New SufA Protease Inhibitors and Activity-based Probes: Design, Synthesis, and Antibacterial Activity Assessment.

BACKGROUND: The growing threat of bacterial drug resistance compels researchers to pursue new therapeutic strategies. Finegoldia magna secretes SufA, a subtilisin- like protease that plays a critical role in virulence by degrading host defense molecules such as fibrinogen and LL-37. This study aimed to investigate the stereoselective inhibition of SufA by pure diastereoisomers of peptidyl 1-aminoalkylphosphonate diaryl esters and to evaluate their ability to reduce bacterial virulence and growth. METHODS: We synthesized and purified the diastereoisomers and assessed their ability to inhibit SufA using a fluorescent enzymatic assay. Antibacterial activity was evaluated against Gram-positive and Gram-negative bacteria. SDS-PAGE was used to examine the inhibition of SufA-mediated degradation of fibrinogen and LL-37. Molecular docking was performed, and binding was confirmed via Western blot. RESULTS: Diastereoisomer XIIIA inhibited SufA activity by 63% at 50 μM, protected fibrinogen and LL-37 from degradation, and significantly suppressed the growth of F. magna and E. coli. In contrast, XIIIB showed minimal activity. Molecular docking revealed key binding interactions between XIIIA and the SufA active site, which was further confirmed by Western blot analysis. DISCUSSION: These results demonstrate that stereochemistry critically influences SufA inhibition, with XIIIA showing superior potency and functional protection of host defense molecules. This supports its potential as an antivirulence agent. The validated probe also provides a tool for identifying serine protease targets in other pathogens. CONCLUSION: Compound XIIIA exhibits potent stereoselective inhibition of SufA and notable antimicrobial effects, supporting its development as a novel therapeutic targeting bacterial virulence.