SummaryTo 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.
The
essential enzyme CYP121 is a target for drug development against
antibiotic resistant strains of Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1 was identified to
bind to CYP121 using a cascade of biophysical assays. Synthetic merging
and optimization of 1 produced a 100-fold improvement
in binding affinity, yielding lead compound 2 (KD = 15 μM). Deconstruction of 2 into its component retrofragments allowed the group efficiency of
structural motifs to be assessed, the identification of more LE scaffolds
for optimization and highlighted binding affinity hotspots. Structure-guided
addition of a metal-binding pharmacophore onto LE retrofragment scaffolds
produced low nanomolar (KD = 15 nM) CYP121
ligands. Elaboration of these compounds to target binding hotspots
in the distal active site afforded compounds with excellent selectivity
against human drug-metabolizing P450s. Analysis of the factors governing
ligand potency and selectivity using X-ray crystallography, UV–vis
spectroscopy, and native mass spectrometry provides insight for subsequent
drug development.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.