Domperidone inhibits dengue virus infection by targeting the viral envelope protein and nonstructural protein 1

Abstract

Dengue is a mosquito-borne disease caused by dengue virus (DENV) infection, which remains a major public health concern worldwide owing to the lack of specific treatments or antiviral drugs available. This study investigated the potential repurposing of domperidone, an antiemetic and gastrokinetic agent, to control DENV infection. Domperidone was identified by pharmacophore-based virtual screening as a small molecule that can bind to both the viral envelope (E) and the nonstructural protein 1 (NS1) of DENV. Molecular dynamics (MD) simulations and surface plasmon resonance (SPR) analysis were subsequently performed to determine specific interactions of domperidone with the DENV E and NS1 proteins and their binding affinity. Treatment of immortalized human hepatocyte-like cells (imHC) with domperidone could inhibit DENV production and NS1 secretion in a dose-dependent manner following infection with DENV serotype 2. These inhibitory effects were mediated by reduction in viral RNA replication and viral E and NS1 protein expression, but not by interference with virus entry into cells or NS1 oligomerization. The suppression of DENV production and NS1 secretion by domperidone was observed across all four DENV serotypes to varying degrees between different virus strains. The findings from our study suggest viral target-based repurposing of domperidone for modulating DENV.

Keywords: DENV production; Dengue virus; Domperidone; E and NS1 proteins; MD simulation; NS1 secretion.

Fig. 1

Molecular interactions between domperidone and DENV E protein. MD simulations of the domperidone–DENV E complex were performed three times (300 ns each). A representative tertiary structure of the domperidone–DENV E complex obtained from MD simulation 1 (final structure) is shown: (a) side view; (b) top view; (c) close-up view of the potential core binding sites of domperidone on DENV E protein; (d) molecular interactions between amino acid residues of DENV E proteins and specific positions of the domperidone structure with 5 aromatic rings (Aro, A–D) within the potential core binding region. HSD designates a histidine protonated at its δN-atom on the DENV E protein: chain A (blue) and chain B (green). Affinity of the domperidone–DENV E protein complex was analyzed by a surface plasmon resonance assay using Biacore T200. Single-cycle kinetics of binding responses at varying concentrations of domperidone over time (three cycles each) were shown (e). The equilibrium disassociation constant (K_D) of domperidone binding to DENV E was determined by Biacore T200 Evaluation Software with the steady-state affinity model and shown as mean ± SEM from three cycles of analyte injection (f).

Fig. 2

Molecular interactions between domperidone and DENV NS1 protein. MD simulations of the domperidone–DENV NS1 complex were performed three times (300 ns in each run). A representative tertiary structure of the domperidone–DENV NS1 complex obtained from MD simulation 1 (final structure) is shown: (a) side view; (b) top view; (c) close-up view for the potential core binding sites of domperidone in the β-roll region of DENV NS1; (d) molecular interactions between amino acid residues of DENV NS1 proteins and specific positions of the domperidone structure with 5 aromatic rings (Aro, A–D) within the potential core binding region (β-roll). Dimeric DENV NS1 protein: chain A (blue) and chain B (green). Affinity of the domperidone–DENV NS1 protein complex was analyzed by a surface plasmon resonance assay using Biacore T200. Single-cycle kinetics of binding responses at varying concentrations of domperidone over time (three cycles each) were shown (e). The equilibrium disassociation constant (K_D) of domperidone binding to DENV NS1 was determined by Biacore T200 Evaluation Software with the steady-state affinity model and shown as mean ± SEM from three cycles of analyte injection (f).

Fig. 3

Effects of domperidone on DENV-2 infection. (a) Determination of the 50% cytotoxic concentration (CC50) of domperidone in imHC cell cultures by luminescence-based ATP detection assay and nonlinear regression analysis. The percent cell viability was determined relative to non-treated cell control. (b) Experimental strategies for domperidone treatment in DENV-infected imHC cell cultures. Briefly, cells were infected with DENV-2 and treated with domperidone or DMSO (vehicle control) at nontoxic concentrations during 2 h of virus inoculation (during virus entry) or after 2 h of virus inoculation (after virus entry), and DENV-infected cells and culture supernatants were collected at 24 h post-infection. (c) Determination of infectious DENV production in culture supernatant by FFU assay. (d) Evaluation of the viability of DENV-infected imHC cells by the trypan blue exclusion dye method. The results show the mean ± SEM (4 independent experiments for determination of CC50 or 3 independent experiments for determination of DENV production and cell viability). Statistical analysis for significant difference (****, P-value < 0.0001) was performed between the groups treated with DMSO or domperidone using an unpaired t-test.

Fig. 4

Effects of domperidone on DENV-2 NS1 secretion and oligomerization. (a) imHC cells were infected with DENV-2 and treated with domperidone or DMSO (vehicle control) at the indicated concentrations during or after virus entry. Culture supernatants were collected at 24 h post-infection to determine DENV NS1 secretion by NS1 ELISA. The results show the mean ± SEM of 3 independent experiments. Statistical analysis for significant differences (****, P-value < 0.0001; *, P-value = 0.0294) was performed between the groups treated with DMSO or domperidone using an unpaired t-test. (b) Culture supernatants from DMSO (0.06%) or domperidone (31.25 µM) treatment after virus entry were further analyzed for DENV NS1 oligomerization by native polyacrylamide gel electrophoresis. The results are representative of 3 independent experiments.

Fig. 5

Effects of domperidone on viral RNA replication and intracellular dengue viral protein expression. imHC cells were infected with DENV-2 and treated with domperidone or DMSO (vehicle control) at the indicated concentrations after virus entry and collected at different time points post-infection. (a) Immunoblotting analysis for DENV E and NS1 protein expression in duplicate samples at 24 h post-infection. (b) Relative expression of the DENV E and NS1 proteins based on normalization of their protein band intensities to that of human GAPDH, an internal control protein. (c) Determination of intracellular DENV NS1 expression by NS1 ELISA using the same set of samples at 24 h post-infection. (d) Determination of DENV E and NS1 RNA levels in DENV-2 infected imHC by real-time RT-PCR. imHC cells that had been treated with domperidone (31.25 µM) or DMSO (0.06%, vehicle control) after virus entry were harvested at 0, 6, 12, and 24 h post-infection and processed for RNA extraction. Total RNA was subjected to quantitative real-time RT-PCR using DENV E-specific primers (right panel) or DENV NS1-specific primers (left panel) as described in the Methods. The viral RNA copy numbers in the samples were determined by comparing with the known amount of standard RNA. The results show the mean ± SEM (3 independent experiments for immunoblotting analysis and NS1 ELISA or 2 independent experiments for real-time RT-PCR; duplicate samples in each experiment). Statistical analysis for significant differences was performed between the groups treated with DMSO or domperidone using an unpaired t-test (****, P-value < 0.0001; ***, P-value = 0.0002 and 0.0006 for DENV NS1 expression determined by immunoblotting analysis and NS1 ELISA; *, P-value = 0.0365 for DENV E expression, P-value = 0.0231 and 0.0106 for viral NS1 RNA levels at 6 and 12 h, and P-value = 0.0283 for viral E RNA levels).

Fig. 6

Effects of domperidone against all four serotypes of DENV production and DENV NS1 secretion. imHC cells were infected with DENV-1 (strains Hawaii and 16007), DENV-2 (strains 16681 and D87-011), DENV-3 (strains H87 and 16562), and DENV-4 (strains H241 and 1036) and treated with domperidone (31.25 µM) or DMSO (0.06%, vehicle control) after virus entry. The virus-infected cells and culture supernatants were harvested at 24 h post-infection (a) Determination of infectious DENV production in culture supernatants by FFU assay. (b) Assessment of DENV NS1 secretion by NS1 ELISA. The results show the mean ± SEM of 3 independent experiments. Statistical analysis for significant differences (****, P-value < 0.0001; ***, P-value = 0.0001 for virus production of DENV-3 strain 16562 and P-value = 0.0005 and 0.001 for NS1 secretion of DENV-3 strain H87 and DENV-4 strain H241; **, P-value = 0.0052 for virus production of DENV-1 strain 16007 and P-value = 0.0021 and 0.0061 for NS1 secretion of DENV-2 strain 16681 and DENV-4 strain 1036) was performed using an unpaired t-test.