Identification of novel non-covalent Mpro inhibitors against SARS-CoV-2 by the computational and experimental approaches

The COVID-19 pandemic, caused by SARS-CoV-2, has posed significant global health challenges and there is an urgent need for effective therapeutic agents. The main protease (M^pro) plays a crucial role in viral replication, making it an attractive target for the development of antiviral drugs. In this study, by screening over 8.06 million compounds obtained from Enamine, Vitas-M, ChemDiv, and TargetMol (USA) databases, 52 top-ranking compounds were obtained as promising candidates through molecular docking, followed by Uni-QSAR modeling and ADMET predictions to evaluate their binding affinities and pharmacokinetic properties. Biological activity assays confirmed the efficacy of four standout candidates L17, L26, L37, and L50 with IC₅₀ values of 5.61 ± 0.58 μM, 6.00 ± 0.63 μM, 4.21 ± 0.89 μM, and 2.84 ± 1.20 μM, respectively, comparable to that of reference ML188 (2.41 ± 0.70 μM). Further insights were gained through density functional theory (DFT) analyses, which provided valuable information regarding the electronic and structural properties of the candidate compounds. Additionally, extensive molecular dynamics (MD) simulations were conducted, revealing critical information about the stability and binding interactions of the compounds within the M^pro active site over a 500 ns simulation period. Besides, the results of binding free energy calculations demonstrated that compounds had higher binding affinity than ML188, and dcTMD simulations further revealed that L26, L37, and L50 followed more favorable and cooperative unbinding pathways with higher energy barriers and lower dissipation compared to ML188. Overall, the results highlight the therapeutic potential of these compounds as effective M^pro inhibitors, laying a solid foundation for further development into novel antiviral agents against SARS-CoV-2.