Caseinolytic protease P (ClpP) is not only a central component of bacterial proteostasis but is also involved in the regulation of virulence factors in prominent human pathogens, like methicillin-resistant Staphylococcus aureus (MRSA) [1]. Genetic knockouts and chemical inhibition of ClpP with beta-lactones have been shown to reduce MRSA pathogenicity in murine model systems [1-2]. Furthermore, ClpP was found to be essential for proliferation of acute myeloid lymphoma (AML) cells and recently a beta-lactone compound has been used to target AML [3].
Beta-lactones, however, suffer from their limited potency, selectivity and stability [2]. Therefore, we utilized an unbiased screen of 137000 compounds to identify a new, potent and highly selective set of inhibitors of ClpP. Among the six hit compounds were five phenyl esters and one structurally related triazole amide moiety. Their potency largely exceeded that of previous beta-lactone scaffolds. We used a diverse set of biochemical assays, activity-based protein profiling (ABPP) and structure-activity relationship (SAR) studies to examine the mode of action, target selectivity and fine tuning of compound stability. We found phenyl esters to target the active site serine, inhibit ClpP by stalling the protease at the acyl-enzyme intermediate and trigger deoligomerization of the tetradecameric ClpP into inactive heptamers. ABPP studies revealed a unique target selectivity for ClpP in living S. aureus cells. SAR studies were applied to increase stability of the acyl-enzyme intermediate as well as compound stability in human blood plasma. Stereoselective introduction of a methyl group in alpha position to the ester revealed a previously unknown stereogenic switch for protease deoligomerization that will help dissect the mechanism of ClpP activity.
Our study introduces phenyl esters as a new set of highly potent ClpP inhibitors with unprecedented target selectivity. Our findings elucidate their mechanism of action and revealed a stereogenic switch for deoligomerization in ClpP [4].