Allosteric inhibition of SARS-CoV-2 3CL protease by colloidal bismuth subcitrate

Abstract

The SARS-CoV-2 3-chymotrypsin-like protease (3CLpro or Mpro) is a key cysteine protease for viral replication and transcription, making it an attractive target for antiviral therapies to combat the COVID-19 disease. Here, we demonstrate that bismuth drug colloidal bismuth subcitrate (CBS) is a potent inhibitor for 3CLpro in vitro and in cellulo. Rather than targeting the cysteine residue at the catalytic site, CBS binds to an allosteric site and results in dissociation of the 3CLpro dimer and proteolytic dysfunction. Our work provides direct evidence that CBS is an allosteric inhibitor of SARS-CoV-2 3CLpro.

Fig. 1. (A) Identification of inhibitory metallodrugs for SARS-CoV-2 3CLpro. Bar charts show the relative protease activities of 3CLpro after incubation with 50 μM sodium stibogluconate (SSG), colloidal bismuth subcitrate (CBS), bismuth gluconate, cis-platinum, Auranofin, AuP(CH₂CH₃)₃Cl and four Au(i)-based complexes. (B) Inhibition of the protease activities of 3CLpro by CBS at varying concentrations in vitro. Dose–response curves for half-maximum inhibitory concentration (IC₅₀) values were determined by nonlinear regression. (C) Inhibition of SARS-CoV-2 proteases by CBS in cellulo. Schematic illustration of the cell-based luciferase reporter assay. (D) The cells that expressed 3CLpro are treated with varying concentrations of CBS. NC represents negative control. Bar charts show the relative luciferase activity of cell lysate. Each experiment were performed in triplicates. The data are shown in mean ± sd. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2. SARS-CoV-2 3CLpro dimer dissociation induced by colloidal bismuth subcitrate (CBS). (A) Structure of SARS-CoV-2 3CLpro dimer is shown in cartoon and surface model (PDBID: 6M2Q). The cysteine residues with the highest solvent accessibility are highlighted in red. Bar chart shows the Bi(iii)-binding capability of wild-type 3CLpro and mutants. Each experiment were performed in triplicates. The data are shown in mean ± sd. Size exclusion chromatography analysis of (B) 3CLpro and (C) 3CLpro^C300S mutant after incubation with different molar ratios of Bi(iii).

Fig. 3. A snapshot of 3CLpro dimer in MD simulations in the presence of Bi(iii) and Na(i). The C-terminal helical segment was colored in yellow cartoon and Bi(iii) ion was shown in red sphere. (A) Both the hydrogen bonds between R298-M6 and R4-E290* are disrupted in the presence of Bi(iii). (B) The hydrogen bonds between R298-M6 and R4-E290* are well maintained in the presence of Na(i). The distances between two residues are labeled in Å.

Fig. 4. Inhibition of SARS-CoV-2 replication in Vero E6 cell by colloidal bismuth subcitrate (CBS). (A) Representative immunofluorescence staining images of Vero E6 cells infected with SARS-CoV-2. The nucleocapsid protein of SARS-CoV-2 (N protein) antigens and cell nuclei (DAPI) were stained in green and blue, respectively. (B) Inhibition of SARS-CoV-2 replication and cytotoxicity of increasing concentrations of colloidal bismuth subcitrate (CBS). Each experiment was performed in triplicates. The data are shown in mean ± sd.

Fig. 5. Schematic illustration of the inhibition of SARS-CoV-2 replication in cell. Bismuth-binding induced dissociation of 3CLpro dimer leads to the collapse of substrate-binding site and proteolytic dysfunction.