Structure of Staphylococcus aureus ClpP Bound to the Covalent Active‐Site Inhibitor Cystargolide A

Abstract

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural β‐lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti‐virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by β‐lactones, the predominant class of ClpP inhibitors.

Characterization of the cystargolide biosynthetic gene cluster and functional analysis of the methyltransferase CysG

“…Most of these are designed to covalently bind the catalytic subunits and inactivate the active site in a reversible or irreversible manner ( 10 ). Cystargolides A and B were originally isolated from Kitasatospora cystarginea NRRL B16505 and were found to inhibit the human proteasome and the caseinolytic protease ClpP in the micromolar range ( 11 , 12 , 13 ). The cystargolides feature a β-lactone warhead and a dipeptide backbone similar to the well-characterized belactosins ( Fig.…”