Discovery of a new SARS-CoV-2 Mpro inhibitor from a natural product and natural product-like compound database using in silico and in vitro studies.

This current study employs structure-guided virtual screening to identify potential inhibitors from 69,075 natural product or natural product-like compounds in the InterBioscreen (IBS) database against the SARS-CoV-2 Main protease (Mpro), a well-validated target. From high-throughput structure-guided virtual screening, subsequently followed by more rigorous re-docking, top seven compounds were chosen as hits based on their high binding affinity and specific H-bond interactions with the active-site residues of Mpro. All these compounds were new for this target as well as the virus. The stability of the seven protein-ligand complexes was further assessed using molecular dynamics (MD) simulations and the MM-GBSA method. The ADME/Tox profile of these seven compounds was also predicted theoretically. After MD simulation, only three compounds, viz. STOCK1N-86169, STOCK1N-88750, and STOCK1N-65657 were identified as high-potential candidates for Mpro inhibition. To confirm the in silico analysis results, an in vitro FRET-based Mpro inhibition assay and an ELISA-based SARS-CoV-2 inhibition assay were performed. From these in vitro investigations, STOCK1N-86169 was identified as the only potent Mpro inhibitor, exhibiting an IC50 of 3.8 µM. Independent confirmation of STOCK1N-86169 binding to Mpro was obtained by differential scanning calorimetry (DSC), which revealed an increase of approximately 15 °C in the melting temperature (Tm) of the Mpro-STOCK1N-86169 complex relative to the apo-protein. In the cytotoxicity assay, it showed CC50 > 100 µM for HeLa, ~ 100 µM for the Huh-7 cell line, and ~ 33.5 µM for the Vero E6 cell line. In the Vero cell-based SARS-CoV-2 infection assay, it showed an EC50 of ~ 7 µM, with a reasonable Selective Index (S.I.) of ~ 5-fold. Our study suggests that the natural product like compound STOCK1N-86169, which presents a new scaffold for Mpro inhibition and also SARS-CoV-2, may be a promising candidate for further development as an effective inhibitor of SARS-CoV-2 Mpro.