Mechanistic insights into dengue virus inhibition by a clinical trial compound NITD-688

Abstract

Dengue, caused by the dengue virus (DENV), presents a significant public health challenge with limited effective treatments. NITD-688 is a potent panserotype DENV inhibitor currently in Phase II clinical trials. However, its mechanism of action is not fully understood. Here, we present the molecular details of how NITD-688 inhibits DENV. NITD-688 binds directly to the nonstructural protein 4B (NS4B) with nanomolar affinities across all four DENV serotypes and specifically disrupts the interaction between NS4B and nonstructural protein 3 (NS3) without significantly changing the interactions between NS4B and other viral or host proteins. NS4B mutations that confer resistance to NITD-688 reduce both NITD-688 binding to NS4B and disruption of the NS4B/NS3 interaction. Specifically, NITD-688 blocks the interaction of NS3 with a cytosolic loop within NS4B. This inhibits the formation of new NS4B/NS3 complexes and disrupts preexisting complexes in vitro and DENV-infected cells, ultimately inhibiting viral replication. Consistent with this mechanism, NITD-688 retains greater potency in cellular assays with delayed treatment compared to JNJ-1802, another NS4B inhibitor that has been studied in Phase II clinical trials. Together, these findings provide critical insights into the mechanism of action of NITD-688, facilitating the development of novel flavivirus NS4B inhibitors and informing future clinical interventions against DENV.

Keywords: DENV; NITD-688; NS4B; antiviral; flavivirus.

Fig. 1.

Selection and characterization of NITD-688 resistant DENV. (A) Scheme for resistance selection. (B) EC₅₀ of NITD-688 against P15 viruses. Averaged EC₅₀ values from independent experiments (n = 2 for P15 viruses; n = 6 for WT DENV-2) performed with three technical replicates are shown. Fold change was calculated by normalizing the EC₅₀ of each mutant to that of WT. ^#amino acid changes with >90% frequencies. (C) Membrane topology of NS4B. Orange, mutations unique in NITD-688-selected P15 viruses are colored in orange. Blue, JNJ-1802-resistant mutations, V91A and L94F. (D) NS4B mutations emerged in P4, P8, and P15 viruses. (E) EC₅₀ of NITD-688 against recombinant DENV-2 NS4B mutants. Averaged EC₅₀s (from two independent experiments) are shown. (F) Occurrence of NITD-688-resistant NS4B mutations in the general population of DENVs. n, numbers of DENV genome sequences used for analysis.

Fig. 2.

NITD-688 directly binds to DENV NS4B. (A) ITC analysis of NITD-688 binding to NS4B across DENV four serotypes. The Top panel shows the raw ITC data. The Bottom panel shows the fitting curves (binding model 1:1). (B) ITC analysis of NITD-688 binding to DENV-2 NS4B mutants A193V or W205L. (C) ITC analysis of NITD-688 binding to DENV-2 NS4B mutants T195A, T215A, or A222V. (D) ITC analysis of NITD-688 binding to DENV-2 NS4B mutants A193V/A222V or T195A/A222V/S238F. (E) Summary of K_D values estimated in panels A–D. All mutations were made in the context of DENV-2 NS4B. (F) Alignment of the Alphafold2-predicted structures of DENV-2 and ZIKV NS4B regions at amino acid positions 180 to 240. Residues A222 and T195 in DENV-2 NS4B are labeled in orange, while residues A197 and C224 in ZIKV NS4B are colored in green. (G) ITC analysis of NITD-688 binding to WT ZIKV NS4B or mutant A197T/C224A. (H) Antiviral activity of NITD-688 against WT ZIKV-Nluc or A197T/C224A mutant in A549 cells at 48 h postinfection. Mutant A197T/C224A was engineered into the backbone of Dakar strain-derived reporter ZIKV Dakar-Nluc. (I) Summary of binding K_D and EC₅₀ values of NITD-688 against WT ZIKV or mutant A197T/C224A.

Fig. 3.

NITD-688 disrupts the NS4B/NS3 interaction. (A) ITC analysis of DENV-2 NS4B binding to NS3 in the presence or absence of NITD-688. DENV-2 NS4B was preincubated with indicated molar ratios of NITD-688 or 0.5 % DMSO. (B) ITC analysis of NITD-688’s impact on NS4B mutant A222V binding to NS3. (C) Summary of NS4B/NS3 binding K_D values estimated from panels A–D. (D) Scheme for BLI analysis of inhibitors’ impact on the dissociation of NS3/NS4B complex. Streptavidin (SA) biosensors were presoaked in biotinylated NS4B and followed by incubation with NS3. The dissociation signals of NS3 from preformed NS4B/NS3 complexes were measured in 0.5% DMSO buffer or inhibitors. See details in Materials and Methods. (E) BLI curves show the NITD-688’s impact on the dissociation of NS3/NS4B complexes. (F) BLI curves show the JNJ-1802’s impact on the dissociation of NS3/NS4B complexes. (G) Summary of k_on and k_off values calculated from panels E and F. (H) Dose-k_off curves. NITD-688 and JNJ-1802 were colored in blue and orange, respectively. (I) Scheme of the co-IP experiments for analyzing NS4B/NS3 interaction in cells. Plasmids encoding NS2B-NS3 and 2 K-NS4B with a C-terminal Flag tag (2 K-NS4B-Flag) were cotransfected into HEK-293 T cells. Cells were exposed to inhibitors at 4 h (early treatment) or 24 h (delayed treatment) posttransfection. At 48 h posttransfection, cells were harvested for immunoprecipitation. (J) Western blot analysis of NS3 co-IPed with WT NS4B-Flag. k Western blot analysis of NS3 co-IPed with NS4B mutant T195A/A222V/S238F. (J and K) Representative plots from three independent Co-IP experiments. (L) Quantification of NS3 co-IPed by WT NS4B. The NS3 co-IPed with NS4B from DMSO-treated cells was set at 1.0. (M) Quantification of NS3 co-IPed with NS4B mutant T195A/A222V/S238F. The NS3 co-IPed with NS4B from DMSO-treated cells was set at 1.0. (L and M) Means and SD from three independent experiments are shown. One-way ANOVA with Dunnett’s multiple comparison correction was used for group comparison.

Fig. 4.

NITD-688 disrupts NS4B/NS3 complexes in DENV-infected cells. (A) Cartoon of NTID-688’s impact on NS4B/NS3 complexes in DENV-infected cells. (B) Scheme of DENV-2 infection with delayed treatment. (C) Intracellular viral protein expression. The protein level in DMSO-treated cells was set at 1.0. (D) Extracellular viral RNA levels. The RNA level in DMSO-treated cells was set at 100%. e-f Representative images of NS4B and NS3 staining in WT DENV-2-infected cells when NS4B was probed by mAb 10-3-7 (E) or 44-4-7 (F). (Scar bar, 20 μm.) The fluorescence ratiometric color scale ranges from 0 (dark purple) to 9.0 (bright yellow), representing the increase in fluorescence ratios of NS4B over NS3. (G and H) Cytofluorogram analysis of NS3 and NS4B within WT DENV-2-infected cells. NS4B was probed with mAb 10-3-7 (G) or 44-4-7 (H). Each dot represents the fitted cytofluorogram slope from an individual cell (>24 cells per group). (I and J) Representative images of NS4B and NS3 staining in mutant DENV-2 T195A/A222V/S238F-infected cells. NS4B was probed by (I) mAb 44-4-7 or (J) 10-3-7. The fluorescence ratiometric color scale ranges from 2.0 (dark purple) to 6.0 (bright yellow). (K and L) Cytofluorogram analysis of NS3 and NS4B in T195A/A222V/S238F-infected cells. NS4B was probed by mAb 10-3-7 (K) or 44-4-7 (L). Each dot represents the cytofluorogram slope of an individual cell (50 to 60 cells per group). The one-way ANOVA Dunn’s with multiple comparisons was used for statistical analysis.

Fig. 5.

Antiviral activity of NITD-688 in cell cultures. (A) Scheme for DENV-2 infection followed by inhibitor treatment at various times of addition. Cells were infected with DENV-2 NGC containing a Nanoluciferase reporter (NGC-Nluc). At indicated time points, a series of diluted inhibitors was added. EC₅₀s of each inhibitor at each time of addition were determined at 24 h postinfection. (B) Dose–response curves of inhibitors against NGC-Nluc. (C) Summary of EC₅₀s values and fold changes of inhibitors against NGC-Nluc. For each inhibitor, the fold change in EC₅₀ was calculated by comparing EC₅₀ values at each given time point to that at 1 h postinfection. (D) Scheme for the time of addition after transfection of DENV-2 replicon RNA. Cells were electroporated with in vitro transcribed DENV-2 NGC-Rluc replicon RNAs (57), in which viral structural proteins were replaced with a Renilla luciferase gene (NGC-Rluc replicon). At the indicated time points, a series of diluted inhibitors was added. At 34 h postinfection, EC₅₀s of each inhibitor at each given time of addition were determined. (E) Dose–response curves of inhibitors against transient NGC-Rluc replicon. (F) Summary of EC₅₀ values and fold change of inhibitors against transient NGC-Rluc replicon. For each inhibitor, the fold change in EC₅₀ was calculated by comparing the EC₅₀ values at each given time point to that of immediate treatment (0 h) after electroporation. (G) A proposed working model for NITD-688.