Investigation of the Interaction Between 4‐Thiazolidinone Derivatives and SARS‐CoV‐2 Mpro Using Spectroscopy and Microscale Thermophoresis

Abstract Since the outbreak of COVID‐19, one of the strategies used to search for potential drugs has been to screen inhibitors of SARS‐CoV‐2 Mpro. This work investigated the inhibitory effects of 4‐thiazolidinone derivatives on main protease (Mpro) using various methods, including enzyme activity assays, enzyme kinetics, spectroscopy experiments, molecular docking simulations, microscale thermophoresis, and cytotoxicity tests. Compound 3a demonstrated the best inhibition effect among the compounds tested, with IC 50 = 2.36 ± 0.36 µM and K i = 1.72 µM. The trifluoromethyl group in 3a might enhance its inhibition ability. Interactions between the 4‐thiazolidinone derivatives and Mpro resulted in fluorescence quenching primarily through static quenching. Thermodynamic analysis indicated that the interaction forces are mainly hydrogen bonds and van der Waals forces. Spectral results revealed that the 4‐thiazolidinone derivatives affected the microenvironment of aromatic amino acid residues in Mpro and altered its secondary structure. Differential scanning fluorimetry was used to confirm the interaction between the 4‐thiazolidinone derivatives and Mpro. Results of microscale thermophoresis indicated that the K d between 3a and Mpro was 1.56 ± 0.24 µM. Molecular docking simulations provided a more intuitive visualization of the interaction forces. This study provides clues for screening novel Mpro inhibitors.