Methotrexate-resistant Variants of Human Dihydrofolate Reductase with Substitutions of Leucine 22

Abstract

Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (kcat) at pH 7.65 (except for the arginine 22 variant), but Km for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.

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“…The key interactions involved in binding are summarised in Table 13 with reference to ligand 1ohk and Table 12 shows the corresponding features present in each ligand [33][34][35][36][37]. The target pharmacophore consists of D1, A1, H1, and H2.…”

Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells

“…Alternative dominant selectable markers that are well-defined and widely utilized comprise the mutant variants of the human dihydrofolate reductase (DHFR) gene which maintain a role in folate metabolism when introduced into cells, but confer drug resistance against the cytotoxicity of antifolates, such as methotrexate and trimetrexate. 44,45 Several investigators have successfully accomplished in vitro drug selection of mutant DHFRtransduced cells, 7 including MSCs. 6,7 However, despite selection in saturating concentrations of antifolate drug in vitro, up to 20% of primary untransduced MSCs will survive, thereby somewhat limiting the ability to remove contaminating, non-engineered cells.…”

Increased resistance to nitrogen mustards and antifolates following in vitro selection of murine fibroblasts and primary hematopoietic cells transduced with a bicistronic retroviral vector expressing the rat glutathione S-transferase A3 and a mutant dihydrofolate reductase

“…Mutations at amino acids 22 and 31 of DHFR, two critical sites for substrate binding, have produced variants that are highly resistant to the folate analogs MTX and TMTX. 57,58 These mutants were shown to protect HSCs in vivo against high doses of antifolate drugs. 5,6 Enrichment of DHFR-transduced hematopoietic cells with antifolate treatment was demonstrated both in vitro [34][35][36] and in vivo.…”