New small molecule blocks key enzyme in tuberculosis

Researchers are using new screening strategies to find antibiotics that work against Mycobacterium tuberculosis. One such strategy is PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which profiles how chemical compounds affect a pooled library of Mtb hypomorphs — strains in which each essential gene is partially depleted. Deborah T. Hung is the corresponding author on a study that used PROSPECT screening data to hunt for compounds that show stronger activity against specific genetically weakened strains. From those data the team identified a novel N-oxolan-3-yl pyrazole carboxamide inhibitor named BRD1554. BRD1554 showed increased, selective activity against strains depleted of polyketide synthase 13 (Pks13), an essential enzyme in mycolic acid synthesis, and against strains depleted of Rv2581c, an uncharacterized protein similar to glyoxylase II enzymes. By starting with a chemical-genetic view — asking which compounds hurt which genetically sensitized strains — the researchers could prioritize compounds that point directly to vulnerable pathways in the bacterium.

To move from a hit to a likely mechanism, the team used Perturbagen CLass (PCL) analysis, a reference-based approach within PROSPECT, which predicted Pks13 as the probable target and implicated its thioesterase domain. The group synthesized a more active analogue and determined the absolute stereochemistry of the active diastereomer, yielding 1554-06-3R,4S with an MIC 90 of 3.0 µM against Mtb H37Rv. Treating bacteria with 1554-06 caused upregulation of the pks13 operon, the iniBAC operon, and other genes linked to mycolic acid synthesis, consistent with disruption of that pathway. Resistant mutants isolated after exposure to 1554-06 carried single nucleotide polymorphisms in the thioesterase domain of Pks13. The researchers then biochemically confirmed that 1554-06 inhibits the activity of recombinant Pks13 thioesterase domain, and computational docking of 1554-06 stereoisomers was consistent with the stereospecific activity observed in whole cell assays. They also found unique chemical genetic interactions between inhibitors of different Pks13 domains and different detoxifying enzymes, revealing novel gene-gene interactions.

These findings show how PROSPECT can rapidly reveal the mechanism of action of whole-cell active compounds with domain-level precision. By linking a compound like BRD1554 and its optimized analogue 1554-06-3R,4S to Pks13 and specifically to the thioesterase domain, the approach integrates biological insight into compound triage and accelerates early development decisions. The observed upregulation of the pks13 operon and iniBAC operon and the identification of resistance mutations in the thioesterase domain provide multiple, independent lines of evidence tying the compound to mycolic acid biosynthesis. Beyond a single compound, the discovery of distinct chemical genetic interactions between inhibitors of different Pks13 domains and distinct detoxifying enzymes suggests the platform can illuminate gene-gene relationships that matter for drug combinations. That information could be used to predict synergy or avoid antagonism when designing antitubercular regimens, helping researchers prioritize compounds and combinations most likely to succeed in later development.