Repurposing AGI-6780 to Activate ClpP and Target Gram-Positive Bacteria

The global rise of antibiotic-resistant pathogens has created an imperative to discover novel antimicrobial strategies. Traditional antibiotics predominantly target essential bacterial processes, such as cell division, DNA replication, transcription, and translation, but few new agents targeting these pathways have emerged in recent decades. We explored an alternative approach by identifying small molecules that hyperactivate the bacterial ClpP protease, thereby inducing uncontrolled proteolysis and ultimately leading to bacterial cell death. Leveraging the known binding interactions of the peptide antibiotic ADEP4 with ClpP, we performed high-throughput <i>in silico</i> screening. Molecular docking simulations prioritized compounds based on predicted binding affinity (kcal/mol), complemented by structural chemistry evaluation and <i>in silico</i> pharmacokinetic profiling. AGI-6780 emerged as a lead compound with high predicted affinity for the ClpP active site. <i>In vitro</i> assays showed that AGI-6780 effectively inhibits a panel of Gram-positive bacteria by targeting ClpP. It also exhibits synergy with the antibiotic rifampicin and has minimal cytotoxicity on human cell lines. AGI-6780 is a promising antimicrobial agent that uniquely exploits ClpP, an unconventional bacterial target.