Structure-guided design and triplicate molecular dynamics evaluation of mutant peptide inhibitors targeting SARS-CoV-2 main protease (Mpro).

Unlabelled: The global health crisis caused by SARS-CoV-2 underscores the urgent need for effective antiviral therapeutics. The SARS-CoV-2 main protease (Mpro) is a crucial enzyme in viral replication, making it a prime target for drug development. In this study, we designed and evaluated peptide inhibitors targeting Mpro by introducing systematic mutations in the Nsp10/11 cleavage site peptide (QLMPER). A library of 214 mutant peptides was generated, from which 25 single-mutant and 70 multi-mutant peptides exhibited strong interactions with Mpro. The top four multi-mutant peptides were selected based on docking scores, molecular dynamics (MD) simulations, and MM-GBSA (Molecular Mechanics-Generalized Born Surface Area) binding free energy calculations. Triplicate 100 ns molecular dynamics simulations assessed the stability of these complexes, revealing that M3 exhibited the highest structural stability and lowest binding free energy (- 34 kcal/mol), outperforming the wild-type peptide (- 4.28 kcal/mol). Computational infrared (IR) spectral analysis confirmed structural modifications induced by mutations, while HOMO-LUMO analysis indicated enhanced reactivity for M3 (FLFPFR). These findings suggest that M3 (FLFPFR) is a promising candidate for SARS-CoV-2 inhibition, highlighting the potential of rationally designed peptide inhibitors in antiviral drug discovery.

Graphical Abstract: Computational workflow for design and evaluation of mutant peptides against SARS-CoV-2 Mpro, highlighting the workflow, key interactions, and improved binding of mutants at S2 and S3/S4 subsites.

Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-025-00409-2.