Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance

Abstract

Methotrexate is a slow, tight-binding, competitive inhibitor of human dihydrofolate reductase (hDHFR), an enzyme that provides key metabolites for nucleotide biosynthesis. In an effort to better characterize ligand binding in drug resistance, we have previously engineered hDHFR variant F31R/Q35E. This variant displays a >650-fold decrease in methotrexate affinity, while maintaining catalytic activity comparable to the native enzyme. To elucidate the molecular basis of decreased methotrexate affinity in the doubly substituted variant, we determined kinetic and inhibitory parameters for the simple variants F31R and Q35E. This demonstrated that the important decrease of methotrexate affinity in variant F31R/Q35E is a result of synergistic effects of the combined substitutions. To better understand the structural cause of this synergy, we obtained the crystal structure of hDHFR variant F31R/Q35E complexed with methotrexate at 1.7-Å resolution. The mutated residue Arg-31 was observed in multiple conformers. In addition, seven native active-site residues were observed in more than one conformation, which is not characteristic of the wild-type enzyme. This suggests that increased residue disorder underlies the observed methotrexate resistance. We observe a considerable loss of van der Waals and polar contacts with the p-aminobenzoic acid and glutamate moieties. The multiple conformers of Arg-31 further suggest that the amino acid substitutions may decrease the isomerization step required for tight binding of methotrexate. Molecular docking with folate corroborates this hypothesis.

Selectively weakened binding of methotrexate by human dihydrofolate reductase allows rapid ex vivo selection of mammalian cells

“…Variants of region 31/34/35 are in filled symbols while variants of position 115 are empty symbols. Point substituents F31R and Q35E (included in the doubly-substituted F31R/Q35E variant) were previously created as controls rather than selected (Volpato et al, 2009). The diagonal line indicates a theoretical 1:1 correlation as a reference.…”

“…Specifically, we propose that high selectivity between the substrate and the inhibitor may provide advantageous properties for cellular selection. We have recently reported a number of point mutants and multiply-mutated variants of human hDHFR that were identified in a simple bacterial selection assay (Volpato et al, 2007;Fossati et al, 2008;Volpato et al, 2009). The assay requires that the variants concomitantly provide substrate binding and turnover along with weakened inhibitor binding, in addition to the usual requisite properties such as good expression and stable folding.…”

Chapter 7: Pharmacogenomics

“…Dihydrofolate typically fits into DHFR in a known conformation (Figure 1C), but a phenylalanine to arginine mutation changes this binding conformation (Figure 1D–E). This mutation is hypothesized to confer methotrexate resistance in individuals with this variant [8].…”

“…These variants have decreased affinity to methotrexate, relative to dihydrofolate. Reprinted with permission from [8].…”

Microwave Assisted Synthesis of Pyrimidines in Ionic Liquid and Their Potency as Non‐Classical Malarial Antifolates

“…As an answer of the resistance adapted by the parasite and failure of the current chemotherapeutics, new targets which are employed in the crucial biochemical pathways in the parasite must be identified and besieged. Many strategies are being executed to accomplish the aim for development of target‐based antimalarial drug molecules, one of them is to develop antifolates targeting Plasmodium falciparum dihydrofolate reductase ( Pf ‐DHFR) . The enzyme catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent reduction of 7,8‐dihydrofolate (DHF) to 5,6,7,8‐tetrahydrofolate (THF), a fundamental cofactor in the synthesis of some amino acids and nucleotides, such as thymidylate .…”