Combining virtual screening with cis-/trans-cleavage enzymatic assays effectively reveals broad-spectrum inhibitors that target the main proteases of SARS-CoV-2 and MERS-CoV

The main protease (M(pro)) of SARS-CoV-2 is essential for viral replication, which suggests that the M(pro) is a critical target in the development of small molecules to treat COVID-19. This study used an in-silico prediction approach to investigate the complex structure of SARS-CoV-2 M(pro) in compounds from the United States National Cancer Institute (NCI) database, then validate potential inhibitory compounds against the SARS-CoV-2 M(pro) in cis- and trans-cleavage proteolytic assays. Virtual screening of ∼280,000 compounds from the NCI database identified 10 compounds with highest site-moiety map scores. Compound NSC89640 (coded C1) showed marked inhibitory activity against the SARS-CoV-2 M(pro) in cis-/trans-cleavage assays. C1 strongly inhibited SARS-CoV-2 M(pro) enzymatic activity, with a half maximal inhibitory concentration (IC(50)) of 2.69 μM and a selectivity index (SI) of >74.35. The C1 structure served as a template to identify structural analogs based on AtomPair fingerprints to refine and verify structure-function associations. M(pro)-mediated cis-/trans-cleavage assays conducted with the structural analogs revealed that compound NSC89641 (coded D2) exhibited the highest inhibitory potency against SARS-CoV-2 M(pro) enzymatic activity, with an IC(50) of 3.05 μM and a SI of >65.57. Compounds C1 and D2 also displayed inhibitory activity against MERS-CoV-2 with an IC(50) of <3.5 μM. Thus, C1 shows potential as an effective M(pro) inhibitor of SARS-CoV-2 and MERS-CoV. Our rigorous study framework efficiently identified lead compounds targeting the SARS-CoV-2 M(pro) and MERS-CoV M(pro).