Discovery of Antivirulence ClpP Inhibitors by Self‐Resistance Gene‐Guided Mining Coupled with Dual Functional Screening

Abstract The global threat of MRSA demands innovative anti‐virulence strategies. Caseinolytic peptidase P (ClpP), a central virulence regulator in MRSA, represents an attractive yet underexploited target. Here, we developed a discovery platform integrating self‐resistance gene‐guided genome mining with dual functional screening, combining fluorometric‐based assay and counter‐screening against ADEP‐induced ClpP activation. This led to the discovery of streptoclipamides A–G, novel hybrid polyketide‐nonribosomal peptide ClpP inhibitors from str BGC, validated via heterologous expression and gene knockout. Structure–activity relationship studies enabled by engineered analogues identified key pharmacophores. Streptoclipamide A potently inhibits ClpP (IC 50 = 480 nM) by engaging Thr72 via its C‐21 hydroxyl group, confirmed by biophysics and self‐resistance‐conferring T72P mutation. Streptoclipamide A suppressed MRSA virulence in vitro by reducing critical toxin production, including α‐hemolysin, and demonstrated protection in Galleria mellonella and murine pneumonia models. This work expands chemical diversity of ClpP‐targeting agents, and establishes a genome mining‐driven platform for discovering new therapeutics against antibiotic‐resistant pathogens.