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.