Non-Catalytic Structural Regions as Targets for the Inhibition of Staphylococcal and Mammaliicoccal Exfoliative Toxins

Exfoliative toxins (ETs), produced by species of the genera Staphylococcus and Mammaliicoccus, are glutamyl endopeptidases (GEPs) with high specificity for desmoglein-1 (Dsg1), capable of causing skin lesions and various clinical manifestations in mammals. This study investigated three ETs of significant clinical and economic relevance: ETA, ExhC, and ETE. Despite their importance, no specific inhibitors of these toxins are currently available, and the structural mechanisms responsible for modulating their activity remain poorly understood. Objectives: To evaluate the inhibitory potential of curcumin, heparin, and suramin, as well as to investigate the role of secondary structural regions located outside the catalytic site as regulatory elements and potential targets for enzyme inhibition. Methodology: The esterolytic activity of wild-type ETs, the ETEΔL1 and ExhCmut variants, and V8 protease was characterized. Subsequently, inhibition assays, dose–response curves, tests with mutant proteins, thermal stability analyses using circular dichroism (CD), and in silico studies were performed. Results: Suramin and heparin consistently inhibited the ETs, whereas V8 protease showed low susceptibility to these compounds, indicating that inhibition depends on specific structural features. The dose–response curves displayed a biphasic profile, suggesting the occurrence of more than one binding event between the compounds and their targets. Furthermore, point mutations in the necrosis-associated region of ExhC and deletion of the L1 loop in ETE significantly reduced the inhibition promoted by both ligands. In the presence of heparin and suramin, the ETs also exhibited increased thermal stability. In silico analyses revealed preferential interactions between the ligands and positively charged sites corresponding to these structural regions, whereas the equivalent region in V8 protease exhibited a predominantly negative electrostatic potential. Conclusion: These findings identify non-catalytic structural regions as potential targets for the development of selective inhibitors and broaden the understanding of the structural basis underlying ET inhibition.
This research was funded by the São Paulo Research Foundation (FAPESP), grants Nº. 2020/08615-8 and Nº. 2025/08276-2, and by the Brazilian National Council for Scientific and Technological Development (CNPq), grant Nº. 307129/2025-0.