The Mechanism of Caseinolytic Protease (ClpP) Inhibition

摘要

Maintaining homeostasis at the protein level is an important prerequisite for cellular viability for which prokaryotes exhibit several proteolytic machineries, including ClpXP. In 2008, we reported the first small-molecule inhibitor for the proteolytic subunit ClpP and demonstrated that the inhibition of the enzyme in living bacteria significantly attenuates their capability to produce virulence factors, such as life-threatening toxins. Although ClpP has been extensively studied by biochemical and structural methods, the mechanism of small-molecule inhibition of this enzyme is currently poorly understood. Because chemical inhibition may lead to a novel antibacterial therapy, it is important to systematically analyze the binding site, the mechanism of inhibition, the stereogenic preference of the enzyme for inhibitors, the chemical space of putative inhibitors, and how other members of the ClpP family can be inhibited. One major step towards these aims was accomplished by the recently solved crystal structure of homotetradecameric ClpP from Staphylococcus aureus (SaClpP) in its active conformation. With the structural data at hand, we herein report an in-depth mechanistic analysis of S. aureus ClpP inhibition by β-lactones. A screen of a focused library of enantiopure β-lactones revealed the S,S-stereopreference of the protease, which was rationalized by molecular docking. Docking experiments also gave insight into a hitherto unnoted deep hydrophobic pocket next to the active site that accommodates β-lactone substituents in the exposition to the carbonyl group. The binding hypothesis was verified by binding studies with model compounds, detailed kinetic analysis, and protein mutagenesis studies. Furthermore, the replacement of the β-lactone core by other scaffolds resulted in the loss of inhibitory potency, thereby highlighting the importance of a β-lactone moiety for mechanism-based ClpP inhibition. Taken together, these results open intriguing perspectives in the mechanistic understanding of ClpP inhibition and provide direction for the design of potent and pharmacologically optimized inhibitors.