Inhibition of dengue virus polymerase by blocking of the RNA tunnel

Abstract

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)(20)]. Chemical modification of the initial "hit" improved the compound potency to an IC(50) (that is, a concentration that inhibits 50% RdRp activity) of 0.7 microM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC(50) of 5 microM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC(50)s of >100 microM against hepatitis C virus RdRp and human DNA polymerase alpha and beta. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

FIG. 1.

Specific inhibition of DENV-2 NS5 RdRp activity. (Α) Structure of NITD-1 and NITD-2. NITD-1 was identified from the RdRp HTS. NITD-2 was obtained after the SAR study. (B) Inhibition of DENV-2 NS5 RdRp. Various concentrations of NITD-1 and NITD-2 were incubated with the RdRp reaction containing poly(C)/oligo(G)₂₀ as a template. A SPA-based detection method was used to quantify the incorporation of [³H]GTP. The relative RdRp activities are presented; signals from the mock-treated reaction are set as 100%. (C) Anti-polymerase activity of NITD-2 against HCV RdRp and human DNA polymerase α and β. The inhibition curve of DENV-2 NS5 from panel B is included in this plot to show the relative sensitivity of the different polymerases to the compounds. (D) Inhibition of different polymerases using control inhibitors. Control inhibitors with known activities against HCV RdRp (LCY967), polymerases α (aphidicolin), and polymerases β (lithocholic) were included. See the experimental details in Materials and Methods. The results were derived from ≥3 independent experiments. Error bars represent the standard deviations.

FIG. 2.

Inhibition of RNA elongation in a single cycle RNA synthesis assay. (A) Heparin titration (0 to 96 ng/ml) in the standard SPA reaction. Heparin was added to 60-min preincubated NS5-RNA complex. The mixture was incubated for another 15 min before addition of [³H]GTP to initiate the reaction. The reaction was allowed to proceed for 60 min. (B) Analysis of IC₅₀s for NITD-2. (C) Analysis of IC₅₀s for 3′ddGTP. For each compound, the IC₅₀s were determined in the presence or absence of heparin (50 ng/ml). The experimental procedures were described in Materials and Methods. Average results from three experiments are presented.

FIG. 3.

Inhibition of de novo RNA synthesis of DENV RdRp. (A) Recombinant proteins of full-length NS5. Full-length NS5 from DENV-2 (1 μg), DENV-4 (2 μg), and WNV (2 μg) were analyzed by SDS-PAGE and stained with Coomassie blue. Molecular masses of protein markers are labeled. (B) DENV de novo RNA synthesis. Subgenomic RNA of DENV-2 was incubated with recombinant DENV NS5 in the presence or absence of NITD-2. The RdRp products were analyzed on a denaturing PAGE and quantified by using a PhosphorImager. The relative amount of RNA product was indicated below the autoradiograph of the PAGE gel. The concentrations of NITD-2 are labeled above the autoradiograph. As a maker of the input RNA template, the subgenomic RNA was 5′-end ³³P-labeled (12) and analyzed on the PAGE. (C) WNV de novo RNA synthesis. A subgenomic RNA of WNV was incubated with WNV NS5 in the presence indicated concentrations of NITD-2. The RdRp products were similarly analyzed as described in panel B. See experimental details in Materials and Methods.

FIG. 4.

Photoreactive inhibitor of DENV-2 NS5 RdRp. (Α) Structure of the photoreactive inhibitor NITD-29. (B) Inhibition of DENV-2 NS5 RdRp. An RdRp SPA assay was performed using poly(C)/oligo(G)₂₀ as a RNA template. The experiment procedure was identical to that described in the legend to Fig. 1B. Average results from three independent experiments are shown. Error bars represent standard deviations. (C) Cytotoxicity of NITD-29 in Vero cells. Cytotoxicity was examined by incubation of Vero cells with the indicated concentrations of NITD-29. Cell viability at 48 h posttreatment was measured by an MTT assay kit (American Type Culture Collection) and presented as a percentage of colorimetric absorbance derived from the compound-treated cells compared to that from the mock-treated cells (with 1% DMSO). (D) Virus titer reduction assays. Vero cells were infected with indicated viruses (MOI of 0.1) and treated immediately with compound at 6 and 17 μM. For DENV-2 and YFV, culture medium were collected at 44 h p.i.; for chikungunya virus, culture medium was collected at 22 h postinfection. Virus titers of all samples were determined by plaque assays on BHK cells.

FIG. 5.

MS analysis of UV-cross-linked DENV-2 NS5 and NITD-29 complex. (A) Partial MALDI-MS traces of compound-cross-linked (left) and control (right) NS5 tryptic digests showing the differential peak at m/z 2,048.87. (B) MSMS spectrum of m/z 2,048.87 with insets to illustrate compound cross-linking (top) and peak assignment of the observed fragment ions (bottom).

FIG. 6.

Binding site of NITD-29 in the DENV RdRp domain. (A) Three-dimensional model of NITD-29 in complex with DENV RdRp. The model was built on the results of UV cross-linking, MS analysis, and the crystal structure of DENV-3 RdRp (43). Three RdRp subdomains (thumb, fingers, and palm) are indicated. The RNA tunnel and NTP entrance are circled with dotted lines. NITD-29 is shown in green. Three critical residues for interaction with the compound are labeled in red: M343 was cross-linked to NITD-29; R737 and T413 are within a hydrogen bond distance from the compound. (Left panel) Surface view; (right panel) ribbon view; (middle panel) magnified surface view of the compound binding site (the RdRp protein was set to partial transparency to view the complete molecule of NITD-29). The images were produced by using PyMOL software (6). (B) IC₅₀s from RdRp assays with different orders of reagent assembly. Different orders of reagent addition (RNA template and NITD-2 inhibitor) during reaction assembly are depicted. The IC₅₀s were determined for each experimental scheme. The experiments were performed using poly(C)/oligo(G)₂₀ as a template in the SPA format. The abbreviations are indicated. See Materials and Methods for reagent concentrations.