Identification of aminopyrimidine-sulfonamides as potent modulators of Wag31-mediated cell elongation in mycobacteria

There is an urgent need to discover new antitubercular agents with novel mechanisms of action in order to tackle the scourge of drug-resistant tuberculosis. Here, we report the identification of such a molecule - an AminoPYrimidine-Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1-resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1-resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time-lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.

Metabolic modeling and experimental studies on carbon and energy metabolism of Mycobacterium tuberculosis

Muhittin Emre Özdemir

Tuberculosis is an ancient human disease affecting millions of people every year. The common causative agent, Mycobacterium tuberculosis, is a peculiar pathogen that can persist in the host tissues for decades. Many of the key attributes of M. tuberculosis can be traced to its metabolism. Therefore, novel insights into the metabolism of this bacterium could lead to the identification of new drug targets as well as better methods to understand the mechanism of action of anti-tuberculosis drugs. Analysis of the metabolic network of this bacterium using a genome-scale model offers an unconventional approach to understanding utilization of metabolic pathways of this pathogen during growth and non-replicating persistence. Metabolic adaptation of a pathogen is central to its life style in vivo. To understand the capacity and limitations of M. tuberculosis, we developed an updated and thermodynamically curated genome scale model – iOS783. This model has been verified with respect to the observed phenotypes for targeted gene deletion studies reported previously. Evaluation of iOS783 with thermodynamics based flux balance analysis (TFBA) improved overall reliability of the model results and led to identification of possible bottleneck reactions. In addition, several new hypotheses were generated with iOS783, involving the role of isocitrate lyase enzymes, propionate toxicity, and energy metabolism of this pathogen. Analysis of iOS783 for conditional gene essentiality emphasizes the importance of the carbon source available to the pathogen. Several genes show a conditional impact on biomass production rate of the model, depending on the availability of carbohydrates or fatty acids as the carbon substrate. Conditional essentiality is even more pronounced when ATP replenishment capacity is used as a measure of a pathogen’s ability to survive under stress in a non-growing state. As expected, ATP synthase plays a major role in estimations of ATP replenishment capacity based on iOS783. Curiously, computational analysis of the metabolic network with ATP synthase activity blocked indicates that the damaged network can use substrate-level phosphorylation reactions to sustain ATP production to fulfill non-growth-associated ATP requirements when glucose is available. In order to establish the dependence of M. tuberculosis on ATP synthase, we attempted to construct a deletion of the chromosomal atp operon. Our results show that the native copy of these genes can be deleted only when a second set of genes are introduced elsewhere in the genome, indicating essentiality of the ATP synthase for growth. Although our results indicate that ATP synthase is essential for growth, we observed carbon source specific efficacy of an ATP synthase inhibitor, bedaquiline, on M. tuberculosis. These experimental results are consistent with ATP turnover estimations based on modeling results. Furthermore, analysis of iOS783 suggests a novel role in energy metabolism for recently described glycine dehydrogenase (GDH) on energy metabolism. Deletion of ald gene responsible for GDH activity reduces the ATP replenishment capacity of iOS783, especially during metabolism of fatty acid derivative carbon sources. Consistent with these modeling results, wetlab experiments designed to test this hypothesis show that bedaquiline has a stronger bactericidal effect on an M. tuberculosis ∆ald mutant, compared to the wild-type parental strain, when cells are cultured in medium containing acetate as the carbon source. Metabolic modelling is already well established as an invaluable tool for engineering of strains with desired characteristics. For pathogenic species, however, the application of this approach has so far been quite limited. The work presented in this thesis exemplifies an interdisciplinary approach combining computational modeling and experimental studies of M. tuberculosis metabolism. First, iOS783 was utilized to interpret existing experimental results and to generate novel hypotheses, thereby providing the impetus for new wetlab experiments. Conversely, results from iOS783-inspired wetlab experiments were used to further refine and expand the iOS783 model. This cyclic exchange between computational modeling and wetlab experimentation illustrates how a close interdisciplinary partnership can create a positive feedback loop that enriches both disciplines.

EPFL2013

In Vitro Combination Studies of Benzothiazinone Lead Compound BTZ043 against Mycobacterium tuberculosis

Stewart Cole, Ruben Hartkoorn, Benoit Lechartier

Benzothiazinones (BTZ) are a new class of drug candidates to combat tuberculosis that inhibit decaprenyl-phosphoribose epimerase (DprE1), an essential enzyme involved in arabinan biosynthesis. Using the checkerboard method and cell viability assays, we have studied the interaction profiles of BTZ043, the current lead compound, with several antituberculosis drugs or drug candidates against Mycobacterium tuberculosis strain H37Rv, namely, rifampin, isoniazid, ethambutol, TMC207, PA-824, moxifloxacin, meropenem with or without clavulanate, and SQ-109. No antagonism was found between BTZ043 and the tested compounds, and most of the interactions were purely additive. Data from two different approaches clearly indicate that BTZ043 acts synergistically with TMC207, with a fractional inhibitory concentration index of 0.5. TMC207 at a quarter of the MIC (20 ng/ml) used in combination with BTZ043 (1/4 MIC, 0.375 ng/ml) had a stronger bactericidal effect on M. tuberculosis than TMC207 alone at a concentration of 80 ng/ml. This synergy was not observed when the combination was tested on a BTZ-resistant M. tuberculosis mutant, suggesting that DprE1 inhibition is the basis for the interaction. This finding excludes the possibility of synergy occurring through an off-target mechanism. We therefore hypothesize that sub-MICs of BTZ043 weaken the bacterial cell wall and allow improved penetration of TMC207 to its target. Synergy between two new antimycobacterial compounds, such as TMC207 and BTZ043, with novel targets, offers an attractive foundation for a new tuberculosis regimen.

Amer Soc Microbiology2012

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG

Stewart Cole, Ruben Hartkoorn

To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl) thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl) carba-mothioyl] propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

Elsevier2015

Metabolic modeling and experimental studies on carbon and energy metabolism of Mycobacterium tuberculosis

Muhittin Emre Özdemir

EPFL2013