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Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate (H2folate) to tetrahydrofolate (H4folate). This enzyme is necessary for maintaining adequate levels of H4folate and its derivatives, which are essential for the biosynthesis of purines, thymidylate and several amino acids. This enzyme is therefore a target for anticancer drugs such as methotrexate (MTX). Cancer patients receiving high dose MTX as part of their therapy frequently develop clinical resistance and molecular mechanisms of such resistance are of interest.

Since dihydrofolate reductase (DHFR) is responsible for maintaining intracellular folate pools in their biochemically active reduced state, its inhibition has been the target of continuing antifolate development to improve selectivity, penetration into target cells, and effectiveness against tumors with either intrinsic or acquired antifolate resistance1. For example, investigation of patients with methotrexate (MTX)-resistance has shown that in some tumors the resistance is due to defects in cellular transport or polyglutamation and that new, second generation, antifolate drugs such as 10-ethyl-10-deazaminopterin (10-EDAM) or the lipophilic antifolates piritrexim (PTX) and trimetrexate (TMQ), are more effective than methotrexate (MTX)2 . In other tumors it was found that DHFR encoded mutations which were responsible for the MTX resistance. Studies of a highly MTX-resistant subline of the HCT-8 human colon carcinoma cell line showed that DHFR possessed a phenylalanine to serine mutation at residue 313–5 . Kinetic studies further showed that the Ki for MTX was 100-fold higher than wild type DHFR while a mutation of phenylalanine 34 to serine resulted in over 20,000-fold increase in the Ki for MTX. Similar changes in Ki were noted for trimetrexate as well.

Oligonucleotide-directed mutagenesis has been used by many laboratories to obtain information regarding the significance of the structural features of various enzymes. When an amino acid residue, or group of residues, is deleted from the enzyme structure, or is replaced by other residues by directed mutagenesis, inferences can be drawn regarding the relation of that portion of the structure to the catalytic activity of the enzyme. This approach has been applied extensively to the study of dihydrofolate reductase (DHFR) by Benkovic and his collaborators.

Dihydrofolate reductase (DHFR) catalyzes NADPH-dependent reduction of 7,8-dihydrofolate (H2folate) into 5,6,7,8-tetrahydrofolate (H4folate), which is essential for the synthesis of thymidylate and hence the synthesis of DNA. Since rapidly growing cells can be killed by inhibition of hDHFR by methotrexate (MTX), this and other inhibitory drugs are used in the chemotherapy of cancer. A serious problem in such chemotherapy with MTX is development of MTX-resistance in tumor cells. Spontaneous mutation in the DHFR gene is suspected to be one of the causes for such resistance.

Fluorimetric titration has been used to measure the dissociation constants for the complexes of folate, pteroyltriglutamate and pteroylheptaglutamate with dihydrofolate reductase purified from Lactobacillus casei, Streptococcus faecium (isoenzyme 2) and bovine liver. Effects of pH, temperature, salt concentration and second ligands have been examined. The method is shown to be unsuitable for methotrexate complexes.

The considerable cytotoxicity of 2-chloro-2⌂-deoxyadenosine (2-CldAdo) and 2-bromo-2′-deoxyadenosine (2-BrdAdo) is accounted for by their relatively high resistance to adenosine deaminase (1) and consequently more prolonged effect on cell metabolism compared with that of 2′-deoxyadenosine (dAdo) unless the latter is protected by an inhibitor of the deaminase like EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine]. Significant therapeutic activity for mice with L1210 leukemia has been reported for both 2-CldAdo (2) and 2-BrdAdo (3), and in a phase I clinical trial in patients with intractable malignancies 2-CldAdo had significant antileukemic activity (4).