Discovery of thiosemicarbazone derivatives as promising SARS-CoV-2 Mpro inhibitors by spectroscopy and microscale thermophoresis.

Since the outbreak of COVID-19, one key strategy has been to screen inhibitors of the SARS-CoV-2 main protease (Mpro). Mpro is critical for viral replication and a key target for therapeutic development. In this work, we report the design and synthesis of 29 thiosemicarbazone derivatives. Enzymatic inhibition assays showed that 27 compounds exhibited inhibitory activity against Mpro, among which 4 compounds (1e, 2c, 2f, 3a) demonstrated significant inhibitory potency, with IC50 values of 5.97 ± 0.98 μM, 4.43 ± 1.67 μM, 10.99 ± 2.39 μM, and 28.88 ± 3.57 μM, respectively. Fluorescence spectroscopy, molecular docking, microscale thermophoresis and molecular dynamics simulations were used to analyze the interaction mechanisms between thiosemicarbazone derivatives and Mpro. Fluorescence results indicated that the fluorescence quenching mechanism of the compounds on Mpro was predominantly static quenching. Hydrogen bonding and hydrophobic interactions were observed in the binding process. Microscale thermophoresis results confirmed that compounds 1e, 2c and 2f exhibit significant binding affinity for Mpro, with Kd values of 4.21 ± 0.35 μM, 3.24 ± 0.28 μM, and 24.26 ± 1.82 μM, respectively. This study provides valuable information for understanding the interaction mechanism between thiosemicarbazone derivatives and Mpro and offers insights into novel Mpro inhibitors.