Tuberculosis is a serious global health problem caused by the bacterium Mycobacterium tuberculosis . There is an urgent need for discovery and development of new treatments, but this can only be accomplished through rapid and reproducible M. tuberculosis assays designed to identify potent inhibitors. We developed an automated 96-well assay utilizing a recombinant strain of M. tuberculosis expressing a far-red fluorescent reporter to determine the activity of novel compounds; this allowed us to measure growth by monitoring both optical density and fluorescence. We determined that optical density and fluorescence were correlated with cell number during logarithmic phase growth. Fluorescence was stably maintained without antibiotic selection over 5 days, during which time cells remained actively growing. We optimized parameters for the assay, with the final format being 5 days’ growth in 96-well plates in the presence of 2% w/v DMSO. We confirmed reproducibility using rifampicin and other antibiotics. The dual detection method allows for a reproducible calculation of the minimum inhibitory concentration (MIC), at the same time detecting artefacts such as fluorescence quenching or compound precipitation. We used our assay to confirm anti-tubercular activity and establish the structure activity relationship (SAR) around the imidazo[1,2- a ]pyridine-3-carboxamides, a promising series of M. tuberculosis inhibitors.

Mycobacterium tuberculosis is a pathogen of major global importance. Validated drug targets are required in order to develop novel therapeutics for drug-resistant strains and to shorten therapy. The Clp protease complexes provide a means for quality control of cellular proteins; the proteolytic activity of ClpP in concert with the ATPase activity of the ClpX/ClpC subunits results in degradation of misfolded or damaged proteins. Thus, the Clp system plays a major role in basic metabolism, as well as in stress responses and pathogenic mechanisms. M. tuberculosis has two ClpP proteolytic subunits. Here we demonstrate that ClpP1 is essential for viability in this organism in culture, since the gene could only be deleted from the chromosome when a second functional copy was provided. Overexpression of clpP1 had no effect on growth in aerobic culture or viability under anaerobic conditions or during nutrient starvation. In contrast, clpP2 overexpression was toxic, suggesting different roles for the two homologs. We synthesized known activators of ClpP protease activity; these acyldepsipeptides (ADEPs) were active against M. tuberculosis . ADEP activity was enhanced by the addition of efflux pump inhibitors, demonstrating that ADEPs gain access to the cell but that export occurs. Taken together, the genetic and chemical validation of ClpP as a drug target leads to new avenues for drug discovery.

Two-component regulatory systems (2CRSs) are widely used by bacteria to sense and respond to environmental stimuli with coordinated changes in gene expression. Systems are normally comprised of a sensory kinase protein that activates a transcriptional regulator by phosphorylation. Mycobacteria have few 2CRSs, but they are of key importance for bacterial survival and play important roles in pathogenicity. Mycobacterium tuberculosis has 12 paired two-component regulatory systems (which include a system with two regulators and one sensor, and a split sensor system), as well as four orphan regulators. Several systems are involved in virulence, and disruption of different systems leads to attenuation or hypervirulence. PhoPR plays a major role in regulating cell wall composition, and its inactivation results in sufficient attenuation of M. tuberculosis that deletion strains are live vaccine candidates. MprAB controls the stress response and is required for persistent infections. SenX3-RegX3 is required for control of aerobic respiration and phosphate uptake, and PrrAB is required for adaptation to intracellular infection. MtrAB is an essential system that controls DNA replication and cell division. The remaining systems (KdpDE, NarL, TrcRS, TcrXY, TcrA, PdtaRS, and four orphan regulators) are less well understood. The structure and binding motifs for several regulators have been characterized, revealing variations in function and operation. The sensors are less well characterized, and stimuli for many remain to be confirmed. This chapter reviews our current understanding of the role of two-component systems in mycobacteria, in particular M. tuberculosis.

The phenoxy alkyl benzimidazoles (PABs) have good antitubercular activity. We expanded our structure–activity relationship studies to determine the core components of PABs required for activity. The most potent compounds had minimum inhibitory concentrations against Mycobacterium tuberculosis in the low nanomolar range with very little cytotoxicity against eukaryotic cells as well as activity against intracellular bacteria. We isolated resistant mutants against PAB compounds, which had mutations in either Rv1339, of unknown function, or qcrB, a component of the cytochrome bc₁ oxidase of the electron transport chain. QcrB mutant strains were resistant to all PAB compounds, whereas Rv1339 mutant strains were only resistant to a subset, suggesting that QcrB is the target. The discovery of the target for PAB compounds will allow for the improved design of novel compounds to target intracellular M. tuberculosis.