Joshua Odingo

Validation of the Essential ClpP Protease in Mycobacterium tuberculosis as a Novel Drug Target

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.

A High-Throughput Screen against Pantothenate Synthetase (PanC) Identifies 3-Biphenyl-4-Cyanopyrrole-2-Carboxylic Acids as a New Class of Inhibitor with Activity against Mycobacterium tuberculosis

The enzyme pantothenate synthetase, PanC, is an attractive drug target in Mycobacterium tuberculosis. It is essential for the in vitro growth of M. tuberculosis and for survival of the bacteria in the mouse model of infection. PanC is absent from mammals. We developed an enzyme-based assay to identify inhibitors of PanC, optimized it for high-throughput screening, and tested a large and diverse library of compounds for activity. Two compounds belonging to the same chemical class of 3-biphenyl-4- cyanopyrrole-2-carboxylic acids had activity against the purified recombinant protein, and also inhibited growth of live M. tuberculosis in manner consistent with PanC inhibition. Thus we have identified a new class of PanC inhibitors with whole cell activity that can be further developed.

Identification of Phenoxyalkylbenzimidazoles with Antitubercular Activity.

We conducted an evaluation of the phenoxyalkylbenzimidazole series based on the exemplar 2-ethyl-1-(3-phenoxypropyl)-1H-benzo[d]imidazole for its antitubercular activity. Four segments of the molecule were examined systematically to define a structure-activity relationship with respect to biological activity. Compounds had submicromolar activity against Mycobacterium tuberculosis; the most potent compound had a minimum inhibitory concentration (MIC) of 52 nM and was not cytotoxic against eukaryotic cells (selectivity index = 523). Compounds were selective for M. tuberculosis over other bacterial species, including the closely related Mycobacterium smegmatis. Compounds had a bacteriostatic effect against aerobically grown, replicating M. tuberculosis, but were bactericidal against nonreplicating bacteria. Representative compounds had moderate to high permeability in MDCK cells, but were rapidly metabolized in rodents and human liver microsomes, suggesting the possibility of rapid in vivo hepatic clearance mediated by oxidative metabolism. These results indicate that the readily synthesized phenoxyalkylbenzimidazoles are a promising class of potent and selective antitubercular agents, if the metabolic liability can be solved.

Synthesis and evaluation of the 2,4-diaminoquinazoline series as anti-tubercular agents.

The 2,4-diaminoquinazoline class of compounds has previously been identified as an effective inhibitor of Mycobacterium tuberculosis growth. We conducted an extensive evaluation of the series for its potential as a lead candidate for tuberculosis drug discovery. Three segments of the representative molecule N-(4-fluorobenzyl)-2-(piperidin-1-yl)quinazolin-4-amine were examined systematically to explore structure-activity relationships influencing potency. We determined that the benzylic amine at the 4-position, the piperidine at 2-position and the N-1 (but not N-3) are key activity determinants. The 3-deaza analog retained similar activity to the parent molecule. Biological activity was not dependent on iron or carbon source availability. We demonstrated through pharmacokinetic studies in rats that good in vivo compound exposure is achievable. A representative compound demonstrated bactericidal activity against both replicating and non-replicating M. tuberculosis. We isolated and sequenced M. tuberculosis mutants resistant to this compound and observed mutations in Rv3161c, a gene predicted to encode a dioxygenase, suggesting that the compound may act as a pro-drug.

Synthesis and Evaluation of the 2-Aminothiazoles as Anti-Tubercular Agents.

The 2-aminothiazole series has anti-bacterial activity against the important global pathogen Mycobacterium tuberculosis. We explored the nature of the activity by designing and synthesizing a large number of analogs and testing these for activity against M. tuberculosis, as well as eukaryotic cells. We determined that the C-2 position of the thiazole can accommodate a range of lipophilic substitutions, while both the C-4 position and the thiazole core are sensitive to change. The series has good activity against M. tuberculosis growth with sub-micromolar minimum inhibitory concentrations being achieved. A representative analog was selective for mycobacterial species over other bacteria and was rapidly bactericidal against replicating M. tuberculosis. The mode of action does not appear to involve iron chelation. We conclude that this series has potential for further development as novel anti-tubercular agents.

Improved Phenoxyalkylbenzimidazoles with Activity against Mycobacterium tuberculosis Appear to Target QcrB

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.

The synthesis and evaluation of triazolopyrimidines as anti-tubercular agents

Graphical abstract

Synthesis and anti-tubercular activity of 3-substituted benzo[b]thiophene-1,1-dioxides

We demonstrated that the 3-substituted benzothiophene-1,1-dioxide class of compounds are effective inhibitors of Mycobacterium tuberculosis growth under aerobic conditions. We examined substitution at the C-3 position of the benzothiophene-1,1-dioxide series systematically to delineate structure–activity relationships influencing potency and cytotoxicity. Compounds were tested for inhibitory activity against virulent M. tuberculosis and eukaryotic cells. The tetrazole substituent was most potent, with a minimum inhibitory concentration (MIC) of 2.6 µM. However, cytotoxicity was noted with even more potency (Vero cell TC50 = 0.1 µM). Oxadiazoles had good anti-tubercular activity (MICs of 3–8 µM), but imidazoles, thiadiazoles and thiazoles had little activity. Cytotoxicity did not track with anti-tubercular activity, suggesting different targets or mode of action between bacterial and eukaryotic cells. However, we were unable to derive analogs without cytotoxicity; all compounds synthesized were cytotoxic (TC50 of 0.1–5 µM). We conclude that cytotoxicity is a liability in this series precluding it from further development. However, the series has potent anti-tubercular activity and future efforts towards identifying the mode of action could result in the identification of novel drug targets.

Synthesis and biological evaluation of aryl-oxadiazoles as inhibitors of Mycobacterium tuberculosis

Graphical abstract

Synergy between Colistin and the Signal Peptidase Inhibitor MD3 Is Dependent on the Mechanism of Colistin Resistance in Acinetobacter baumannii

ABSTRACT Synergy between colistin and the signal peptidase inhibitor MD3 was tested against isogenic mutants and clinical pairs of Acinetobacter baumannii isolates. Checkerboard assays and growth curves showed synergy against both colistin-susceptible strains (fractional inhibitory concentration index [FICindex] = 0.13 to 0.24) and colistin-resistant strains with mutations in pmrB and phosphoethanolamine modification of lipid A (FICindex = 0.14 to 0.25) but not against colistin-resistant Δlpx strains with loss of lipopolysaccharide (FICindex = 0.75 to 1). A colistin/MD3 combination would need to be targeted to strains with specific colistin resistance mechanisms.