Identification of low micromolar SARS-CoV-2 Mpro inhibitors from hits identified by in silico screens

Abstract

Mpro, also known as 3CLpro, is the main protease of the SARS-CoV-2 coronavirus and, as such, is essential for the viral life cycle. Two studies have each screened and ranked in silico more than one billion chemical compounds in an effort to identify putative inhibitors of Mpro. More than five hundred of the seven thousand top-ranking hits were synthesized by an external supplier and examined with respect to their activity in two biochemical assays: a protease activity assay and a thermal shift assay. Two clusters of chemical compounds with Mpro inhibitory activity were identified. An additional five hundred molecules, analogues of the compounds in the two clusters described above, were also synthesized and characterized in vitro. The study of the analogues revealed that the compounds of the first cluster acted by denaturing Mpro and might denature other proteins as well. In contrast, the compounds of the second cluster targeted Mpro with much greater specificity and enhanced its melting temperature, consistent with the formation of stable Mpro-inhibitor complexes. The most active compounds of the second cluster exhibited IC50 values between 4 and 7 M and their chemical structure suggests that they could serve as leads for the development of potent Mpro inhibitors.