Design, Syntheses, and Anti-TB Activity of 1,3-Benzothiazinone Azide and Click Chemistry Products Inspired by BTZ043.

Electron deficient nitroaromatic compounds such as BTZ043 and its closest congener, PBTZ169, and related agents are a promising new class of anti-TB compounds. Herein we report the design and syntheses of 1,3-benzothiazinone azide (BTZ-N3) and related click chemistry products based on the molecular mode of activation of BTZ043. Our computational docking studies indicate that BTZ-N3 binds in the essentially same pocket as that of BTZ043. Detailed biochemical studies with cell envelope enzyme fractions of Mycobacterium smegmatis combined with our model biochemical reactivity studies with nucleophiles indicated that, in contrast to BTZ043, the azide analogue may have a different mode of activation for anti-TB activity. Subsequent enzymatic studies with recombinant DprE1 from Mtb followed by MIC determination in NTB1 strain of Mtb (harboring Cys387Ser mutation in DprE1 and is BTZ043 resistant) unequivocally indicated that BTZ-N3 is an effective reversible and noncovalent inhibitor of DprE1.

Mitochondrial Carriers Link the Catabolism of Hydroxyaromatic Compounds to the Central Metabolism in Candida parapsilosis.

The pathogenic yeast Candida parapsilosis metabolizes hydroxyderivatives of benzene and benzoic acid to compounds channeled into central metabolism, including the mitochondrially localized tricarboxylic acid cycle, via the 3-oxoadipate and gentisate pathways. The orchestration of both catabolic pathways with mitochondrial metabolism as well as their evolutionary origin is not fully understood. Our results show that the enzymes involved in these two pathways operate in the cytoplasm with the exception of the mitochondrially targeted 3-oxoadipate CoA-transferase (Osc1p) and 3-oxoadipyl-CoA thiolase (Oct1p) catalyzing the last two reactions of the 3-oxoadipate pathway. The cellular localization of the enzymes indicates that degradation of hydroxyaromatic compounds requires a shuttling of intermediates, cofactors, and products of the corresponding biochemical reactions between cytosol and mitochondria. Indeed, we found that yeast cells assimilating hydroxybenzoates increase the expression of genes SFC1, LEU5, YHM2, and MPC1 coding for succinate/fumarate carrier, coenzyme A carrier, oxoglutarate/citrate carrier, and the subunit of pyruvate carrier, respectively. A phylogenetic analysis uncovered distinct evolutionary trajectories for sparsely distributed gene clusters coding for enzymes of both pathways. Whereas the 3-oxoadipate pathway appears to have evolved by vertical descent combined with multiple losses, the gentisate pathway shows a striking pattern suggestive of horizontal gene transfer to the evolutionarily distant Mucorales.

Development of 3,5-Dinitrobenzylsulfanyl-1,3,4-oxadiazoles and Thiadiazoles as Selective Antitubercular Agents Active Against Replicating and Nonreplicating Mycobacterium tuberculosis.

Herein, we report the discovery and structure-activity relationships of 5-substituted-2-[(3,5-dinitrobenzyl)sulfanyl]-1,3,4-oxadiazoles and 1,3,4-thiadiazoles as a new class of antituberculosis agents. The majority of these compounds exhibited outstanding in vitro activity against Mycobacterium tuberculosis CNCTC My 331/88 and six multidrug-resistant clinically isolated strains of M. tuberculosis, with minimum inhibitory concentration values as low as 0.03 µM (0.011-0.026 µg/mL). The investigated compounds had a highly selective antimycobacterial effect because they showed no activity against the other bacteria or fungi tested in this study. Furthermore, the investigated compounds exhibited low in vitro toxicities in four proliferating mammalian cell lines and in isolated primary human hepatocytes. Several in vitro genotoxicity assays indicated that the selected compounds have no mutagenic activity. The oxadiazole and thiadiazole derivatives with the most favorable activity/toxicity profiles also showed potency comparable to that of rifampicin against the nonreplicating streptomycin-starved M. tuberculosis 18b-Lux strain, and therefore, these derivatives, are of particular interest.

DprE1 Is a Vulnerable Tuberculosis Drug Target Due to Its Cell Wall Localization.

The flavo-enzyme DprE1 catalyzes a key epimerization step in the decaprenyl-phosphoryl d-arabinose (DPA) pathway, which is essential for mycobacterial cell wall biogenesis and targeted by several new tuberculosis drug candidates. Here, using differential radiolabeling with DPA precursors and high-resolution fluorescence microscopy, we disclose the unexpected extracytoplasmic localization of DprE1 and periplasmic synthesis of DPA. Collectively, this explains the vulnerability of DprE1 and the remarkable potency of the best inhibitors.

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

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)carbamothioyl]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.