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

M ^pro , also known as 3CL ^pro , 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 M ^pro . 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 M ^pro 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 M ^pro and might denature other proteins as well. In contrast, the compounds of the second cluster targeted M ^pro with much greater specificity and enhanced its melting temperature, consistent with the formation of stable M ^pro -inhibitor complexes. The most active compounds of the second cluster exhibited IC ₅₀ values between 4 and 7 μM and their chemical structure suggests that they could serve as leads for the development of potent M ^pro inhibitors.