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

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 (IC₅₀ = 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.