Potency boost of a <i>Mycobacterium tuberculosis</i> dihydrofolate reductase inhibitor by multienzyme F₄₂₀H₂-dependent reduction.

Abstract

Triaza-coumarin (TA-C) is a <i>Mycobacterium tuberculosis</i> (Mtb) dihydrofolate reductase (DHFR) inhibitor with an IC₅₀ (half maximal inhibitory concentration) of ∼1 µM against the enzyme. Despite this moderate target inhibition, TA-C shows exquisite antimycobacterial activity (MIC₅₀, 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 <i>thyA</i>, which sensitizes Mtb to DHFR inhibition, rather than in DHFR itself. We observed <i>thyA</i> mutations, consistent with TA-C interfering with the folate pathway. A second resistance mechanism involved biosynthesis of the redox coenzyme F₄₂₀ Thus, we hypothesized that TA-C may be metabolized by Mtb F₄₂₀-dependent oxidoreductases (FDORs). By chemically blocking the putative site of FDOR-mediated reduction in TA-C, we reproduced the F₄₂₀-dependent resistance phenotype, suggesting that F₄₂₀H₂-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 IC₅₀ 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.