Revitalizing antifolates through understanding mechanisms that govern susceptibility and resistance

Kordus, Shannon Lynn; Baughn, Anthony D.

doi:10.1039/c9md00078j

Cited by 18 publications

(15 citation statements)

References 157 publications

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“…Since we demonstrated PAS can act in lieu of PABA in folate biosynthesis, we wanted to determine any resulting drug-drug interactions. Exogenous PABA can antagonize the activity of many FolP inhibitors such as sulfonamides and diaminodiphenyl sulfones 11,12,17,18 (Figure 2 and Extended Data Figure 4). Unlike FolP inhibitors, exogenous PABA cannot antagonize the activity of trimethoprim (TMP), a FolA inhibitor 12,19 .…”

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confidence: 99%

“…Exogenous PABA can antagonize the activity of many FolP inhibitors such as sulfonamides and diaminodiphenyl sulfones 11,12,17,18 (Figure 2 and Extended Data Figure 4). Unlike FolP inhibitors, exogenous PABA cannot antagonize the activity of trimethoprim (TMP), a FolA inhibitor 12,19 . HIV/AIDS-infected individuals are given a lifelong prophylactic combination therapy of SMX-TMP in addition to receiving anti-retroviral therapy 3 .…”

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confidence: 99%

“…This suggests that PAS can only antagonize the synergistic activity of SMX and TMP, resulting in an effect similar as treating with TMP alone. This data suggests treatment with PAS and SMX in patients Humans lack many of the de novo enzymes required to make folates and must acquire folates from diet or the environment, specifically by the commensal microbiota 12 Since we determined PAS can be utilized as a substrate for one-carbon metabolism, we hypothesized bacteria in the human gastrointestinal tract could produce a hydroxy-folate species. We reasoned that this hydroxy-folate species could result in toxicity as many patients discontinue PAS treatment because of the severe side effects, which largely encompasses gastrointestinal distress.…”

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Mechanism of selectivity reveals novel antifolate drug interactions

Kordus

Lamont

Howe

et al. 2020

Preprint

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Antimicrobial agents that target a specific pathogen of interest is the gold standard in drug design. para-Aminosalicylic acid (PAS), remains a cornerstone therapy, in the treatment against Mycobacterium tuberculosis, owing to its high level of selectivity. Despite its high level of selectivity, PAS has been reassigned to treat drug-resistant strains of M. tuberculosis because it causes severe gastrointestinal (GI) distress that results in poor patient compliance. We have previously shown PAS inhibits the folate biosynthetic pathway specifically inhibiting dihydrofolate reductase1,2. In this study, we sought to determine the mechanistic basis of PAS selectivity and determined that PAS can be utilized in folate biosynthesis by other bacterial pathogens. The utilization of PAS ultimately led to the antagonism of key antibiotics, specifically the sulfonamides, used to prophylactically treat individuals with HIV-AIDS3. In addition, we found many bacteria in the GI tract could also utilize PAS to make a hydroxy-folate species which resulted in GI toxicity. Using sulfonamides as a tool to prevent PAS associated toxicity in the GI tract, we discovered that the sulfonamides antagonized the antimycobacterial activity of PAS. These findings indicate a new need for understanding the mechanisms of selective therapies and more important, that HIV-AIDS/M. tuberculosis co-infected individuals should avoid co-treatment of PAS and sulfonamides.

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Mechanism of selectivity reveals novel antifolate drug interactions

Kordus

Lamont

Howe

et al. 2020

Preprint

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“…The protein catalyzes the NADPHdependent conversion of dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the synthesis of many cellular building blocks including thymidylate, purines, and certain amino acids. Several DHFR inhibitors are in clinical use for the treatment of various infectious diseases and cancer (3,4). However, approved DHFR inhibitors have only weak or no activity against Mtb, and there are no DHFR inhibitors used clinically for the treatment of TB (5).…”

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“…Here, our aim was to confirm DHFR inhibition as the primary mechanism of action of TA-C and to shed light on the peculiar on-target/whole-cell potency disconnect. Resistance to the prodrug DHFR inhibitor PAS has been extensively studied and recently discussed in comprehensive reviews by Baughn and colleagues (4,16). PAS resistance can emerge via multiple mechanisms that include preventing efficient bioactivation within the folate synthesis pathway, mitigating the impact of target inhibition, and limiting drug accumulation within the bacilli.…”

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

Aragaw

Lee

Yang

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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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.

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