Reactivation of dihydrofolate reductase inhibited by methotrexate or aminopterin

Jackson, Robert; Niethammer, D.; Hart, L. I.

doi:10.1016/0003-9861(77)90545-8

Cited by 37 publications

(16 citation statements)

References 17 publications

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“…Owing to this, achieving complete suppression of this small fraction of DHFR requires levels of drug far in excess of the K i . Inhibition is, in fact, competitive with DHF (Jackson et al, 1977;White, 1979;White and Goldman, 1981).…”

Section: Polyglutamation Of Antifolates -A Key Determinant Of Antifolmentioning

confidence: 97%

“…The basis for the difference in the kinetics of inhibition of the isolated enzyme versus the enzyme in cells was clarified experimentally and with the application of computer modeling systems (Jackson and Harrap, 1973;Jackson et al, 1977;White, 1979;White and Goldman, 1981). Unlike the cell-free system, folate metabolism in cells is a dynamic process in which the levels of THF-cofactors and DHF vary with the activities of enzymes and flows in this system.…”

Section: Polyglutamation Of Antifolates -A Key Determinant Of Antifolmentioning

confidence: 99%

“…The ratio of DHF : MTX is high, and this results in net displacement of a small amount of MTX from DHFR by DHF as the drug cycles on and off the enzyme. Following this, free MTX leaves the cell and the enzyme is reactivated (Jackson et al, 1977;White, 1979). As this occurs, DHF is consumed, normal rates of THF synthesis are restored, DHF returns to its usual very low levels, and the remaining MTX remains essentially irreversibly bound to B95% of the enzyme, a level that cannot be distinguished experimentally from the total enzyme-binding capacity.…”

Section: Polyglutamation Of Antifolates -A Key Determinant Of Antifolmentioning

confidence: 99%

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Resistance to antifolates

2003

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Section: Polyglutamation Of Antifolates -A Key Determinant Of Antifolmentioning

confidence: 97%

Section: Polyglutamation Of Antifolates -A Key Determinant Of Antifolmentioning

confidence: 99%

Section: Polyglutamation Of Antifolates -A Key Determinant Of Antifolmentioning

confidence: 99%

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Resistance to antifolates

2003

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“…Allowance must therefore be made for the reduction of free inhibitor concentration which occurs as a result of its binding to the enzyme [2]. In general, it is tight-binding inhibitors which are of therapeutic value, but no quantitative relationship equivalent to Eqn (2) has been developed for this more complex case although several authors [5,6] have acknowledged the importance of substrate accumulation in response to drug administration in vivo. In this paper, we present the equation which describes metabolic resistance to tight-binding inhibitors and demonstrate the use of this equation in describing published experimental data [4] for the interaction of PAcAsp with aspartate transcarbamoylase.…”

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confidence: 99%

Metabolic Resistance: The Protection of Enzymes against Drugs which are Tight‐Binding Inhibitors by the Accumulation of Substrate

Christopherson

Duggleby

1983

European Journal of Biochemistry

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Blockade of a metabolic pathway by interaction of a drug with a particular 'target enzyme' results in depletion of essential end-products of the pathway and accumulation of intermediates prior to the blockade. Metubolic resistance to a particular drug can arise if the substrate of the inhibited enzyme accumulates to levels sufficiently high to compete effectively with the inhibitor, leading to restoration of full activity of the metabolic pathway after a transitory delay.Such resistance has recently been demonstrated in vitro for the interaction of the tight-binding inhibitor N-phosphonacetyl-L-aspartate (PAcAsp) with the aspartate transcarbamoylase activity of the trifunctional protein which initiates pyrimidine biosynthesis in mammals [Christopherson, R. I. and Jones, M. E. (1980) J . Biol. Chem. 255, 11 381 -11 3951. Carbamoyl phosphate, the product of the carbamoyl phosphate synthetase activity of this trifunctional protein, accumulates to a sufficiently high concentration that the inhibitory effect of PAcAsp is effectively abolished. We have developed a theoretical model for metabolic resistance which quantitatively accounts for these experimental data. This model has been used to simulate the interaction between the following potential or proven anti-cancer drugs and their target enzyme, under conditions similar to those which would occur in vivo: PAcAsp with aspartate transcarbamoylase; various OMP analogues [the 5'-monophosphates of 6-azauridine, pyrazofurin and I-(P-D-ribofuranosy1)-barbituric acid] with OMP decarboxylase; 5-fluorodeoxyUMP with thymidylate synthase; methotrexate with dihydrofolate reductase; and deoxycoformycin with adenosine deaminase.

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“…INTRODUCTION Methotrexate (MTX-Glul)', the 2-4-diamino, 10-methyl analogue of folic acid, is thought to exert its antitumor effects through inhibition of dihydrofolate reductase (DHFR) (1). MTX-Glul is a tight-binding but reversible inhibitor in the presence of NADPH, with slow but definite drug dissociation from the enzyme (2,3). Consequently, MTX-Glul must bind all enzyme catalytic sites (bound drug) and in addition must be pres-' Abbreviations used in this paper: DHFR, dihydrofolate reductase; dI, deoxyinosine; dT, thymidine; FCS, fetal calf serum; FH2, dihydrofolic acid; HPLC, high-pressure liquid chromatography; IMEM, improved minimal essential medium; MTX-Glul, methotrexate; MTXPG, MTX polyglutamates.…”

mentioning

confidence: 99%

Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells. Selective retention and less dissociable binding of 4-NH2-10-CH3-pteroylglutamate4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase.

Jolivet¹,

Chabner²

1983

J. Clin. Invest.

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A B S T R A C T Methotrexate (MTX-Glul) exerts its antitumor effects through its potent inhibition of dihydrofolate reductase (DHFR), the enzyme responsible for maintaining the cellular pool of reduced folates. Since the drug-enzyme complex (bound drug) is slowly dissociable, an excess of drug (unbound or free drug) above that required to bind all enzyme sites is required in order to compete with substrate for sites made available by enzyme-drug dissociation. We have examined the role of the polyglutamyl metabolites of MTX-Glul containing two to five glutamyl (MTX-Glu2-5) groups in gamma peptide linkage in maintaining an intracellular pool of free drug and in forming slowly dissociable complexes with DHFR. During 24-h incubations of ZR-75-B human breast cancer cells with 2 uM MTX-Glu,, we observed the progressive formation of derivatives with two to five glutamyl groups, which rapidly replaced the parent compound on enzyme binding sites and represented 85% of both unbound and bound intracellular drug at the end of incubation. When cells were then placed in drug-free medium, the rates of disappearance of drug and metabolites from the intracellular bound and free fractions decreased with increasing glutamyl chain length. Over 90% of both bound and free MTX-Glul left the cells within 1 h, >90% of MTX-glu2 left within 6 h, and >90% of MTX-Glu3 left the bound and free fractions within 24 h. In contrast, free MTX-Glu4 fell by only 63% and bound by only 23% affter 24 h, while free MTX-Glu5 increased by 52% after 6 h in drug-free tively, while -Glu3, -Glu4, and -Glu5 had t½ of 102, 108, and 120 min. These experiments demonstrated that the longer chain polyglutamates have prolonged intracellular retention and can be dissociated less readily than MTX-Glu2 from DHFR, properties likely to make them more efficient DHFR inhibitors than the parent drug and of potential importance in extending the duration of drug action in tumor cells.

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Reactivation of dihydrofolate reductase inhibited by methotrexate or aminopterin

Cited by 37 publications

References 17 publications

Resistance to antifolates

Resistance to antifolates

Metabolic Resistance: The Protection of Enzymes against Drugs which are Tight‐Binding Inhibitors by the Accumulation of Substrate

Intracellular pharmacokinetics of methotrexate polyglutamates in human breast cancer cells. Selective retention and less dissociable binding of 4-NH2-10-CH3-pteroylglutamate4 and 4-NH2-10-CH3-pteroylglutamate5 to dihydrofolate reductase.

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