Antibacterial Activity of and Resistance to Small Molecule Inhibitors of the ClpP Peptidase

Abstract

There is rapidly mounting evidence that intracellular proteases in bacteria are compelling targets for antibacterial drugs. Multiple reports suggest that the human pathogen Mycobacterium tuberculosis and other actinobacteria may be particularly sensitive to small molecules that perturb the activities of self-compartmentalized peptidases, which catalyze intracellular protein turnover as components of ATP-dependent proteolytic machines. Here, we report chemical syntheses and evaluations of structurally diverse β-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase. β-lactones with certain substituents on the α- and β-carbons were found to be toxic to M. tuberculosis. Using an affinity-labeled analog of a bioactive β-lactone in a series of chemical proteomic experiments, we selectively captured the ClpP1P2 peptidase from live cultures of two different actinobacteria that are related to M. tuberculosis. Importantly, we found that the growth inhibitory β-lactones also inactivate the M. tuberculosis ClpP1P2 peptidase in vitro via formation of a covalent adduct at the ClpP2 catalytic serine. Given the potent antibacterial activity of these compounds and their medicinal potential, we sought to identify innate mechanisms of resistance. Using a genome mining strategy, we identified a genetic determinant of β-lactone resistance in Streptomyces coelicolor, a non-pathogenic relative of M. tuberculosis. Collectively, these findings validate the potential of ClpP inhibition as a strategy in antibacterial drug development and define a mechanism by which bacteria could resist the toxic effects of ClpP inhibitors.

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

“…Mature M. tuberculosis ClpP1 (residues 7-200) and ClpP2 (residues 13-214) with C-terminal His 6 -tags were expressed and purified as described (35). For active-site labeling experiments, a SUMO domain was cloned between ClpP1 and the C-terminal His 6 -tag, and the protein was purified in the same manner as ClpP1.…”

“…For active-site labeling experiments, a SUMO domain was cloned between ClpP1 and the C-terminal His 6 -tag, and the protein was purified in the same manner as ClpP1. For crystallography, ClpP1 labeled with selenomethionine was prepared by growing a strain containing a plasmid overexpressing ClpP1 in Luria-Bertani medium containing 100 mg/L L-selenomethionine for 5 h at room temperature and was purified as described (35). M. tuberculosis ClpX fused to an N-terminal His 7 -SUMO domain to enhance solubility and H 6 -GFP with a C-terminal ADSHQRDYALAA sequence corresponding to the M. tuberculosis ssrA tag (GFP-ssrA) were expressed and purified as described (23).…”

“…ClpP1P2 variants with active-site mutations in one ring or the other have distinct substrate specificities, and the ClpP2 ring reacts preferentially with mechanism-based β-lactone inhibitors (29,35). These observations can be explained by differences in the substrate-binding clefts of ClpP1 and ClpP2, which also affect Z-Ile-Leu binding.…”

Bacterial proteases, untapped antimicrobial drug targets

“…46,47 Furthermore, β-lactone derivatives (compound 7; Figure 3a) were found to be among the privileged group of compounds able to enter M. tuberculosis in order to effectively inhibit growth with an MIC of 28 μg ml − 1 . 48 Ultimately, however, low plasma stability due to rapid hydrolysis of the cyclic ester precluding further clinical development.…”

Potenciais alvos moleculares para o desenvolvimento de novos fármacos antituberculose

“…A substituição da subunidade β-lactâmica por outros grupos, como carbamato, lactona, éster e oxetano levou à perda da atividade antituberculose devido a restrições no sítio ativo da enzima. 119 Outros produtos naturais capazes de inibir a ClpP também já foram relatados. A lassomicina (47) …”