Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 µM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.

Shin-ichiro Hattori, Nobuyo Higashi-Kuwata, Hironori Hayashi, Srinivasa Rao Allu, Jakka Raghavaiah, Haydar Bulut, Debananda Das, Brandon J. Anson, Emma K. Lendy, Yuki Takamatsu, Nobutoki Takamune, Naoki Kishimoto, Kazutaka Murayama, Kazuya Hasegawa, Mi Li, David A. Davis, Eiichi N. Kodama, Robert Yarchoan, Alexander Wlodawer, Shogo Misumi, Andrew D. Mesecar, Arun K. Ghosh, Hiroaki Mitsuya