Potency boost of a Mycobacterium tuberculosis dihydrofolate reductase inhibitor by multienzyme F(420)H(2)-dependent reduction

Aragaw, Wassihun Wedajo; Lee, Brendon M.; Yang, Xuan; Zimmerman, Matthew D.; Gengenbacher, Martin; Dartois, Véronique; Chui, Wai-Keung; Jackson, Colin J.; Dick, Thomas

Triaza-coumarin (TA-C) is a Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor with an IC(50) (half maximal inhibitory concentration) of ∼1 µM against the enzyme. Despite this moderate target inhibition, TA-C shows exquisite antimycobacterial activity (MIC(50), concentration inhibiting growth by 50% = 10 to 20 nM). Here, we investigated the mechanism underlying this potency disconnect. To confirm that TA-C targets DHFR and investigate its unusual potency pattern, we focused on resistance mechanisms. In Mtb, resistance to DHFR inhibitors is frequently associated with mutations in thymidylate synthase thyA, which sensitizes Mtb to DHFR inhibition, rather than in DHFR itself. We observed thyA mutations, consistent with TA-C interfering with the folate pathway. A second resistance mechanism involved biosynthesis of the redox coenzyme F(420). Thus, we hypothesized that TA-C may be metabolized by Mtb F(420)–dependent oxidoreductases (FDORs). By chemically blocking the putative site of FDOR-mediated reduction in TA-C, we reproduced the F(420)-dependent resistance phenotype, suggesting that F(420)H(2)-dependent reduction is required for TA-C to exert its potent antibacterial activity. Indeed, chemically synthesized TA-C-Acid, the putative product of TA-C reduction, displayed a 100-fold lower IC(50) against DHFR. Screening seven recombinant Mtb FDORs revealed that at least two of these enzymes reduce TA-C. This redundancy in activation explains why no mutations in the activating enzymes were identified in the resistance screen. Analysis of the reaction products confirmed that FDORs reduce TA-C at the predicted site, yielding TA-C-Acid. This work demonstrates that intrabacterial metabolism converts TA-C, a moderately active “prodrug,” into a 100-fold-more-potent DHFR inhibitor, thus explaining the disconnect between enzymatic and whole-cell activity.