Large-Scale Chemical-Genetic Interaction Profiling Identifies a Novel Small-Molecule Inhibitor of Mycobacterium tuberculosis Polyketide Synthase 13

Abstract

PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) is an antimicrobial discovery platform based on chemical-genetic interaction (CGI) profiling of compounds against a pool of Mycobacterium tuberculosis (Mtb) hypomorphs, each depleted of an essential gene. From prior screening data, we have now identified a novel N-oxolan-3-yl pyrazole carboxamide inhibitor (BRD1554) that had increased, selective activity against strains depleted of polyketide synthase 13 (Pks13), an essential enzyme in mycolic acid synthesis, and Rv2581c, an uncharacterized protein similar to glyoxylase II enzymes. Perturbagen CLass (PCL) analysis, a reference-based approach to mechanism of action (MOA) assignment from PROSPECT, predicted Pks13, a polyketide synthase with five catalytic domains responsible for the terminal condensation step in mycolic acid biosynthesis, was the likely target, potentially implicating the thioesterase domain. We synthesized a more active analogue while assigning the absolute stereochemistry of the active diastereomer, resulting in 1554-06-3R,4S with an MIC90 of 3.0 {micro}M against Mtb H37Rv. Exposure to 1554-06 led to the upregulation of the pks13 operon along with the iniBAC operon and other genes linked to mycolic acid synthesis. Isolation of mutants resistant to 1554-06 revealed single nucleotide polymorphisms in the thioesterase domain of Pks13. Finally, we biochemically confirmed that 1554-06 inhibits the activity of recombinant Pks13 thioesterase domain, with computational docking of 1554-06 steroisomers consistent with the stereospecific activity seen in whole cell assays. We found unique chemical genetic interactions between inhibitors of the different Pks13 domains and different detoxifying enzymes of Mtb, thus revealing novel gene-gene interactions. These results highlight how PROSPECT can not only immediately reveal, with domain-level resolution, the MOA of new whole-cell active chemical inhibitors of Mtb, allowing the integration of biological insight into compound triage and accelerated early development, but can also illuminate genetic interactions linked to those mechanisms that could inform predictions of synergy for antitubercular drug development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/704361v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@9a4f38org.highwire.dtl.DTLVardef@c70cd2org.highwire.dtl.DTLVardef@1ac2e1org.highwire.dtl.DTLVardef@f06df0_HPS_FORMAT_FIGEXP M_FIG C_FIG