From ML300 to Novel Non-covalent Inhibitors of SARS‑CoV‑2 Main Protease via Evolutionary De Novo Design, Virtual Screening, Molecular Dynamics, and Retrosynthetic Strategies
Anna O. Geleverya, Alexander V. Kyrychenko, Volodymyr Ivanov, Larysa V. Yevsieieva, Volodymyr Fetyukhin, Sergiy М. Kovalenko, Oleg N. Kalugin · Polycyclic aromatic compounds · 2026
The main protease (Mpro) of the SARS-CoV-2 coronavirus is essential for viral processing and the disruption of the immune response, making inhibitors derived from the ML300 scaffold valuable as potential non-covalent, pan-coronavirus drugs with improved profiles. This study aimed to discover and develop new small-molecule inhibitors of Mpro effective against emerging variants through an integrated computational and synthetic strategy. The methods involved creating virtual libraries of ML300 analogues (2565 and 11367 compounds) using evolutionary algorithms, followed by an in silico screening process that included pharmacophore filtering and molecular docking calculations against both conventional and drug-resistant variants (including Delta and Omicron). Subsequent ADME calculations served as a secondary filter, ultimately leading to a short list of six lead ML300 analogues. The structural stability of the Mpro complex with the best lead was confirmed through all-atom molecular dynamics (MD) simulations in water, validating its inhibitory potency in an aqueous environment. The key finding is the successful identification of these six analogues, which demonstrated superior predicted binding affinity and favorable drug-likeness. In conclusion, this study validated a powerful in silico discovery pipeline, leading to the identification of highly promising Mpro inhibitors; to prepare for future functional testing, we developed a retrosynthetic strategy and synthesized 47 new ML300 analogues, which represent strong candidates for subsequent in vitro and in vivo studies against coronavirus threats.