Human O6-alkylguanine-DNA alkyltransferase (hAGT) is a very important source of the resistance of cancer cells to some chemotherapeutic alkylating agents. AGT inhibitors such as O6-benzylguanine (BG) can be used to overcome this resistance. Several factors may limit the effectiveness of current AGT inhibitors in clinical trial including a lack of selectivity towards tumors and structural alterations in hAGT that may cause resistance to inhibitors. Several common polymorphisms of AGT are known in addition to the rare G160R variant, which we have previously shown to be resistant to BG. In attempts to generate more potent and tumor specific inactivators of AGT, we have synthesized a number of second-generation hAGT inhibitors that are folic acid derivatives i.e. O4-benzylfolic acid, the 3'-O and 5'-O folic acid γ esters of O6-benzyl-2'-deoxyguanosine and the folic acid γ ester of O6-[(4-hydroxymethyl)benzyl]guanine. We have tested these compounds and BG against wild type AGT and the comon variants L84F and I143V/K178R and the rarer variant W65C. The results showed that there was a significant increase in the ED50 for inactivation of the I143V/K178R variant by all of these compounds. There was little or no alteration in the inactivation of the L84F or W65C forms. Further investigation of the difference in inactivation between the wild type and the I143V/K178R variant showed that the effect was reduced but not eliminated in the presence of calf thymus DNA. The I143V and K178R polymorphism are in perfect linkage disequilibrium and always occur together. Tests of mutant AGTs generated in the laboratory with either the I143V or K178R mutation showed that the difference in inactivation by these inhibitors was due exclusively to the I143V change. Although it is not clear that the differences between the I143V/K178R variant and wild type AGT are sufficient to affect the clinical response, these results suggest that the possible occurrence of the I143V/K178R polymorphism in patients should be considered in clinical trials. Supported in part by grant CA-071976.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA