Towards Selective Mycobacterial ClpP1P2 Inhibitors with Reduced Activity against the Human Proteasome.

Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. We recently identified bortezomib (Velcade; compound 1) as a promising antituberculosis (anti-TB) compound. We showed that compound 1 inhibits the mycobacterial caseinolytic proteases P1 and P2 (ClpP1P2) and exhibits bactericidal activity, and we established compound 1 and ClpP1P2 as an attractive lead/target couple. However, compound 1 is a human-proteasome inhibitor currently approved for cancer therapy and, as such, exhibits significant toxicity. Selective inhibition of the bacterial protease over the human proteasome is desirable in order to maintain antibacterial activity while reducing toxicity. We made use of structural data in order to design a series of dipeptidyl-boronate derivatives of compound 1. We tested these derivatives for whole-cell ClpP1P2 and human-proteasome inhibition as well as bacterial-growth inhibition and identified compounds that were up to 100-fold-less active against the human proteasome but that retained ClpP1P2 and mycobacterial-growth inhibition as well as bactericidal potency. The lead compound, compound 58, had low micromolar ClpP1P2 and anti-M. tuberculosis activity, good aqueous solubility, no cytochrome P450 liabilities, moderate plasma protein binding, and low toxicity in two human liver cell lines, and despite high clearance in microsomes, this compound was only moderately cleared when administered intravenously or orally to mice. Higher-dose oral pharmacokinetics indicated good dose linearity. Furthermore, compound 58 was inhibitory to only 11% of a panel of 62 proteases. Our work suggests that selectivity over the human proteasome can be achieved with a drug-like template while retaining potency against ClpP1P2 and, crucially, anti-M. tuberculosis activity.

Target mechanism-based whole-cell screening identifies bortezomib as an inhibitor of caseinolytic protease in mycobacteria.

UNLABELLED: A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy. IMPORTANCE: During the last decade, antibacterial drug discovery relied on biochemical assays, rather than whole-cell approaches, to identify molecules that interact with purified target proteins derived by genomics. This approach failed to deliver antibacterial compounds with whole-cell activity, either because of cell permeability issues that medicinal chemistry cannot easily fix or because genomic data of essentiality insufficiently predicted the vulnerability of the target identified. As a consequence, the field largely moved back to a whole-cell approach whose main limitation is its black-box nature, i.e., that it requires trial-and-error chemistry because the cellular target is unknown. We developed a novel type of antibacterial screening method, target mechanism-based whole-cell screening, to combine the advantages of both approaches. We engineered a mycobacterial reporter strain with a synthetic phenotype allowing us to identify inhibitors of the caseinolytic protease (ClpP1P2) inside the cell. This approach identified bortezomib, an anticancer drug, as a specific inhibitor of ClpP1P2. We further confirmed the specific "on-target" activity of bortezomib by independent approaches including, but not limited to, genetic manipulation of the target level (over- and underexpressing strains) and by establishing a dynamic structure-activity relationship between ClpP1P2 and growth inhibition. Identifying an "on-target" compound is critical to optimize the efficacy of the compound without compromising its specificity. This work demonstrates the feasibility of target mechanism-based whole-cell screening methods, validates ClpP1P2 as a druggable target, and delivers a lead compound for tuberculosis therapy.