Human O6-alkylguanine-DNA alkyltransferase (hAGT) is a major cause of resistance of tumor cells to killing by certain therapeutic methylating and chloroethylating agents. Inactivation of hAGT by O6-benzylguanine (BG) has been shown to sensitize human tumors to these alkylating agents in cell culture and to improve the therapeutic index against human tumor xenografts in nude mice. Clinical trials with BG indicate that this agent will be successful only in limited situations due to the lack of adequate potency and tumor specificity and the facile production of mutants of hAGT resistant to BG. Therefore, more potent and specific inhibitors are needed. The proposal has 3 specific aims to develop such compounds: (1) To understand the mechanism of the inactivation of hAGT by 2-amino- O4-benzylpteridine derivatives. We have shown that some of these compounds including O4-benzylfolic acid (BF) are potent inactivators of hAGT. In this aim, molecular modelling, crystallography, site-directed mutagenesis and powerful selection techniques to obtain informative mutants will be used to understand the basis for the improved activity of these compounds over BG, the reason that the inactivation is interfered with by the presence of DNA and to improve design of this class of compounds. (2) To investigate the relationship between folate transport and the ability of BF to inactivate cellular AGT and overcome AGT-mediated resistance to alkylating agents. These studies will follow up our preliminary studies that have shown that BF is much more effective at inactivating AGT in tumor cell lines that have high levels of folate transport. (3) Preliminary results also indicate that other folate derivatives of BG or O6-benzyl-2'-deoxyguanosine and oligodeoxynucleotides containing multiple BG residues also have properties that would provide improved AGT inhibitors. The ultimate goal of this aim is that by further study and appropriate modifications of these compounds, AGT inactivators can be made that fill the desired goals of increased solubility, increased potency against BG resistant mutants and tumor specificity. The development of resistance to anticancer agents is a major factor in limiting the success of these drugs. These experiments provide a means to reverse a well know source of resistance to alkylating agents that are used in such therapy. As such, they will increase the likelihood that these agents will produce therapeutic responses. ? ? ?
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