Clinical trials with O6-benzylguanine (b6G) are in progress. The success of this therapy may be compromised by: (a) the facile production of mutants of hAGT resistant to b6G; (b) the lack of adequate potency and tumor specificity and (c) the enhanced toxicity of alkylating agent therapy when AGT inhibitors are used. Experiments are proposed to address these issues building on advances made during the previous period of support which include: (i) development of a selection method for b6G-resistant mutants; (ii) studies showing that these mutants can protect mammalian cells from alkylating agents in the presence of b6G, (iii) derivation of the crystal structure of the hAGT protein; and (iv) indications that short oligodeoxynucleotides containing b6G are extremely potent inactivators of hAGT. The proposed research project has three specific aims. The first specific aim is to characterize the interaction between hAGT and b6G and oligos containing b6G. Studies in this aim will test and refine the current models for binding of b6G and related compounds to hAGT by comparing the inactivation by (and binding of) various b6G derivatives to hAGT mutant proteins in which residues thought to be involved in binding are altered. It is also planned to use various mutants of hAGT such as C145S and inhibitors to obtain crystal structures of complexes with hAGT showing how binding occurs. The second specific aim will obtain a full understanding of hAGT mutants with altered sensitivity to b6G and other inhibitors. Many point mutations (and at least one naturally occurring polymorphic variant) in hAGT render the protein resistant to inactivation by b6G. Such mutant forms of hAGT may create problems in the therapeutic use of b6G but also provide an opportunity to protect normal cells such as bone marrow by gene therapy approaches in which a mutant form is expressed. These studies will provide a detailed knowledge of the sites in hAGT protein at which resistant mutants can occur, and will study the stability and activity of these mutants when expressed in cells. Furthermore, the studies in this specific aim are aimed at isolating mutants more sensitive to inactivation by b6G. Finally, studies in Specific Aim 3 will study the inactivation of hAGT and sensitization of cells to alkylating agents brought about by novel agents inactivating hAGT. Studies will be carried out to develop second generation agents and will develop leads obtained in the current grant support period which include: short oligos containing b6G, pro-drugs generating b6G, and better knowledge of the binding pocket into which the target base is flipped by the hAGT.
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