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PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets) is an antimicrobial discovery platform based on chemical-genetic interaction profiling of compounds against a pool of () hypomorphs, each depleted of an essential gene. We now report a novel -oxolan-3-yl pyrazole carboxamide inhibitor, BRD1554, with selective activity against strains depleted of polyketide synthase 13 (Pks13), an essential polyketide synthase in mycolic acid synthesis, and Rv2581c, an uncharacterized protein similar to glyoxylase II enzymes. PCL analysis, our previously described reference-based approach to PROSPECT mechanism of action (MOA) assignment, predicted Pks13 was the likely target, implicating its thioesterase domain. We synthesized a more potent analogue 1554-06 and assigned the absolute stereochemistry of the active 3,4 diastereomer, which had an MIC of 3.0 μM against H37Rv. Expression profiling and the identification of resistance-conferring mutations in the thioesterase domain of Pks13 were consistent with the PCL prediction. Stereoisomers of 1554-06 inhibited recombinant Pks13 thioesterase domain in a stereospecific manner, consistent with their whole cell activity, and computational docking revealed the structural basis for the observed specificity, thereby confirming Pks13 thioesterase domain as the target. We observed unique chemical-genetic interactions between inhibitors of the different Pks13 domains and different detoxifying enzymes, including Rv2581c. These results highlight how PROSPECT can not only immediately reveal, with domain-level resolution, the MOA of new inhibitors, allowing the integration of biological insight into early prioritization, but can also illuminate genetic interactions linked to those mechanisms that could inform synergy predictions for combination therapy.