# STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE

> **NIH NIH R01** · ST. JUDE CHILDREN'S RESEARCH HOSPITAL · 1987 · $124,247

## Abstract

The objective is to study the structure of the enzyme dihydrofolate
reductase (DHFR) which is the target for antifolates used in the treatment
of cancer (e.g., methotrexate, MTX; trimetrexate; etc.), of bacterial
infections (trimethoprim) and parasitic infections (pyrimethamine).  Much
of the proposed work is related to study of how the structural elements of
the enzyme active site interact with the inhibitors, so as to result in
very tight binding of the latter.  Stopped-flow spectrophotometry and
fluorimetry will be used to continue measurements of rate constants for
inhibitor binding to and release from DHFR from Streptococcus faecium (two
isoenzymes, SFDHFR 1 and 2), Lactobacillus casei, Escherichia coli, bovine
liver, chicken liver and WIL2 human lymphoblasts.  These investigations
will also provide information on isomerizations of the initial inhibitor
complexes involving conformational changes that are critical in increasing
binding.  Information on these isomerizations will also be obtained by
studying the kinetics of development of inhibition and changes in circular
doichroism.  Binding of folate, dihydrofolate and 5-deazafolate and
isomerizations of their complexes will be similarly studied.  The effect
that the presence of nucleotide (NADPH or its analogs) in the catalytic
site on these processes will also be studied.  Additional information on
the interrelation of structural features and ligand binding and
conformational changes of complexes will be sought by creating mutants of
wild-type DHFR by oligonucleotide-directed mutagenesis.  Mutants of E. coli
DHFR with replacement of the active site Asp by Asn or Ser are available
and will be used in the above studies.  Mutants of SFDHFR 1 and 2 will be
constructed and used to identify structural features affecting catalytic
efficiency and substrate specificity.  Other mutants will be used to
enhance interpretation of 13C spectra.  13C labeled folates and inhibitors
will also be synthesized to provide information on the mechanism of
catalysis and of inhibitor binding.  There will also be a search for
variant DHFR in tumors of patients who relapse on MTX therapy.

## Key facts

- **NIH application ID:** 3170027
- **Project number:** 5R01CA031922-07
- **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:** 1987
- **Award amount:** $124,247
- **Award type:** 5
- **Project period:** 1981-09-01 → 1991-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/3170027

## Citation

> US National Institutes of Health, RePORTER application 3170027, STRUCTURAL STUDIES ON DIHYDROFOLATE REDUCTASE (5R01CA031922-07). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/3170027. Licensed CC0.

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