Bepridil is potent against SARS-CoV-2 in vitro

Abstract

Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC₅₀) value below 100 μM in inhibiting M^pro, and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting M^pro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti-SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC₅₀) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.

Keywords: COVID-19; SARS-CoV-2; bepridil; drug repurposing; main protease.

Fig. 1.

Activity of M^pro. (A) The structures of three substrates. (B) Activity of 50 nM M^pro on 10 μM Sub1. (C) Activity of 50 nM M^pro on 10 μM Sub2 and Sub3. The florescence signals are normalized for easy comparison. (D) Activity of different concentrations of M^pro on 10 μM Sub3 plotted as fluorescence intenisty (fluo. int.) in arbitrary units (a.u.) vs time graph. All experiments were carried with three repeats, and data are presented as the average of three repeats in color, with error bars shown in black.

Fig. 2.

Initial screening of M^pro inhibition by 29 FDA/EMA-approved medicines and rupintrivir; 1 mM (0.14 mM for Itraconazole due to its low solubility in DMSO) was used for each inhibitor to perform the inhibition assay. Fluorescence intensity was normalized with respect to the control that had no small molecule provided. Triplicate experiments were performed for each compound, and the value was presented as mean ± SE.

Fig. 3.

IC₅₀ assays for 18 small-molecule medicines on their inhibition of M^pro. Triplicate experiments were performed for each compound, and the IC₅₀ value was presented as mean ± SE. GraphPad Prism 8.0 was used to perform data analysis.

Fig. 4.

(A) Pimozide, (B) ebastine, (C) bepridil, and (D) their overlay in the active site of M^pro. The protein surface topography in A−C is presented to show the concaved active site.

Fig. 5.

The SARS-CoV-2 inhibition by bepridil in (A) Vero E6 and (B) A549/ACE2 cells. Cells were incubated with different concentrations of bepridil and then infected with 0.5 MOI of SARS-CoV-2. Cells were let grow for 3 d for Vero E6 and 4 d for A549/ACE2 cells before their virus loads were determined. All experiments were performed in triplicate. Average MOIs with their SDs are presented.