Methotrexate reductase

In view of the well-documented inhibition of dihydrofolate reductase by aminopterin (325), methotrexate (326) and related compounds it is generally accepted that this inhibitory effect constitutes the primary metabolic action of folate analogues and results in a block in the conversion of folate and dihydrofolate (DHF) to THF and its derivatives. As a consequence of this block, tissues become deficient in the THF derivatives, and this deficiency has many consequences similar to those resulting from nutritional folate deficiency. The crucial effect, however, is a depression of thymidylate synthesis with a consequent failure in DNA synthesis and arrest of cell division that has lethal results in rapidly proliferating tissues such as intestinal mucosa and bone marrow (B-69MI21604, B-69MI21605). 

Methotrexate (MTX, chemical structure shown in Fig. 1.) competitively inhibits the dehyrofolate reductase, an enzyme that plays an essential role in purine synthesis. The dehydrofolate reductase regenerates reduced folates when thymidine monophosphate is formed from deoxyuridine monophosphate. Without reduced folates cells are unable to synthesize thymine. Administration of N-5 tetrahydrofolate or N-5 formyl-tetrahydrofolate (folinic acid) can bypass this block and rescue cells from methotrexate activity by serving as antidote. 

Methotrexate belongs to the class of antimetabolites. As a derivative of folic acid it inhibits the enzyme dihydrofolate reductase resulting in a decreased production of thymidine and purine bases essential for RNA and DNA synthesis. This interruption of the cellular metabolism and mitosis leads to cell death. 

Matthews DA, Alden RA, Bolin JT, Filman DJ, Freer ST, Hamlin R, Hoi WG, Kisliuk RL, Pastore EJ, Plante FT, Xuong N, Kraut J. Dihydrofolate reductase from Lactobacillus casei. X-ray structure of the enzyme methotrexate-NADPH complex. J Biol Chem 1978 253 6946-54. 

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

Cardiovascular Keep doses of NSAIDs and glucocorticoids low, consider initiation of folic acid to reduce homocysteine level elevations induced by methotrexate, consider initiation of low-dose aspirin and/or HMG-CoA reductase inhibitors (statins), and encourage smokers to discontinue tobacco use and assist with the development of a tobacco-cessation plan.11,12... 

Figure 1.4 Left panel Space filing model of the structure of bacterial dihydrofolate reductase with methotrexate bound to the active site. Right panel Close-up view of the active site, illustrating the structural complementarity between the ligand (methotrexate) and the binding pocket. See color insert. Source Courtesy of Nesya Nevins.

Figure 1.5 Interactions of the dihydrofolate reductase active site with the inhibitor methotrexate (left) and the substrate dihydrofolate (right).

Methotrexate Dihydrofolate reductase Cancer, bacterial infection... 

We saw in Chapter 3 that bisubstrate reactions can conform to a number of different reaction mechanisms. We saw further that the apparent value of a substrate Km (KT) can vary with the degree of saturation of the other substrate of the reaction, in different ways depending on the mechanistic details. Hence the determination of balanced conditions for screening of an enzyme that catalyzes a bisubstrate reaction will require a prior knowledge of reaction mechanism. This places a necessary, but often overlooked, burden on the scientist to determine the reaction mechanism of the enzyme before finalizing assay conditions for HTS purposes. The importance of this mechanistic information cannot be overstated. We have already seen, in the examples of methotrexate inhibition of dihydrofolate, mycophenolic acid inhibiton of IMP dehydrogenase, and epristeride inhibition of steroid 5a-reductase (Chapter 3), how the [5]/A p ratio can influence one s ability to identify uncompetitive inhibitors of bisubstrate reactions. We have also seen that our ability to discover uncompetitive inhibitors of such reactions must be balanced with our ability to discover competitive inhibitors as well. 

Example of Scheme C Inhibition of Dihydrofolate Reductase by Methotrexate... 

We have already used the interactions of methotrexate with dihydrofolate reductase (DHFR) several times within this text to illustrate some key aspects of enzyme inhibition. The reader will recall that methotrexate binds to both the free enzyme and the enzyme-NADPH binary complex but displays much greater affinity for the latter species. The time dependence of methotrexate binding to bacterial DHFR was studied by Williams et al. (1979) under conditions of saturating [NADPH], In the presence of varying concentrations of methotrexate, the progress curves for DHFR activity became progressively more nonlinear (Figure 6.14). The value of kobs from... 

Figure 6.14 Progress curves for the enzymatic reaction of dihydrofolate reductase in the presence of the indicated concentrations of methotrexate.

Figure 6.16 Energy level diagram for the two-step inhibition of dihydrofolate reductase by methotrexate. The AGbinding were calculated at 30°C base on the dissociation constants reported by Williams et al. (1979).

III. The answer is d, (Hardman, pp 1247, L135.) Leucovorin prevents methotrexate from inhibiting dihydrofolate reductase and reverses all of its adverse effects except neurotoxicity... 

U. C. Singh, Probing the salt bridge in the dihydrofolate reductase-methotrexate complex... 

The above model was used to study the binding of methotrexate, and analogues of it, to wild-type and mutant forms of human dihydrofolate reductase (DHFR).29 This turned out to be a particularly difficult test because of the three ionized groups of methotrexate. The overall electrostatic interactions of this inhibitor amount to around -500 kcal/mol and MD trajectories of length more than a ns were required in order to get average... 

Poe, K. Williams, and K. Hoogsteen, Dihydrofolate reductase X-ray structure of the binary complex with methotrexate, Science 197 452 (1977). 

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