Exploring the Structural and Functional role of α,β-unsaturated Ketoesters as Anti-Staphylococcal Agents Targeting Glutathione Peroxidase

Multiple-drug resistant (MDR) Staphylococcus aureus strains (like methicillin-resistant S. aureus or MRSA) uses an arsenal of antioxidant enzymes to mitigate host-induced oxidative stress. Among them the non-canonical Staphylococcal glutathione peroxidase (SaGpx) plays a crucial role in bacterial redox homeostasis by reducing peroxides via thioredoxin-dependent pathways. Thus, enabling oxidative stress mitigation during host infection. Despite its importance in S. aureus, its role in bacterial pathogenesis remains unexplored. This study aimed to elucidate the possible role of SaGpx in Staphylococcal virulence. First, we determined the high-resolution crystal structure of SaGpx (at 1.65 [A] resolution) using X-ray crystallography. Guided by the catalytic cleft architecture of SaGpx, small-molecule based inhibitors were then rationally designed and synthesized. These inhibitors exhibited good binding affinity to SaGpx and complete enzymatic blockade. These inhibitors exhibited potent anti-S. aureus activity (MICs 6.25-31.25 M) along with no cytotoxicity in L929 fibroblast wound-healing assays. Furthermore, the in vivo antibacterial ability of these inhibitors was evaluated using S. aureus-infected skin wound mouse model, where these compounds show potent antibacterial and wound healing ability supported by subsequent histological as well as immunohistochemical analysis. These findings suggest SaGpx as a possible virulence determinant in S. aureus and position these synthesized inhibitors as promising antivirulence therapeutics. HighlightsO_LIThe high-resolution crystal structure of Staphylococcal glutathione peroxidase is solved. C_LIO_LIBased on the SaGpx catalytic site, ,{beta}-unsaturated ketoesters derivatives are synthesized. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives inhibit SaGpx activity and binds the protein at M range. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives show in vitro antibacterial activity against S. aureus at low M range. C_LIO_LISynthesized ,{beta}-unsaturated ketoesters derivatives show in vivo antibacterial and wound healing ability S. aureus-infected skin wound mouse model. C_LI