Novel Nonnucleoside Inhibitors of Zika Virus Polymerase Identified through the Screening of an Open Library of Antikinetoplastid Compounds

Abstract

Zika virus (ZIKV) is a mosquito-borne pathogen responsible for neurological disorders (Guillain-Barré syndrome) and congenital malformations (microcephaly). Its ability to cause explosive epidemics, such as that of 2015 to 2016, urges the identification of effective antiviral drugs. Viral polymerase inhibitors constitute one of the most successful fields in antiviral research. Accordingly, the RNA-dependent RNA polymerase activity of flavivirus nonstructural protein 5 (NS5) provides a unique target for the development of direct antivirals with high specificity and low toxicity. Here, we describe the discovery and characterization of two novel nonnucleoside inhibitors of ZIKV polymerase. These inhibitors, TCMDC-143406 (compound 6) and TCMDC-143215 (compound 15) were identified through the screening of an open-resource library of antikinetoplastid compounds using a fluorescence-based polymerization assay based on ZIKV NS5. The two compounds inhibited ZIKV NS5 polymerase activity in vitro and ZIKV multiplication in cell culture (half-maximal effective concentrations [EC₅₀] values of 0.5 and 2.6 μM for compounds 6 and 15, respectively). Both compounds also inhibited the replication of other pathogenic flaviviruses, namely, West Nile virus (WNV; EC₅₀ values of 4.3 and 4.6 μM for compounds 6 and 15, respectively) and dengue virus 2 (DENV-2; EC₅₀ values of 3.4 and 9.6 μM for compounds 6 and 15, respectively). Enzymatic assays confirmed that the polymerase inhibition was produced by a noncompetitive mechanism. Combinatorial assays revealed an antagonistic effect between both compounds, suggesting that they would bind to the same region of ZIKV polymerase. The nonnucleoside inhibitors of ZIKV polymerase here described could constitute promising lead compounds for the development of anti-ZIKV therapies and, eventually, broad-spectrum antiflavivirus drugs.

Keywords: RNA polymerases; West Nile virus; Zika virus; allosteric; antiviral; antiviral agents; dengue virus; nonnucleoside inhibitor; polymerase.

FIG 1

Novel ZIKV polymerase inhibitors. (A) Structure of compounds 6 and 15. (B) Real-time fluorescence-based enzymatic activity of ZIKV NS5 RdRp in the absence (black circles) or in the presence of 100 μM compound 6 (blue squares) or 100 μM compound 15 (red triangles). Fluorometric measurements were performed as described in Materials and Methods (n = 4). (C and D) Same as in panel A, but using HCV NS5b and FMDV 3D RdRps, respectively (n = 4). Data are expressed as mean ± SD.

FIG 2

Biochemical characterization of polymerase inhibitors. (A and B) Dose-response curves of ZIKV RdRp against compound 6 (A) and compound 15 (B) (n = 4). (C and D) Enzyme inhibition kinetics of compound 6 (C) and compound 15 (D) against ZIKV RdRp. Fluorescence-based polymerization assays were performed using increasing concentrations of either compound 6 or 15 (n = 3). Experimental conditions, compounds concentrations, and data processing are explained in the corresponding section of Materials and Methods. (E) Combination study between compounds 6 and 15. The combination index (CI) plot of different combination doses of compounds 6 and 15 (n = 4). Data above or below the dotted line (CI = 1) represent antagonism and synergism, respectively. Data are expressed as mean ± SD.

FIG 3

Antiviral activity of compounds 6 and 15 against medically relevant flaviviruses. (A to C) Dose-response curves of ZIKV (A), WNV (B), and DENV-2 (C) against compound 6. (D to F) Dose-response curves of ZIKV (D), WNV (E), and DENV-2 (F) against compound 15. Vero cells were infected (MOI of 1 PFU/cell) and treated with increasing amounts of the compounds, and virus yield in supernatant was determined at 24 hpi (ZIKV and WNV) or 48 hpi (DENV-2). The cytotoxicity of the compounds was estimated in parallel by quantification of cellular ATP in uninfected samples at 24 h (A, B, D, and E) or 48 h (C and F) (n = 2 to 4). Dashed lines denote a 50% reduction. Data are expressed as mean ± SD.

FIG 4

Effect of compounds 6 and 15 on ZIKV infection. (A) Inhibition of ZIKV production by compounds 6 and 15. Vero cells were infected with ZIKV (MOI of 1 PFU/cell) and treated with drug vehicle (DMSO), compound 6 (15 μM), or compound 15 (15 μM), and virus yield in supernatant was determined by plaque assay at the indicated times postinfection (n = 3). (B) Quantification of viral RNA in the supernatant of infected cultures in samples infected and treated as in panel A (n = 3). (C) Lack of virucidal effect of compounds 6 and 15. ZIKV (∼4 × 10⁶ PFU) was treated with drug vehicle (DMSO), EGCG (5 μg/ml), compound 6 (15 μM), or compound 15 (15 μM) for 1 h at 37°C in culture medium. Then, the infectivity in each sample was determined by plaque assay. EGCG was included in the experiments as a positive-control compound with virucidal activity (n = 4). (D) Time-of-addition experiments of compounds 6 and 15. Vero cells were infected (MOI of 1 PFU/cell) and treated with drug vehicle (DMSO), compound 6 (15 μM), or compound 15 (15 μM) from 0, 6, or 12 h after virus inoculation. Virus yield was determined by plaque titration at 24 hpi (n = 3 to 4). (E) Inhibition profiles of ZIKV after 10 serial passages in the presence of compounds 6 and 15. Vero cells were infected (MOI of 1 PFU/cell) with ZIKV stock (initial population) or the viral populations resultant from three-independent series of 10 passages (passage 10) in the presence of the compound 6 (15 μM) or compound 15 (15 μM). Virus yield was determined at 24 hpi by plaque assay (n = 2 to 3). P values were calculated using Dunnet’s test for pairwise comparisons of multiple treatment groups with a single control group in panels C and D or Tukey’s test for pairwise comparisons of the mean of each group with the mean of every other group in panel E. Asterisks denote statistically significant differences between control group (DMSO) and treatments. *, P < 0.05; **, P < 0.005; n.s., nonstatistically significant differences between groups. Data are expressed as mean ± SD. Points indicate independent biological replicates.