Design and Synthesis of Peptidomimetic Inhibitors Targeting the Main Protease (Mpro) of Human Coronavirus NL63 Using Molecular Docking and In Vitro Assays

Human coronavirus NL63 (HCoV-NL63) is a major respiratory pathogen that causes severe upper and lower respiratory tract infections, particularly in pediatric, geriatric, and immunocompromised populations. Currently, there are no approved antiviral therapies specifically targeting HCoV-NL63. The viral main protease (Mpro) is an attractive target for drug development due to its indispensable role in processing the viral polyproteins required for replication. In this study, we designed and synthesized a series of novel peptidomimetic inhibitors featuring a C-terminal electrophilic warhead (either an aldehyde or a fluoromethyl ketone) to target the active site of HCoV-NL63 Mpro. In silico molecular docking was utilized to optimize interactions within the S1, S2, and S4 subsites, leading to the selection and synthesis of five top-ranking candidate compounds (NL-01 to NL-05). In vitro fluorogenic enzyme assays demonstrated that compound NL-03, harboring a leucine residue at P2 and an aldehyde warhead, exhibited potent inhibitory activity with an IC50 value of 0.84 ± 0.09 µM. Kinetic analysis characterized NL-03 as a competitive, reversible inhibitor with a Ki of 0.42 ± 0.05 µM. These findings provide a solid biochemical framework for the development of highly selective, small-molecule therapeutics against HCoV-NL63 and related coronaviruses.