# STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

> **NIH NIH R01** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 1993 · $231,137

## Abstract

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.

## Key facts

- **NIH application ID:** 3170031
- **Project number:** 5R01CA031922-12
- **Recipient organization:** ST. JUDE CHILDREN'S RESEARCH HOSPITAL
- **SAM.gov UEI:** JL4JHE9SDRR3
- **Principal Investigator:** RAYMOND L BLAKLEY
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 1993
- **Award amount:** $231,137
- **Award type:** 5
- **Project period:** 1981-09-01 → 1996-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3170031

## Citation

> US National Institutes of Health, RePORTER application 3170031, STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE (5R01CA031922-12). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3170031. Licensed CC0.

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