Discovery of Dengue Virus NS4B Inhibitors

Abstract

The four serotypes of dengue virus (DENV-1 to -4) represent the most prevalent mosquito-borne viral pathogens in humans. No clinically approved vaccine or antiviral is currently available for DENV. Here we report a spiropyrazolopyridone compound that potently inhibits DENV both in vitro and in vivo. The inhibitor was identified through screening of a 1.8-million-compound library by using a DENV-2 replicon assay. The compound selectively inhibits DENV-2 and -3 (50% effective concentration [EC50], 10 to 80 nM) but not DENV-1 and -4 (EC50,>20 M). Resistance analysis showed that a mutation at amino acid 63 of DENV-2 NS4B (a nonenzymatic transmembrane protein and a component of the viral replication complex) could confer resistance to compound inhibition. Genetic studies demonstrate that variations at amino acid 63 of viral NS4B are responsible for the selective inhibition of DENV-2 and -3. Medicinal chemistry improved the physicochemical properties of the initial “hit” (compound 1), leading to compound 14a, which has good in vivo pharmacokinetics. Treatment of DENV-2-infected AG129 mice with compound 14a suppressed viremia, even when the treatment started after viral infection. The results have proven the concept that inhibitors of NS4B could potentially be developed for clinical treatment of DENV infection. Compound 14a represents a potential preclinical candidate for treatment of DENV-2- and -3-infected patients.

FIG 1

(A) Schematic diagram of the Renilla luciferase (RLUC)-expressing DENV-2 replicon (New Guinea C strain) and the firefly luciferase (LUC)-expressing HCV replicon. The diagram is not drawn to scale. C22, N-terminal 22 amino acids of the DENV-2 capsid protein; E24, C-terminal 24 amino acids of the E protein; Pu, puromycin N-acetyltransferase gene; FMDV 2A, foot-and-mouth disease virus 2A protein; IRES, internal ribosomal entry site from encephalomyocarditis virus; Ub, ubiquitin; Neo, neomycin resistance gene; UTR, untranslated region. (B) Dual-replicon screening flow chart. Compound libraries were screened at 5 μM by using Renilla luciferase DENV-2 replicon cells. Compounds with >70% inhibition of Renilla luciferase activity in the primary screening were subjected to a dose-response test in Renilla luciferase DENV-2 replicon cells to derive EC₅₀ and CC₅₀ values. The compounds were also tested against the HCV firefly luciferase replicon. Compounds that selectively inhibited the DENV-2 replicon were further examined in a Renilla luciferase reporter cell line to rule out Renilla luciferase inhibitors. The inhibitors were further validated in a viral titer reduction assay using authentic DENV. (C) Chemical structure of compound 1 (racemate). (D) Antiviral activities of compound 1 (Cpd 1). NITD-008, a nucleoside inhibitor of DENV and HCV (12), was included as a positive control. DENV-2 Pu-Renilla luciferase replicon BHK-21 cells or HCV Neo-firefly luciferase replicon Huh-7 cells were cultured in medium containing 3-fold serial dilutions of compound 1 for 2 days at 37°C. ViviRen (Promega) and Britelite (PerkinElmer) were added to the DENV-2 and HCV replicons, respectively. The luciferase signal was detected with a Clarity 4.0 plate reader, followed by cell viability detection by the addition of CellTiter-Glo (Promega). The luciferase assay and the cell viability assay were performed according to the manufacturers' protocols. Antiviral activity and cell viability are presented as the left y axis and right y axis, respectively.

FIG 2

In vitro antiviral profile of the spiropyrazolopyridone compounds. (A) Structures of spiropyrazolopyridone compound 1a (R), compound 1b (S), and compound 14a (R). (B) Antiviral activities of two enantiomers (compounds 1a and 1b) of compound 1 against DENV-2. A549 cells were infected with DENV-2 (New Guinea C strain) at an MOI of 0.5 in the presence of 2-fold serial dilutions of compounds. After incubation at 37°C for 48 h, cell culture fluids were harvested for a plaque assay. The data are plotted as logarithm (log₁₀) values of average viral titers from triplicates versus the compound concentration. The error bars represent standard deviations (n = 3). (C) Antiviral spectra of compound 1a and its analog compound 14a. A549 cells were infected with DENV-1, -2, -3, or -4 (MOI of 0.5). Vero cells were infected with WNV, YFV, JEV, POW, WEEV, or VSV (MOI of 0.1). EC₅₀s were calculated by nonlinear regression analysis using Prism software (GraphPad). See Materials and Methods for assay details. (D) Transient-replicon assay. A549 cells were transfected with 10 μg of DENV-2 luciferase replicon RNA. The transfected cells were immediately treated with different concentrations of compound 1a or 0.9% DMSO (as a negative control). At the indicated time points p.i., cells were assayed for luciferase signals (quantified as relative light units [RLU]). The log₁₀ values of average luciferase signals and standard deviations are presented (n = 3). (E) Time-of-addition analysis. A549 cells were infected with DENV-2 (New Guinea C strain) at an MOI of 2 at 4°C for 1 h. After three washes with PBS to remove unbound viruses, cells were incubated at 37°C. At the indicated time points, compound 1a (1 μM) was added to the infected cells. As controls, the infected cells were treated with 0.5% DMSO. At 24 h p.i., the culture medium was collected, and viral titers were determined by a plaque assay. Average results and standard errors (n = 3) are presented. (F) Antiviral activity in mosquito C6/36 cells. C3/36 cells were infected with DENV-2 at an MOI of 1. Compound 1a was added at the indicated concentrations to cells immediately after infection. The culture supernatant was collected at 48 h p.i., and viral titers were quantified by a plaque assay.

FIG 3

Characterization of resistant viruses. (A) Scheme of selection and validation of DENV-2 that is resistant to compound 1a inhibition. WT DENV-2 was used for resistance selection. The virus was cultured in A549 cells with increasing concentrations of compound 1a. The EC₅₀ for each selected isolate was measured at passage 16 (P16) by using qRT-PCR. The P16 viral RNA from each of the six independent selections was extracted for whole-genome sequencing. The extracted RNA was amplified by using SuperScript One-Step RT-PCR with Platinum Taq (Life Technologies). The RT-PCR products were sequenced. The identified mutations were introduced into a DENV-2 infectious cDNA clone to generate recombinant viruses. The resistance levels of recombinant viruses were evaluated by a viral titer reduction assay in A549 cells. (B) Resistance profile of the DENV-2 escape mutant. Mutations at position V63 of DENV-2 NS4B were consistently recovered from six independent resistant virus selections. DMSO (0.9%) was used as a negative control during resistance selection. Mixed mutations of V63 were obtained from selections I to IV. The sensitivities of the escape viruses to compound 1a (indicated by EC₅₀s) were compared to that of the WT virus. Fold resistance was calculated as the EC₅₀ for the resistant virus divided by the EC₅₀ for the WT virus. (C) Membrane topology and sequence alignment of the DENV NS4B protein. The identified V63 mutation is located in predicted transmembrane domain 2 (pTMD2) of the NS4B topology (22). An amino acid sequence alignment of the pTMD2 regions (residues 60 to 83) of different flavivirus NS4B proteins is shown. The amino acid positions of NS4B are numbered according to DENV-2 numbering (GenBank accession number AY037116). The V63 mutation in DENV-2 NS4B is highlighted; the equivalent position in DENV-1 NS4B is I64, as indicated. (D) Characterization of DENV-2 NS4B V63 mutants. BHK-21 cells were transfected with 10 μg of WT or NS4B mutant genome-length RNA of DENV-2. The transfected cells were monitored for viral E protein expression by IFA at days 2 and 4 p.t. (Top) Anti-E monoclonal antibody 4G2 and Alexa Fluor 488 goat anti-mouse IgG were used as the primary and secondary antibodies, respectively. (Middle) Plaque morphology of recombinant WT and mutant viruses. (Bottom) Viral titers in the culture fluids on day 5 p.t. were quantitated by a plaque assay. (E) Summary of resistance of the recombinant DENV-2 V63 mutants. Six recombinant DENV-2 V63 mutants were prepared and tested against compound 1a. The EC₅₀s and fold resistance values are shown.

FIG 4

Characterization of the recombinant DENV-1 I64V mutant. (A) BHK-21 cells were transfected with 10 μg of WT or NS4B mutant genome-length RNA of DENV-1 (West Pacific strain). The transfected cells were monitored for viral E protein expression by IFA at days 2 and 4 posttransfection. (Left) Anti-E monoclonal antibody 4G2 and Alexa Fluor 488 goat anti-mouse IgG were used as the primary and secondary antibodies, respectively. (Middle) Plaque morphology of WT DENV-1 and the I64V mutant. (Right) Viral titers in the culture fluids at day 5 p.t. were determined by a plaque assay. (B) Conversion of DENV-1 to sensitivity to compound 1a inhibition by an I64V mutation. Recombinant DENV-1 containing the I64V mutation was generated. The effect of compound 1a on the DENV-1 I64V mutant was determined by a viral titer reduction assay in A549 cells. The logarithm (log₁₀) values of average viral titers from triplicates are plotted against the compound 1a concentration. Average results and standard deviations (n = 3) are presented.

FIG 5

Cross-resistance analysis of distinct NS4B inhibitors. (A) Mapping of resistance mutations within NS4B. Amino acid changes (P104L and A119) associated with NITD-618 resistance are depicted in circles. (B) Analysis of cross-resistance between NITD-618 and spiropyrazolopyridone compounds. A549 cells containing the DENV-2 WT or P104L A119T mutant luciferase replicon were treated with compound 1a (top), compound 14a (middle), and NITD-618 (bottom) at the indicated concentrations for 48 h. The inhibition of viral replication was measured by the luciferase activity. EC₅₀s are shown.

FIG 6

Binding of ³H-labeled compound 14a to recombinant NS4B. (A) SDS-PAGE analysis of recombinant DENV-2 NS4A and NS4B proteins. Full-length NS4A and the N-terminal 125 amino acids of DENV-2 NS4B were expressed and purified by using an E. coli expression system (18). The molecular masses (kilodaltons) of protein standards (marker) are indicated on the left and right. (B) Gel filtration analysis of binding of ³H-labeled compound 14a to the NS4B protein. ³H-labeled compound 14a was mixed with the indicated proteins and centrifuged through a Micro BioSpin 6 gel filtration column. Protein-bound ³H-labeled compound-14a was quantitated in the eluent by using a beta scintillation counter (PerkinElmer Life Sciences). NS4A served as a negative control. Analysis of each data point was carried out in triplicate. ***, P < 0.001 for comparison of binding of the compound to WT and V63I mutant NS4B proteins. (C) Effect of compound 1a on the induction of the ISRE-Luc reporter gene after treatment with IFN. A plasmid containing the coding region of DENV-2 2K-NS4B was cotransfected into HEK-293T cells together with the ISRE-Luc plasmid. At 24 h p.t., the cells were stimulated with 1,000 U of IFN-β in the presence or absence of 1 μM compound 1a. The cells were further incubated for 24 h, followed by quantification of luciferase expression. Results show the mean percentages of luciferase activity for each treatment (n = 3).

FIG 7

In vivo pharmacokinetics and efficacy of compound 14a. (A) AG129 mice (8 to 12 weeks old; 6 mice per treatment group) were infected with 1.5 × 10⁶ PFU of DENV-2 (strain TSV01) on day 0. The mice were treated orally with 5, 25, 50, or 100 mg/kg compound 14a BID. The peak viremia on day 3 p.i. was quantified by a plaque assay. A one-way ANOVA Tukey-Kramer test indicates that, compared with the vehicle control, the reduction in viremia in all treatment groups, except for the 5-mg/kg group, is statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001. LOD, limit of detection (250 PFU/ml). Values below the limit of detection were set equal to 250 PFU/ml. The antiviral activities with treatment doses of 50 and 100 mg/kg were recently reported (19) (B) Plasma concentrations of compound 14a after oral administration to DENV-2-infected AG129 mice at a dosage of 100 mg/kg (n = 3 per time point). The EC₉₀ (0.2 μM) of compound 14a is indicated by the dotted line. (C) Peak viremia of mice with a delayed start of treatment after infection with 1.5 × 10⁷ PFU of DENV-2 (strain TSV01) (dosed at 100 mg/kg).