change in IC₅₀ as measured with TCRV GP-pseudotyped HIV, relative to wild type, and averaged across at least four independent experiments. “Allele” refers to the common name for the mutant GP.

FIG. 5.

Amino acid alignment of a portion of the arenavirus GP2 subunit. Amino acid numbering is as in Fig. 4. Residues identified as ST-193 sensitivity determinants (Fig. 4) are in bold, and the predicted TMD is indicated. The highlighted residues at positions 421 and 425 are sensitivity determinants for which some sequence divergence is observed across the Arenaviridae family. Abbreviations (GenBank accession numbers) are as follows: LASV, LASV strain Josiah (J04324) (5); LCMV, LCMV strain Armstrong 53b (AY847350) (29); MACV, MACV strain Carvallo (AY619643); JUNV, JUNV strain MC2 (D10072); TCRV, TCRV strain TRVL 11598 (P31840) (3); GTOV, Guanarito virus (AY129247) (13); SABV, Sabiá virus (U41071) (28); Chapare, Chapare virus (EU260463) (14); LATV, Latino virus (AF485259) (4); PICV (U77602); PIRV, Pirital virus (AF485262) (4); and WWAV, Whitewater Arroyo virus (AF228063) (12). LASV and LCMV are Old World arenaviruses, while New World arenaviruses are represented by PICV and PIRV (clade A); MACV, JUNV, TCRV, GTOV, SABV, and Chapare virus (clade B); and LATV (clade C). WWAV is likely a recombinant of clades A and B.

FIG. 6.

Site-directed mutagenesis of arenavirus GPs identifies important ST-193 sensitivity determinants. Pseudotype infectivity was measured as in Fig. 1 (representative experiments are shown in panels B to D). (A) List of GP constructs and their sensitivity to ST-193 (*, numbering based on alignment with the corresponding TCRV GP amino acid). Data shown are averages from at least four experiments. (B) LASV GP is much less sensitive when either site 1 or 2 is converted to the corresponding LCMV amino acid, and all arenavirus-specific ST-193 activity is lost when both sites are changed. (C) LCMV GP does not gain ST-193 sensitivity when the reciprocal substitutions are introduced (sites 1 and 2 converted to LASV amino acids). (D) PICV GP becomes significantly more sensitive to ST-193 when valine is substituted at site 1 (to match LASV GP). WT, wild type.

FIG. 7.

Dilution of antiviral activity. LASV GP (A) or TCRV GP (B) pseudotype infectivity was measured after addition of ST-193 to 80 nM (A) or of ST-294 to 2 μM (B) and following two serial 20-fold (A) or 10-fold (B) dilutions (shown as open squares, with error bars designating standard deviations). The line in each graph indicates the standard inhibition curve assembled from 10 independent experiments (dots are averages of three replicates from one experiment) using the XLfit dose-response one-site model. ST-193 activity is diluted as predicted, while ST-294 inhibitory activity is retained.