Discovery of Semicarbazone and Thiosemicarbazone Analogs as Competitive SARS-CoV-2 Virus Main Protease (M pro ) Inhibitors

Despite the development of vaccines and antivirals, coronavirus disease 2019 (COVID-19) continues to affect populations worldwide. Given the high mutation rate of the SARS-CoV-2 virus and reports of drug resistance, there is a continued need for new therapeutic options. SARS-CoV-2 main protease (M pro ) is essential for viral replication and is a conserved target among coronaviruses. Most known M pro inhibitors target the active site, although allosteric sites have already been identified. In this study, we conducted a virtual screening of 2,060 compounds targeting an allosteric site of SARS-CoV-2 M pro . From this screen, 41 computational hits and analogs were selected and evaluated using biochemical assays against SARS-CoV-2 M pro . Among them, compound 25 , a semicarbazone, demonstrated a half-maximal inhibitory concentration (IC 50 ) of 99 μM. Additionally, two thiosemicarbazone analogs (compounds 50 and 51 ) inhibited SARS-CoV-2 M pro with IC 50 values of 61 μM and 70 μM. Biochemical assays suggest that these compounds act as noncovalent competitive inhibitors of SARS-CoV-2 M pro . Molecular dynamics simulations revealed that compound 25 is unstable at the allosteric site of SARS-CoV-2 M pro but forms stable and favorable interactions at the active site, supporting its potential as a competitive inhibitor, a finding subsequently confirmed by biochemical assays. Our structure-based computational and biochemical approach identified semicarbazone and thiosemicarbazone scaffolds as promising candidates for the development of reversible SARS-CoV-2 M pro inhibitors.