Dihydrofolate reductase (DHFR) is the target for """"""""antifolate"""""""" drugs that are clinically useful in treatment of cancer, malaria, and bacterial infections. Efficacy of such treatment of protozoal and bacterial infections has been decreased by the appearance of resistant organisms having variant DHFRs, for at least some of which the dissociation constant (K/d) for the inhibitor is greatly increased compared with wild-type DHFR (wt) while K/m for dihydrofolate (H2folate), and k(cat) are much less affected. Similar resistant forms of DHFR have also been purified from mammalian cells exposed to antifolates in culture. All known mutations conferring resistance on DHFR from any species occur at one of eight sites in the protein sequence, but the effect of only one of a few mutations has been explored at most of these sites. In this project we will explore the effect of other mutations at these sites. Specifically, we will examine in human DHFR (hDHFR) affects of such mutations on sensitivity to methotrexate (MTX). The cassette replacement method will be used to introduce mutations into hDHFR cDNA in a high expression vector that we have already used for the expression of wt hDHFR. Mutant hDHFRs that are highly resistant to MTX will be extensively characterized with respect to kinetics and thermodynamics of the binding of substrates, products and MTX, the kinetics of catalysis, and kinetics and thermodynamics of protein stability. Collaborative arrangements have been made for X-ray crystallography of complexes of these mutants. We will also investigate whether any inhibitors other than MTX bind tightly to the modified active site of these mutant hDHFRs. In another part of the project, we will examine hDHFR from leukemic cells from pediatric patients who have relapsed on standard therapy that includes repeated treatment with high dose MTX. Reverse transcription from mRNA, followed by PCR from the single strand cDNA, will be used to prepare cDNA for hDHFR and this will be cloned into a high expression vector. the hDHFR obtained will be examined for inhibition by MTX. If enzyme with high Ki is found the mutation responsible will be studied by the methods previously indicated.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Biochemistry Study Section (BIO)
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St. Jude Children's Research Hospital
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Bunting, K D; Flynn, K J; Riberdy, J M et al. (1999) Virus-specific immunity after gene therapy in a murine model of severe combined immunodeficiency. Proc Natl Acad Sci U S A 96:232-7
Sorrentino, B P; Allay, J A; Blakley, R L (1999) In vivo selection of hematopoietic stem cells transduced with DHFR-expressing retroviral vectors. Prog Exp Tumor Res 36:143-61
Bunting, K D; Galipeau, J; Topham, D et al. (1999) Effects of retroviral-mediated MDR1 expression on hematopoietic stem cell self-renewal and differentiation in culture. Ann N Y Acad Sci 872:125-40;discussion 140-1
Mareya, S M; Sorrentino, B P; Blakley, R L (1998) Protection of CCRF-CEM human lymphoid cells from antifolates by retroviral gene transfer of variants of murine dihydrofolate reductase. Cancer Gene Ther 5:225-35
Cody, V; Galitsky, N; Luft, J R et al. (1998) Comparison of ternary crystal complexes of F31 variants of human dihydrofolate reductase with NADPH and a classical antitumor furopyrimidine. Anticancer Drug Des 13:307-15
Allay, J A; Galipeau, J; Blakley, R L et al. (1998) Retroviral vectors containing a variant dihydrofolate reductase gene for drug protection and in vivo selection of hematopoietic cells. Stem Cells 16 Suppl 1:223-33
Allay, J A; Persons, D A; Galipeau, J et al. (1998) In vivo selection of retrovirally transduced hematopoietic stem cells. Nat Med 4:1136-43
Allay, J A; Spencer, H T; Wilkinson, S L et al. (1997) Sensitization of hematopoietic stem and progenitor cells to trimetrexate using nucleoside transport inhibitors. Blood 90:3546-54
Cody, V; Galitsky, N; Luft, J R et al. (1997) Comparison of two independent crystal structures of human dihydrofolate reductase ternary complexes reduced with nicotinamide adenine dinucleotide phosphate and the very tight-binding inhibitor PT523. Biochemistry 36:13897-903
Patel, M; Sleep, S E; Lewis, W S et al. (1997) Comparison of the protection of cells from antifolates by transduced human dihydrofolate reductase mutants. Hum Gene Ther 8:2069-77

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