Dihydrofolate inducible

The concept that oestrogens stimulate cell proliferation directly arises mainly from the observation that physiological concentrations of oestrogens stimulate both the de novo and salvage pathways of DNA synthesis as well as inducing a number of enzymes intimately involved in DNA synthesis and including DNA polymerase, thymidine and uridine kinases, thymidilate synthetase and dihydrofolate reductase. There is evidence that some of these enzymes may be regulated at the transcrip-... 

Oral use of sulfonamides, pyrimethamine, and trimethoprim can canse blood dyscrasias such as hemolytic anemia, aplastic anemia, lenkopenia, and agran-nlocytosis. Becanse these blood changes are due to a drug-induced folic acid deficiency, administering folinic (not folic) acid can coimteract the toxicity. Use of folinic acid bypasses the need fc>r dihydrofolate reductase by supplying the fully reduced folate. 

Methotrexate is an antagonist of folic acid and is used for treating neoplastic diseases and non-neoplastic diseases such as rheumatoid arthritis and psoriasis. As methotrexate reduces the activity of dihydrofolate reductase, supplementation with folic acid and especially with folinic acid could reduce the beneficial effects of methotrexate. This assumption has been supported by the results of an intervention study (40). Patients with rheumatoid arthritis treated with methotrexate (15 g/week) had an increase in symptoms. An open intervention trial of folinic acid 45 mg/week also showed an increase in arthritis symptoms (41). However, other trials showed no effects of this sort, and administration of folic acid or folinic acid is important in preventing methotrexate-induced blood dyscrasias. 

Dihydrofolate reductase has been used extensively in translocation experiments. A fusion protein with diphtheria toxin A-fragment was shown to be translocated to the cytosol (Klingenberg and Ols-nes, 1996). The translocation was inhibited by methotrexate, which induces a tight folding of the protein. A fusion with a mutated dihydrofolate reductase that does not bind methotrexate tightly was also translocated, and in this case methotrexate was not able to prevent the translocation. This indicates that not only must the toxin A-fragment be unfolded, but the passenger protein must also be able to unfold for translocation to occur. 

The dihydrofolate reductase inhibitor, methotrexate (Fig. 8.47), was developed as an anticancer drug, whose inhibition of formation of folic acid coenzymes would block purine synthesis. In other words, it was designed to induce a folic acid deficiency. Notice in Figs. 8.50 and 8.51 that formation of dTMP, FGAR, and AICAR also causes the oxidation of tetra-hydrofolate to dihydrofolate. The latter must be reduced by dihydrofolate reductase to tet-rahydrofolate before active coenzyme can form again. Thus, not only does methotrexate inhibit the initial formation of the tetrahydro-folate moiety, it blocks regeneration of the coenzyme form. 

FIGURE 99-4. Drug induced megaloblastosis. DHF, dihydrofolate 5-MTHF, 5-methyl-tetrahydrofolate 5,10-MTHF, 5,10-methyl-tetrahydrofolate THF THF, tetrahydrofolate. 

Several drugs (e.g., sulfasalazine, trimethoprim-sulfamethoxazole, and methotrexate) have been reported to cause a fohc acid deficiency megaloblastic anemia. These drugs either interfere with folate absorption or inhibit the dihydrofolate reductase enzyme necessary for conversion of dihydrofolate to its active tetrahydrofolate form (see Chap. 102, on drug-induced blood dyscrasias). 

Dietary folates must be chemically reduced to their tetrahy-dro forms, with four hydrogens on the pteridine ring, to be active. The enzyme responsible for this reduction is dihydrofolate reductase (DHFR), a key enzyme whose actions are inhibited by methotrexate and other antifolates. The result of this inhibition is depletion of intracellular pools of reduced folates (tetrahydrofolates) essential for thymidylate and purine synthesis. Lack of either thymidine or purines prevents synthesis of DNA. The DHFR-mediated effects of antifolate drugs on normal and probably also on cancerous cells may be neutralized by supplying reduced folates exogenously. The reduced folate used clinically for rescue is leucovorin (folinic acid), which bypasses the metabolic block induced by DHFR inhibitors. ... 

Carothers, A.M., Yuan, W., Hingerty, B.E., Broyde, S., Grunberger, D., and Snyderwine, E.G. (1994) Mutation and repair induced by the car-dnogen 2- (hydroxyamino) -1 -methyl-6-phenylimidazo[4,5 -fejpyridine (N-OH-PhIP) in the dihydrofolate reductase gene of Chinese hamster ovary cells and conformational modeling of the dG-C8-PhIP adduct in DNA. Chem. Res. Toxicol., 7, 209-218. 

Tang, M.S., Pao, A., and Zhang, X.S. (1994) Repair ofbenzo(a)pyrene diol epoxide- and UV-induced DNA damage in dihydrofolate reductase and adenine phosphoribosyltransferase genes of CHO cells./. Biol. Chem., 269, 12749-12754. 

FIGURE 6-4 Complementary shapes of a substrate and its binding site on an enzyme. The enzyme dihydrofolate reductase with its substrate NADP" (red), unbound (top) and bound (bottom). Another bound substrate, tetrahydrofolate (yellow), is also visible (PDB ID 1 RA2).The NADP binds to a pocket that is complementary to it in shape and ionic properties. In reality, the complementarity between protein and ligand (in this case substrate) is rarely perfect, as we saw in Chapter 5. The interaction of a protein with a ligand often involves changes in the conformation of one or both molecules, a process called induced fit. This lack of perfect complementarity between enzyme and substrate (not evident in this figure) is important to enzymatic catalysis. 

J. C. Hu, Chemically induced dimerization of dihydrofolate reductase by a homobifunctional dimer of methotrexate, Chem. Biol. 2000, 7, 313-321. 

There is a very large area of enzyme targets as illustrated in Table 4.2. For example, dihydrofolate reductase (DHFR) catalyses the NADPH-linked reduction of dihydrofolate to tetrahydrofolate. The tetrahydrofolate are cofactors for the biosynthesis of nucleic acids and aminoacids. The reduction of their level induces a limitation of cell growth. ... 

Cody, V, Galitsky, N., Rak, D., Luft, J.R., Pangborn, W., and Queener, S.F. (1999) Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP( + ). Biochemistry, 38, 4303-4312. 

Bystroff, C., Kraut, J. 1991. Crystal structure of unhganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding. 

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