Identification of Quinolinones as Antivirals against Venezuelan Equine Encephalitis Virus

Abstract

Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.

Keywords: alphavirus; antiviral agents.

FIG 1

Structure-activity relationship studies with SRI-33394, a quinolinone with antiviral activity against VEEV. (A) Lead compound SRI-33394 has potent antiviral activity against VEEV. (B) Structure-activity-relationship (SAR) study to replace sulfur in SRI-33394 with oxygen. (C) Analogs with substitutions at R¹, R², R⁴, and R⁵. (D) SRI-36959 with cyclization of the ethyl-N,N-dimethyl moiety to pyrrolidines at R⁴. (E) SRI-34329 with hydrogen on the quinolinone ring switched to a methyl group. (F) Analogs with substitutions at R¹ to R⁵.

FIG 2

Antiviral activity of SRI-33394 and SRI-34329. At 1 h prior to infection, (A) normal human dermal fibroblasts (NHDFs) or (B) Aedes albopictus C6/36 mosquito cells were pretreated with dimethyl sulfoxide (DMSO) or 2-fold serial dilutions of SRI-33394 or SRI-34329, ranging from 100 μM to 0.2 μM. Treated cells were infected with VEEV_TC-83 at a multiplicity of infection (MOI) of 1 PFU/cell. At 48 hpi, infectious virus in the supernatant was quantified by plaque assay, and the IC₉₀ was determined by nonlinear regression analysis. (C) For cytotoxicity assays, NHDFs were treated with dilutions of quinolinone compounds. At 24 h posttreatment, cells were analyzed using the CellTiter-Glo luminescent cell viability assay. Fifty percent cytotoxic concentration (CC₅₀) values were determined by nonlinear regression analysis. (D) NHDFs were pretreated for 1 h with DMSO, 10 μM SRI-33394, or 10 μM SRI-34329. Treated cells were then infected with VEEV_ZPC738 at an MOI of 3 PFU/cell. At 48 h postinfection (hpi), infectious virus in the supernatant was quantified by plaque assay. Data are combined from two independent experiments. Each bar represents the mean ± standard error of the mean (SEM). P values were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. ****, P < 0.001.

FIG 3

Antiviral spectrum of quinolinones against selected alphaviruses. NHDFs were pretreated for 1 h with 2-fold serial dilutions of (A) SRI-33394 or (B) SRI-34329, ranging from 100 μM to 0.2 μM. Treated cells were then infected with CHIKV, MAYV, ONNV, RRV, or UNAV at an MOI of 3 PFU/cell. At 48 hpi, infectious virus in the supernatant was quantified by plaque assay.

FIG 4

Quinolinones inhibit VEEV_TC-83 replication at a postentry step. (A) NHDFs were infected with VEEV_TC-83 at an MOI of 3 PFU/cell and treated with DMSO or 10 μM SRI-34329 at the indicated time points. Infectious virus in the supernatant at 48 hpi was quantified by plaque assay. Data are derived from two independent experiments. **, P < 0.01; Mann-Whitney test. (B) NDHFs were pretreated for 1 h with DMSO, 10 μM SRI-33394, or 10 μM SRI-34329 and then infected with VEEV_TC-83 (MOI = 3 PFU/cell). At 12 hpi, total RNA was evaluated by Northern blotting analysis with digoxigenin (DIG)-labeled RNA probes specific for VEEV or human β-actin. Data shown are representative of two independent experiments.

FIG 5

Serial passage of VEEV_TC-83 in the presence of SRI-34329 yields an escape mutant. (A) VEEV_TC-83 was repeatedly passaged in triplicate through normal human dermal fibroblasts (NHDFs) in the presence of DMSO or 10 μM SRI-34329. (B) Titer of supernatant collected from the third passage of triplicate (3A to 3C) DMSO- or SRI-34329-treated cells was determined by plaque assay in the absence of drug. (C) NHDFs treated with DMSO or 10 μM SRI-34329 1 h prior to infection with VEEV_TC-83 or with replicate passage 3C samples from either DMSO or SRI-34329 serial passages at an MOI of 3 PFU/cell. At 48 hpi, virus in the supernatant was evaluated by plaque assay. P values were determined by unpaired t tests. **, P < 0.01; ***, P < 0.001.

FIG 6

VEEV_TC-83 nsP2 Y102C mutation confers resistance to SRI-33394 and SRI-34329. (A) Following a 1 h pretreatment with DMSO, 10 μM SRI-33394, or 10 μM SRI-34329, normal human dermal fibroblasts (NHDFs) were infected with wild-type (WT) or nsP2 Y102C VEEV_TC-83 at an MOI of 1 PFU/cell. At the indicated times postinfection, infectious virus in the supernatant was quantified by plaque assay. P values were determined by two-way ANOVA followed by Bonferroni’s multiple-comparison test. ***, P < 0.001; ****, P < 0.0001. (B) NHDFs were pretreated for 1 h with DMSO or with 2-fold serial dilutions of SRI-34329 ranging from 100 μM to 0.2 μM. Treated cells were then infected with WT or nsP2 Y102C VEEV_TC-83 at an MOI of 1 PFU/cell. At 48 hpi, infectious virus in the supernatant was quantified by plaque assay, and the percent infection relative to DMSO control was quantified.

FIG 7

Mutation of CHIKV nsP2 (K102Y) confers sensitivity to quinolinones. (A) Amino acid sequence alignment of alphavirus nsP2 residues 90 to 118 identifies a natural single-nucleotide polymorphism (SNP) at amino acid position 102 (red box). (B) An nsP2-K102Y mutation was introduced into the cDNA clone for CHIKV_181/25. NHDF cells were infected with WT and CHIKV_181/25 nsP2-K102Y virus at an MOI of 1 PFU/cell. Culture supernatants were collected at the indicated times, and titer was determined by plaque assay. (C) Triplicate wells of NHDFs were pretreated for 1 h with 2-fold serial dilutions of SRI-34329, ranging from 140 μM to 0.078 μM. Treated cells were then infected with WT CHIKV_181/25 or CHIKV_181/25 nsP2-K102Y at an MOI of 1 PFU/cell. At 24 hpi, infectious virus was quantified by plaque assay, and IC₉₀ values were determined by nonlinear regression analysis. Data are representative of two independent experiments.

FIG 8

Docked poses of lead SRI-34329 at VEEV and CHIKV nsP2 WT and position 102 mutants. Docked ligand is colored in green. Salt bridges, pi-pi stacking, and hydrophobic contacts are indicated by dashed cyan, green, and orange lines, respectively. (A) Pose at VEEV nsP2 WT. (B) Superposition of the ligands from docked poses of VEEV nsP2 Y102C (cyan) and Y102S (orange) onto the docked pose of VEEV nsP2 WT. (C) Pose at CHIKV nsP2 K102Y. (D) Superposition of the ligand from docked pose of CHIKV nsP2 K102Y (turquoise) onto the docked pose of CHIKV nsP2 WT.

FIG 9

Docking results of SRI-34329 analogs. (A) Docked pose of compound 3d (green) at VEEV nsP2 WT. Hydrogen bonds, pi-pi stacking, and hydrophobic contacts are indicated by dashed black, green, and orange lines, respectively. (B) Summary of the SAR in line with docking results. Analogs belonging to each case are listed in either black (active and retention of certain interaction in docked poses) or red (inactive and loss of certain interaction in docked poses).

FIG 10

Zoomed-out view of the docked pose of SRI-34329 at the VEEV nsP2 helicase. The helicase domain is represented in ribbons. Region 1B is colored in yellow. The single-strand RNA 14-mer and ADP from the CHIKV nsP2 helicase crystal structure (PDB identifier 6JIM) were superimposed to the analogous positions at the VEEV nsP2 helicase in silico homolog to indicate the RNA binding groove and ATPase active site.