Abstract
ABSTRACTMycobacterium 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.
Highlights
Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent
An allelic-exchange substrate (AES) with homology to the flanking regions of the prcAB locus was constructed using stitch-PCR (Fig. 2A) and electroporated into a strain of M. smegmatis previously transformed with the plasmid pJV53. pJV53 carries the genes encoding the recombinase gp60 and the resolvase gp61, which mediate the homologous recombination of the AES
The M. smegmatis ⌬prcAB strain was subsequently transformed with a plasmid carrying an SsrA-tagged red fluorescent protein (RFP)
Summary
Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. 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 mycobacterialgrowth inhibition as well as bactericidal potency. We made use of this mechanism and employed this ClpP1P2specific degradation tag to develop a fluorescence-based synthetic phenotype to detect and measure intracellular ClpP1P2 inhibition. Using this approach, we identified compound 1 as the first mycobacterial caseinolytic protease inhibitor with whole-cell bactericidal activity and as a promising lead candidate against TB [11]. The hydrophobic side chain of compound 1 is consistent with the protease P1 site preference for a hydrophobic residue [9, 12]
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