Dual Inhibition of Mycobacterial Fatty Acid Biosynthesis and Degradation by 2-Alkynoic Acids

Summary2-Hexadecynoic acid and 2-octadecynoic acid have cidal activity against Mycobacterium smegmatis and Mycobacterium bovis BCG. At subinhibitory concentrations, M. smegmatis rapidly transformed [1-14C]-2-hexadecynoic acid into endogenous fatty acids and elongated them into mycolic acids. Toxic concentrations of 2-hexadecynoic acid resulted in accumulation of 3-ketohexadecanoic acid, which blocked fatty acid biosynthesis, and 3-hexadecynoic acid, an inhibitor of fatty acid degradation. The combination of these two metabolites is necessary to achieve the inhibition of M. smegmatis. We conclude that 2- and 3-hexa/octadecynoic acids inhibit mycolic acid biosynthesis, fatty acid biosynthesis, and fatty acid degradation, pathways of significant importance for mycobacteria

Inactivation of the inhA-Encoded Fatty Acid Synthase II (FASII) Enoyl-Acyl Carrier Protein Reductase Induces Accumulation of the FASI End Products and Cell Lysis of Mycobacterium smegmatis

xii, 391 hlm., 24 c

Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability

Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in Vmax for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs

Mycobacterium tuberculosis Dihydrofolate Reductase Is Not a Target Relevant to the Antitubercular Activity of Isoniazid

Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro, and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INH-resistant clinical isolates known to lack mutations in inhA and katG, but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH

An Anaerobic-Type α-Ketoglutarate Ferredoxin Oxidoreductase Completes the Oxidative Tricarboxylic Acid Cycle of Mycobacterium tuberculosis

Aerobic organisms have a tricarboxylic acid (TCA) cycle that is functionally distinct from those found in anaerobic organisms. Previous reports indicate that the aerobic pathogen Mycobacterium tuberculosis lacks detectable α-ketoglutarate (KG) dehydrogenase activity and drives a variant TCA cycle in which succinyl-CoA is replaced by succinic semialdehyde. Here, we show that M. tuberculosis expresses a CoA-dependent KG dehydrogenase activity, albeit one that is typically found in anaerobic bacteria. Unlike most enzymes of this family, the M. tuberculosis KG: ferredoxin oxidoreductase (KOR) is extremely stable under aerobic conditions. This activity is absent in a mutant strain deleted for genes encoding a previously uncharacterized oxidoreductase, and this strain is impaired for aerobic growth in the absence of sufficient amounts of CO2. Interestingly, inhibition of the glyoxylate shunt or exclusion of exogenous fatty acids alleviates this growth defect, indicating the presence of an alternate pathway that operates in the absence of β-oxidation. Simultaneous disruption of KOR and the first enzyme of the succinic semialdehyde pathway (KG decarboxylase; KGD) results in strict dependence upon the glyoxylate shunt for growth, demonstrating that KG decarboxylase is also functional in M. tuberculosis intermediary metabolism. These observations demonstrate that unlike most organisms M. tuberculosis utilizes two distinct TCA pathways from KG, one that functions concurrently with β-oxidation (KOR-dependent), and one that functions in the absence of β-oxidation (KGD-dependent). As these pathways are regulated by metabolic cues, we predict that their differential utilization provides an advantage for growth in different environments within the host

Contribution of Efflux to the Emergence of Isoniazid and Multidrug Resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment

Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å