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}.
【저자키워드】 SARS-CoV-2, main protease, MERS-CoV, cis-/trans-cleavage assay, In-silico prediction, National cancer institute database,