Discovery of Novel Peptidomimetic Boronate ClpP Inhibitors with Noncanonical Enzyme Mechanism as Potent Virulence Blockers and .

Caseinolytic protease P (ClpP) is considered as a promising target for the treatment of infections. In an unbiased screen of 2632 molecules, a peptidomimetic boronate, MLN9708, was found to be a potent suppressor of ClpP function. A time-saving and cost-efficient strategy integrating position scanning, multistep miniaturized synthesis, and bioactivity testing was deployed for optimization of this hit compound and led to fast exploration of structure-activity relationships. Five of 150 compounds from the miniaturized synthesis exhibited improved inhibitory activity. Compound was the most active inhibitor and showed reversible covalent binding to ClpP while did not destabilize the tetradecameric structure of ClpP. The crystal structure of -ClpP complex provided mechanistic insight into the covalent binding mode of peptidomimetic boronate and ClpP. Furthermore, could bind endogenous ClpP in cells and exhibited significant efficacy in attenuating virulence and .