Identification and biochemical properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells.

Baram, J; Chabner, Bruce A.; Drake, James C.; Fitzhugh, Anthony L.; Sholar, Pam W.; Allegra, Carmen J.

doi:10.1016/s0021-9258(18)68611-9

Cited by 39 publications

(4 citation statements)

References 27 publications

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“…However, H2folate, and especially its poyglutamates, are powerful inhibitors of several folate enzymes, such as methylenetetrahydrofolate reductase (Matthews & Baugh, 1980), thymidylate synthase (Dolnick & Cheng, 1978;Barum et al, 1988), and amido phosphoribosyltransferase (Sant et al, 1992). Although accurate calculations are not possible from the available data, the concentrations of H2folate necessary for its reduction by Phe34 variants of hDHFR may well be highly toxic to the cell.…”

Section: Discussionmentioning

confidence: 99%

Critical Role of Phenylalanine 34 of Human Dihydrofolate Reductase in Substrate and Inhibitor Binding and in Catalysis

Nakano

Ht²,

Appleman³

et al. 1994

Biochemistry

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Directed mutagenesis has been used to construct five variants of human dihydrofolate reductase in which smaller residues are substituted for phenylalanine 34, a residue participating in the binding of substrate and methotrexate by interaction with their pteridine rings. The variant enzymes are stable and have decreased affinities for methotrexate (by factors of 2700-60000 at pH 7.65) due to a decreased rate of methotrexate association and a much larger increase in the rate constant for dissociation. However, the catalytic efficiencies of the variants are also lowered by factors of 160-5000, so that it is doubtful whether these enzymes are capable of conferring methotrexate resistance on the cells harboring them. High concentrations of dihydrofolate cause marked inhibition of all the variants, which complicates the determination of kinetic parameters. By the use of stopped-flow spectrophotometry and fluorimetry and other methods, it has been shown that, like the wild-type enzyme, the variants have a branched reaction pathway, but in contrast to the wild-type enzyme, the distribution of flux between alternate pathways is dependent on the concentration of dihydrofolate. This different branch point is a consequence of the very rapid dissociation of tetrahydrofolate from the ternary product complexes of the variant enzymes. Inhibition by dihydrofolate is due to its combination with the enzyme-NADP complex and the slow dissociation of NADP from the resulting abortive complex. When steady state kinetics for this model are simulated using the experimentally determined rate and dissociation constants for the alanine 34 variant, most steady state experimental results are closely approximated.

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Section: Discussionmentioning

confidence: 99%

Critical Role of Phenylalanine 34 of Human Dihydrofolate Reductase in Substrate and Inhibitor Binding and in Catalysis

Nakano

Ht²,

Appleman³

et al. 1994

Biochemistry

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“…Structural similarities between methotrexate and folic acid dominate enzyme inhibitions. Inhibition of enzymes, such as dihydrofolate reductase (DHFR), leads to reduction of de novo biosynthesis of the nucleoside thymidine. − Thymidine is required for DNA synthesis. This gives methotrexate its anti-cancer activity.…”

Section: Mie Examplesmentioning

confidence: 99%

Defining Molecular Initiating Events in the Adverse Outcome Pathway Framework for Risk Assessment

Allen

Goodman

Gutsell

et al. 2014

Chem. Res. Toxicol.

152

106

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Consumer and environmental safety decisions are based on exposure and hazard data, interpreted using risk assessment approaches. The adverse outcome pathway (AOP) conceptual framework has been presented as a logical sequence of events or processes within biological systems which can be used to understand adverse effects and refine current risk assessment practices in ecotoxicology. This framework can also be applied to human toxicology and is explored on the basis of investigating the molecular initiating events (MIEs) of compounds. The precise definition of the MIE has yet to reach general acceptance. In this work we present a unified MIE definition: an MIE is the initial interaction between a molecule and a biomolecule or biosystem that can be causally linked to an outcome via a pathway. Case studies are presented, and issues with current definitions are addressed. With the development of a unified MIE definition, the field can look toward defining, classifying, and characterizing more MIEs and using knowledge of the chemistry of these processes to aid AOP research and toxicity risk assessment. We also present the role of MIE research in the development of in vitro and in silico toxicology and suggest how, by using a combination of biological and chemical approaches, MIEs can be identified and characterized despite a lack of detailed reports, even for some of the most studied molecules in toxicology.

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“…(Kim et al, 1993) The resulting polyglutamate metabolites become increasingly potent direct inhibitors of several folate-dependent enzymes. (Allegra et al, 1985a;Allegra et al, 1985b;Baram et al, 1988;Sant et al, 1992) As a result of the short circulating half-life of MTX and the relative abundance of MTX polyglutamate metabolites in tissues, it has been hypothesized that the pharmacological basis for MTX response in autoimmune arthritis is not through the inhibition of DHFR, but rather through the inhibition of the folate-dependent enzymes by the polyglutamate metabolites of MTX. (Chabner et al, 1985) Efforts to identify clinical biomarkers of MTX efficacy have primarily focused on establishing the relationship between concentrations of the polyglutamate metabolites of MTX in patient erythrocytes and clinical response.…”

Section: Introductionmentioning

confidence: 99%

Methotrexate disposition, anti-folate activity and efficacy in the collagen-induced arthritis mouse model

Singh

Haandel

Kiptoo

et al. 2019

European Journal of Pharmacology

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Methotrexate (MTX) efficacy in autoimmune arthritis is variable and unpredictable resulting in the need for the identification of biomarkers to guide drug therapy. This study utilizes the collageninduced arthritis mouse model to investigate erythrocyte MTX disposition and anti-folate activity as biochemical markers of efficacy in autoimmune arthritis. Following induction of arthritis, DBA/1J mice were treated with once-weekly subcutaneous MTX at varying doses over a period of

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Identification and biochemical properties of 10-formyldihydrofolate, a novel folate found in methotrexate-treated cells.

Cited by 39 publications

References 27 publications

Critical Role of Phenylalanine 34 of Human Dihydrofolate Reductase in Substrate and Inhibitor Binding and in Catalysis

Critical Role of Phenylalanine 34 of Human Dihydrofolate Reductase in Substrate and Inhibitor Binding and in Catalysis

Defining Molecular Initiating Events in the Adverse Outcome Pathway Framework for Risk Assessment

Methotrexate disposition, anti-folate activity and efficacy in the collagen-induced arthritis mouse model

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