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.
| Gerson, S L; Schupp, J; Liu, L et al. (1999) Leukocyte O6-alkylguanine-DNA alkyltransferase from human donors is uniformly sensitive to O6-benzylguanine. Clin Cancer Res 5:521-4 |
