A widespread family of bacterial gene clusters produces ClpP inhibitors

Abstract Intracellular proteolytic complexes play an essential role in modeling the proteome in both bacteria and eukaryotes. ClpP is the protease subunit of one such highly conserved proteolytic complex that, despite its potential, remains unexploited as a drug target. Here we describe a target-directed genome mining strategy to identify ClpP targeting compounds from the bacterial order Actinomycetales. By searching for biosynthetic gene clusters that contain duplicated copies of ClpP as putative antibiotic resistance genes, we identify a family of ClpP-associated clusters that are widespread across phyla, including environmental and pathogenic bacteria. While numerous bacterial pyrrolizidine alkaloids produced by these gene clusters are known, their connection to ClpP has never been made. We show that these previously characterized molecules do not affect ClpP function but are shunt metabolites derived from the genuine product of these gene clusters, a reactive covalent ClpP inhibitor. Focusing on one such cryptic gene cluster from Streptomyces cattleya DSM 46488, we use heterologous expression to purify the relevant ClpP inhibitor, which we name clipibicyclene. We show in vitro and in vivo that clipibicyclene is a potent covalent inhibitor of ClpP and that cluster-associated ClpPs provide resistance. ClpP inhibition results in antibacterial activity against actinobacteria, including Mycobacterium smegmatis, and inhibition of virulence factor production by Staphylococcus aureus. Finally, we solve the crystal structure of clipibicyclene-modified Escherichia coli ClpP. Clipibicyclene’s discovery deconvolutes the actual function of a family of natural products widespread in nature. It provides a novel scaffold for therapeutic ClpP inhibitor development, making these findings significant from the perspective of their discovery and their clinical potential.