Dihydrofolate reductase interaction with methotrexate

Drugs that may interact with folic acid include aminosalicylic acid, oral contraceptives, dihydrofolate reductase inhibitors (eg, methotrexate, trimethoprim), sulfasalazine, hydantoins. 

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

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... 

The chemical structures of four commonly used anticancer drugs are shown in Fig. 15-17. Methotrexate was the first "true anticancer drug, synthesized in 1949, and has been in clinical use for treatment of a variety of cancers since the early 1950s. Methotrexate is a potent inhibitor of dihydrofolate reductase with an inhibition constant (Kt) for interaction with the enzyme of 10 9Af. Inhibition of this enzyme in a cell leads to major accumulation of DHF to concentrations of 2.5 fiM, and minor decreases in THF. Marked decreases in THF may not be seen due to the release of bound THF in methotrexate-treated cells. The high levels of DHF are toxic to the cell, inhibiting the reaction catalyzed by thymidylate synthase,... 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

X-ray diffraction analysis of dihydrofolate reductase (DHFR), co-crystal-lized with methotrexate, has shed much light on the action of this inhibitor. This work, one of the earliest visualizations of a drug interacting with its receptor (Matthews etal., 1977), has since been refined to the remarkably clear resolution of 1.7 A (Bolin et al., 1982). A typical diagram of DHFR, its coenzyme (NADPH), and methotrexate is shown in Fig. 9.4. The enzyme depicted there is from Lactobacillus casei and the same authors also report on DHFR (with cocrystallized methotrexate) from the bacterium E. colt. However, they have not been able to co-crystallize methotrexate with DHFR from any vertebrate source. 

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