Dihydrofolate reductase analysis

The use of pH variation and isotope effects in transient kinetics can be illustrated with a recent study on dihydrofolate reductase. Analysis by steady-state methods had indicated an apparent p/fa of 8.5 that was assigned to an active site aspartate residue required to stabilize the protonated state of the substrate (59). In addition, it was shown that there was an isotope effect on substitution of NADPD (the deuterated analog) for NADPH at high pH but not at low pH, below the apparent p/fa This somewhat puzzling finding was explained by transient-state kinetic analysis. Hydride transfer, the chemical reaction converting enzyme-bound NADPH and dihydrofolate to NAD+ and tetrahydrofolate, was shown to occur at a rate of approximately 1000 sec at low pH. The rate of reaction decreased with increasing pH with a of 6.5, a value more in line with expectations for an active site aspartate residue. As shown in Fig. 14, there was a threefold reduction in the rate of the chemical reaction with NADPD relative to NADPH. Thus direct measurement of the chemical reaction revealed the full isotope effect. 

AJ Hopfinger. A QSAR investigation of dihydrofolate reductase inhibition by Baker triazmes based upon molecular shape analysis. I Am Chem Soc 102 7196-7206, 1980. 

GM Crippen. Quantitative structure-activity relationships by distance geometry Systematic analysis of dihydrofolate reductase inhibitors. I Med Chem 23 599-606, 1980. 

Reaction Free Energy Profiles Using Free Energy Perturbation and Coordinate Coupling Methodologies Analysis of the Dihydrofolate Reductase Catalytic Mechanism... 

Studies using free energy calculations for the design and analysis of potential drug candidates are reviewed in section five. The chapters in this section cover drug discovery programs targeting fructose 1,6-bisphosphatase (diabetes), COX-2 (inflammation), SRC SH2 domain (osteoporosis and cancer), HIV reverse transcriptase (AIDS), HIV-1 protease (AIDS), thymidylate synthase (cancer), dihydrofolate reductase (cancer) and adenosine deaminase (immunosuppression, myocardial ischemia). 

Hopfinger, A. J. (1981) Inhibition of dihydrofolate reductase structure-activity correlations of 2,4-diamino-5-benzylpyrimidines based upon molecular shape analysis. J. Med. Chem. 24, 818-822. 

Crystallographic analysis has been used quite effectively to solidify structural determinations. Several examples include dihydrofolate reductase complexes , nucleosides <1999AXC1335, 1999AXC1560>, zopi-clone <2001CC2204>, and immucillin inhibitors <2003BBR917>. 

JM Wooden, LH Hartwell, B Vasquez, CH Sibley. Analysis in yeast of antimalaria drugs that target the dihydrofolate reductase of Plasmodium falciparum. Mol Biochem Parasitol 85 25 10, 1997. 

Some of the structural properties which have been ascribed to DA receptors appear to deserve attention for their heuristic value, but painfully few should engender much confidence in their reality. A sobering lesson is available from analysis of complexes of dihydrofolate reductase (], 8). Methotrexate is a very close analog of folic acid and is a potent inhibitor of the enzyme, but it is now almost certain that these ligands bind in the enzyme active site in aspects differing by a rota-... 

Another example of the application of lineshape analysis to ligand dynamics is described in Section 4.3 for the drug trimetrexate, which binds to dihydrofolate reductase (DHFR). From that example it is clear that the techniques described above can equally be applied to ligands when bound to their receptors. In some cases significant but highly specific mobility appears to be present at the bound site. 

Kim, S.J., and G.M. Lee. 1999. Cytogenetic analysis of chimeric antibody-producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol Bioeng 64 741-749. 

Electrodes based on the immobilization of enzymes at the electrode surface have been reported for a few pharmaceutical applications. Seegopaul and Rechnitz developed a CO2 electrode that responds to methotrexate due to the inhibition of dihydrofolate reductase by the analyte. Additional applications have included the detection of penicillin consumption by penicillinase as well as urea by urease with a pH electrode and analysis of L-ascorbic acid by ascorbate oxidase.f ... 

Battershell, C., Malhotra, D. and Hopfinger, A.J. (1981). Inhibition of Dihydrofolate Reductase Structure-Activity Correlations of Quinazolines Based upon Molecular Shape Analysis. 

Crippen, G.M. (1980). Quantitative Structure-Activity Relationships by Distance Geometry Systematic Analysis of Dihydrofolate Reductase Inhibitors. J.Med.Chem.,23,599-606. 

Hopfinger, A.J. (1980). A QSAR Investigation of Dihydrofolate Reductase Inhibition by Baker Triazines Based upon Molecular Shape Analysis. J.Am.Chem.Soc., 102, 7196-7206. 

Hopfinger, A.J. (1983). Theory and Application of Molecular Potential Energy Fields in Molecular Shape Analysis A Quantitative Structure-Activity Relationship Study of 2,4-Diamino-5-benzylpyrimidines as Dihydrofolate Reductase Inhibitors. J.Med.Chem.,26, 990-996. 

Ivanciuc, O. (1996). Artificial Neural Networks Applications. 2. Using Theoretical Descriptors of Molecular Structure in Quantitative Structure-Activity Relationships Analysis of the Inhibition of Dihydrofolate Reductase. Rev.Roum.Chim., 41,645-652. 

Silipo, C. and Hansch, C. (1975). Correlation Analysis. Its Application to the Structure-Activity Relationship of Triazines Inhibiting Dihydrofolate Reductase. J.Am.Chem.Soc., 97, 6849-6861. 

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