Abstract
New drugs and new targets are urgently needed to treat tuberculosis. We discovered that d-phenylalanine-benzoxazole Q112 displays potent antibacterial activity against Mycobacterium tuberculosis (Mtb) in multiple media and in macrophage infections. A metabolomic profiling indicates that Q112 has a unique mechanism of action. Q112 perturbs the essential pantothenate/coenzyme A biosynthetic pathway, depleting pantoate while increasing ketopantoate, as would be expected if ketopantoate reductase (KPR) were inhibited. We searched for alternative KPRs, since the enzyme annotated as PanE KPR is not essential in Mtb. The ketol-acid reductoisomerase IlvC catalyzes the KPR reaction in the close Mtb relative Corynebacterium glutamicum, but Mtb IlvC does not display KPR activity. We identified the essential protein Rv3603c as an orthologue of PanG KPR and demonstrated that a purified recombinant Rv3603c has KPR activity. Q112 inhibits Rv3603c, explaining the metabolomic changes. Surprisingly, pantothenate does not rescue Q112-treated bacteria, indicating that Q112 has an additional target(s). Q112-resistant strains contain loss-of-function mutations in the twin arginine translocase TatABC, further underscoring Q112's unique mechanism of action. Loss of TatABC causes a severe fitness deficit attributed to changes in nutrient uptake, suggesting that Q112 resistance may derive from a decrease in uptake.