Targeting the allosteric mechanism of methicillin-resistant Staphylococcus aureus pyruvate kinase: From inhibitor discovery to network reorganization.

Abstract

Pyruvate kinase (PK) is a promising antimicrobial target against Methicillin-resistant Staphylococcus aureus (MRSA) due to its central role in bacterial metabolism. Building on previous virtual screening of three putative/known allosteric sites, we evaluated six FDA-approved compounds for effects on PK-associated enzymatic activity in a bacterial extract system. Four compounds (Dihydroergocristine, Nilotinib, Zanubrutinib, and Ledipasvir) reduced measured activity, whereas Dihydroergotamine and Simeprevir produced modest increases in assay signal. Complementary computational analyses investigated the potential structural and dynamic basis of these activity changes. Elastic network-based AlloSigMA2 indicated similar allosteric responses at the subunit interfaces, while the putative allosteric site was preferentially linked to active-site stabilization. Molecular dynamics (MD) simulations, mutual information and network analyses focused on Zanubrutinib and Simeprevir, with inhibitor 09C and the apo-state as controls. Zanubrutinib remained relatively stable at the inter-subunit interfaces and was associated with inactive-like dynamic features, including strengthened inter-domain coupling and inter-chain correlations. Conversely, Simeprevir was associated with site-dependent active-like dynamic features: binding at the putative allosteric site redistributed correlations toward intra-chain couplings, whereas binding at the large interface markedly reduced long-range correlations, consistent with localized or decoupled communication modes. The apo-state exhibited a pre-organized, effector-centered communication network. Collectively, these findings support a model in which the functional effects of putative allosteric ligands on S. aureus PK may be associated with site-dependent reorganization of its residue communication network. The identified compounds and their predicted communication footprints provide a foundation for further experimental validation and development of species-selective inhibitors as potential next-generation antimicrobial agents.