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

Abstract

Triaza-coumarin (TA-C) is a Mycobacterium tuberculosis (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 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 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.

Nitroimidazopyrazinones with oral activity against tuberculosis and Chagas disease in mouse models of infection

“…In the future, a comparison of activity against Ddn mutants or other homologues such as Rv1261c, Rv1558 and Rv3178 could assess the specificity of this Ddn-mediated bioactivation. As resistance towards nitroimidazoles have been found to already be present due to mutations in Ddn, assessing and developing new compounds that can be activated by more than one F 420 -dependent enzyme would be of great benefit [39,55].…”

In Vitro Resistance against DNA Gyrase Inhibitor SPR719 in Mycobacterium avium and Mycobacterium abscessus

“…Genomic DNA was extracted using the phenol-chloroform method as described previously ( 26 ). Targeted sanger sequencing of gyrA and gyrB was performed by Genewiz Inc. (South Plainfield, NJ, USA) using primers listed in Table S2.…”

The implication of Mycobacterium tuberculosis-mediated metabolism of targeted xenobiotics

“…A new class of 1,3,5-triazaspiro[5.5]undeca-2,4-dienes has been identified as a selective inhibitor series of dihydrofolate reductases (DHFRs), an essential enzyme involved in folate metabolism, with potent whole- Mtb cell activity 17 . Congeners of triaza-coumarin ( 39 ) (Fig.…”

“…The first mechanism was associated with low-frequency mutations in thymidylate synthase ( thyA ), confirming that 39 is involved in the folate pathway. The second mechanism is based on the loss of enzymes (FbiC and Fgd1) required for the biosynthesis of redox cofactor F 420 , which implicated metabolism by Mtb F 420 -dependent oxidoreductases (FDORs) 17 . The latter was corroborated by the generation of mutant strains harbouring loss-of-function mutations in fbiC and fgd1 , which are involved in the biosynthesis of the redox cofactor F 420 H 2 , a bioactivator of pretomanid.…”