Novel Aryl Substituted Pyrazoles as Small Molecule Inhibitors of Cytochrome P450 CYP121A1: Synthesis and Antimycobacterial Evaluation.

Three series of biarylpyrazole imidazole and triazoles are described, which vary in the linker between the biaryl pyrazole and imidazole/triazole group. The imidazole and triazole series with the short -CH2- linker displayed promising antimycobacterial activity, with the imidazole-CH2- series (7) showing low MIC values (6.25-25 µg/mL), which was also influenced by lipophilicity. Extending the linker to -C(O)NH(CH2)2- resulted in a loss of antimycobacterial activity. The binding affinity of the compounds with CYP121A1 was determined by UV-visible optical titrations with KD values of 2.63, 35.6, and 290 µM, respectively, for the tightest binding compounds 7e, 8b, and 13d from their respective series. Both binding affinity assays and docking studies of the CYP121A1 inhibitors suggest type II indirect binding through interstitial water molecules, with key binding residues Thr77, Val78, Val82, Val83, Met86, Ser237, Gln385, and Arg386, comparable with the binding interactions observed with fluconazole and the natural substrate dicyclotyrosine.

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.