INTERACTION OF BG AND RELATED COMPOUNDS WITH AGT

  • Pegg, Anthony (PI)
  • Pegg, Anthony (PI)
  • PEGG, ANTHONY (PI)
  • PEGG, ANTHONY (PI)
  • DOLAN, EILEEN (PI)
  • FRIEDMAN, HENRY (PI)
  • SCHOLD (PI)
  • Gerson, Stanton (PI)
  • DOLAN, EILEEN (PI)
  • FRIEDMAN, HENRY (PI)
  • SCHOLD (PI)
  • Gerson, Stanton (PI)
  • PEGG, ANTHONY (PI)
  • Dolan, M. Eileen (PI)
  • Friedman, Henry S. (PI)
  • Schold, S. (PI)
  • Gerson, Stanton (PI)

Project: Research project

Project Details

Description

Work carried out during the previous period of support has demonstrated
that BG acts as a substrate for AGT leading to the formation of S-
benzylcysteine at the active site and thus inactivating the protein. These
studies have also shown that mutants of AGT can be made that are resistant
to inactivation by BG. Future experiments will be carried out in
Laboratory Program 1 to assist in the design of improved inhibitors. In
order to do this, the experiments will provide a greater understanding of
the mechanism(s) responsible for the BG resistance of mutant AGT proteins
and investigate the interaction a d mechanism of action of AGT with
substrates and inhibitors. The detailed specific aims are: (1) To study
in detail mutations in AGT that lead to resistance to BG. The spectrum of
mutants in human AGT that can lead to resistance to BG will be identified
using the ability of the protein to protect E. coli from killing by MNNG in
the presence of BG as a screen for activity; (2) To design and test
inhibitors that inactivate BG-resistant AGTs. These studies will be
carried out with purified recombinant AGT and the BG-resistant G156A and
P140A AGT mutant proteins and with astrocytoma cells that express these
proteins. The ability of compounds active against these resistant AGTs to
enhance the killing of these cells by BCNU and temozolomide will also be
determined. Additional BG-resistant mutants arising from aim (1) will also
be tested as they become available; (3) To study the interaction of BG and
other known inactivators of AGT with the protein. The ability of the
control and BG-resistant human AGTs and the control and BG-sensitive AGT
with the protein. The ability of the control and BG-resistant human AGTs
and the control and BG-sensitive AdaC alkyltransferases to bind to low
molecular weight substrates including very short oligodeoxynucleotides
containing O6-methyl and O6-benzyl adducts will be determined. Kinetic
measurements of the interaction of the alkyltransferases and the inhibitors
will be made and the ability of the compounds to act as inhibitors of the
formation of [8=3H]guanine from [8-3H]BG will be used as an assay; (4) To
study the structure of AGT (and the related Ad-C alkyltransferase from E.
coli) and mutants with altered reactivity for BG and other inhibitors. The
crystal structure of the AGT protein in the presence and absence of
inhibitors and a DNA substrate will be determined. Some of these studies
will use an inactive C145A mutant AGT which binds substrates but cannot
react with them to form the S-alkylcysteine at the active site. Modeling
and direct determination of the structures of mutant AGT and Ada-C proteins
with altered ability to react with the inhibitors will be carried out.
StatusFinished
Effective start/end date1/1/019/29/01

Funding

  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute

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