Discovery of antiviral SARS-CoV-2 main protease inhibitors by structure-guided hit-to-lead optimization of carmofur.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M) has been targeted for the development of anti-SARS-CoV-2 agents against COVID-19 infection because M processes essential viral polyproteins and plays a key role in SARS-CoV-2 replication. In this study, we report the development of novel SARS-CoV-2 M inhibitors derived from carmofur, a previously identified compound that has shown moderate potency as a covalent inhibitor of SARS-CoV-2 M. To employ a structure-guided drug design strategy, a putative intact binding mode of carmofur at catalytic active site of M was initially predicted by docking simulation. Based on the predicted binding mode, a series of carmofur derivatives aiming to occupy the M substrate binding regions were investigated for structure-activity relationship analysis. As a result, an indole-based derivative, speculated to interact with the S4 binding pocket, 21b (IC = 1.5 ± 0.1 μM) was discovered. Its structure was further modified and evaluated in silico by combining docking simulation, free energy perturbation calculation and subpocket interaction analysis to optimize the interactions at the S2 and S4 binding pockets. Among the newly designed novel derivatives, 21h and 21i showed the best inhibitory potencies against M with IC values of 0.35 and 0.37 μM, respectively. Moreover, their antiviral activities were confirmed with EC values of 20-30 μM in the SARS-CoV-2-infected cell-based assay, suggesting that these novel M inhibitors could be applied as potential lead compounds for the development of substantial anti-SARS-CoV-2 agents.