Purine aminopterin

Inhibition of nucleobase synthesis (2). Tetrahydrofolic acid (THF) is required for the synthesis of both purine bases and thymidine. Formation of THF from folic acid involves dihydrofolate reductase (p. 272). The folate analogues aminopterin and methotrexate (ame-thopterin) inhibit enzyme activity as false substrates. As cellular stores of THF are depleted, synthesis of DNA and RNA building blocks ceases. The effect of these antimetabolites can be reversed Ltillmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of iicense. 

Fusion with the cells compensates for this deficiency. When fused and unfused cells are incubated in the presence of the folic acid antagonist aminopterin, the de novo synthesis of purines and pyrimidines for DNA is blocked. Cells deficient in HGPRT die, whereas hybrid cells are able to bypass aminopterin blockage by metabolism of hypoxanthine and thymidine added to the medium. In the generation of mouse hybridomas, an number of myelomas deficient in HGPRT are available, all originating from MOPC 21, a spontaneous myeloma from the BALB/c mouse strain. 

Methotrexate and aminopterin are believed to inhibit dihydrofolate reductase by blocking the reduction of dihydrofolate to tetrahydrofolate, which is the cofactor in the synthesis of thymine and purines used in DNA synthesis. 

Aminopterin and amethopterin are 4-amino analogues of folic acid (Fig. 11.5) and as such are potent inhibitors of the enzyme dihydrofolate reductase (EC 1.5.1.3) (Blakley, 1969). This enzyme catalyses the reduction of folic acid and dihydrofolic acid to tetrahy-drofolic acid which is the level of reduction of the active coenzyme involved in many different aspects of single carbon transfer. As is clear from Fig. 11.6, tetrahydrofolate is involved in the metabolism of (a) the amino acids glycine and methionine (b) the carbon atoms at positions 2 and 8 of the purine ring (c) the methyl group of thymidine and (d) indirectly in the synthesis of choline and histidine. 

Amino-4-imidazolecarboxamide was isolated from sulfonamide-inhibited cultures of Escherichia coli. - ° This substance also accumulates when Escherichia coli are inhibited by aminopterin, and is found in the culture medium of a purine-requiring mutant of Escherichia coZf. °... 

PICOUNIUM CHLORIDE see AOD175 AMINOPTERIDINE see AMG750 AMINOPTERIN see AMG750 4-AMINOPTEROYLGLUTAMIC ACID see AMG750 6-A NOPURINE see AEHOOO 6-AMINO-lH-PURINE see AEHOOO 6-AMINO-3H-PURINE see AEHOOO 6-AMINO-9H-PURINE see AEHOOO... 

Why then, since such an abundance of metabolic inhibitors is available, do so few of them find practical application Examples are the folic acid reductase inhibitors, such as aminopterin, the purine and pyrimidine analogs used as cytostatics in cancer chemotherapy and known for their high toxicity in a wide variety of species, and the organic phosphates and carbamates used as insecticides but also highly toxic to mammals. Lack of selectivity in the action of metabolic inhibitors is inherent in their mechanism of action due to the universality of biochemical processes and principles throughout nature. Selectivity in action requires species differences in biochemistry. For the antivitamins, for instance, there is not only a lack of species differences in action in addition, the fact that vitamins often serve as cofactors for a variety of enzymes is a serious drawback to endeavors to obtain agents with species-selective action. 

One of the first studies that indicated the possible interference of ametbopterin and aminopterin with purine synthesis was the demonstration by Skipper et al. (S21) that administration of these compounds to mice inhibited the incorporation of labeled formate into nucleic acid purines. Ametbopterin (4-amino-A -methyl-pteroylglutamic acid) has produced some inhibition of uric acid synthesis during the use of this compound for treatment of leukemia (K15, E24). Another inhibitor of... 

HAT Medium - A cell culture medium augmented by hypoxanthine, aminopterin, and thymidine, which selects for cells that have a functional purine salvage pathway. 

HAT Selection - The compounds hypoxanthine, aminopterin (see here), and thymidine (H,A, and T, respectively) can be used to select for cells having functional salvage pathways. Aminopterin inhibits dihydrofolate reductase, which blocks de novo purine and thymidine synthesis. Only cells which can utilize thymidine (pyrimidine salvage) and hypoxanthine (purine salvage) can grow in this medium. 

Methotrexate and aminopterin, a similar compound, are analogs of dihydro folate (DHF) and inhibitors of dihydrofolate reductase, an enzyme that converts DHF to tetrahydro-folate (THF). The thymidylate synthase reaction converts N, N °-methylenetetrahydro-folate to DHF in the process of methylating dUMP to form dTMP In the presence of one of the inhibitors, this reaction functions as a sink that reduces the THF level of the cell by converting THF to DHF. Since THF derivatives are substrates in two reactions of purine metabolism and one of pyrimidine metabolism, both pathways are affected by the inhibitor. 

Aminopterin 4-amino-4-deoxyfolic acid (see Vitamins, folic acid), a cytostatic agent used in the management of some caneers. It inhibits the enzyme dihy-drofolate reductase, which reduces the folate coenzymes required for Purine biosynthesis (see) and thymine production (see Pyrimidine biosynth is), and thus prevents DNA synthesis However, it is toxic to nondividing cells as well, and cannot be tolerated indefinitely. Methotrexate (amethopterin) has similar activity. 

With aminopterin, the reductase forms a complex with a low dissociation constant. Thus, in the presence of aminopterin many of the reductase molecules are trapped in an inactive form. The coenzymes necessary for purine biosynthesis are not formed, and that pathway is blocked. In this manner aminopterin interferes with the progress of leukemia and with the proliferation of normal bone marrow. Unfortunately, the cells of individuals treated for leukemia overcome the metabolic block by building up a resistance to the antimetabolites by increasing the level of the reductase. We will now consider the mechanism of action of each of these coenzymes separately. 

AIC also accumulated when E. coli was grown in the presence of aminopterin (4-aminopteroylglutamic acid), a folic acid antagonist. Since addition of aminopterin or p-aminobenzoic acid antimetabolites e.g., sulfonamides) results in the accumulation of AIC, it is presumed that the latter act by creating a folic acid deficiency. Thus, folic acid or a related derivative, and not p-aminobenzoic acid, may be directly concerned with purine synthesis. This is in agreement with Woods hypothesis that the primary action of sulfonamides is the inhibition of folic acid formation, and that purine synthesis is a secondary event in which p-aminobenzoic acid participates only indirectly. ... 

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