Discovery of Novel Peptidomimetic Boronate ClpP Inhibitors with Noncanonical Enzyme Mechanism as Potent Virulence Blockers <i>in Vitro</i> and <i>in Vivo</i>

Abstract

Caseinolytic protease P (ClpP) is considered as a promising target for the treatment of <i>Staphylococcus aureus</i> infections. In an unbiased screen of 2632 molecules, a peptidomimetic boronate, MLN9708, was found to be a potent suppressor of <i>Sa</i>ClpP function. A time-saving and cost-efficient strategy integrating <i>in silico</i> 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 <b>43Hf</b> was the most active inhibitor and showed reversible covalent binding to <i>Sa</i>ClpP while did not destabilize the tetradecameric structure of <i>Sa</i>ClpP. The crystal structure of <b>43Hf</b>-<i>Sa</i>ClpP complex provided mechanistic insight into the covalent binding mode of peptidomimetic boronate and <i>Sa</i>ClpP. Furthermore, <b>43Hf</b> could bind endogenous ClpP in <i>S. aureus</i> cells and exhibited significant efficacy in attenuating <i>S. aureus</i> virulence <i>in vitro</i> and <i>in vivo</i>.