| Most carcinogens and anticancer agents show little selectivity in their reactions with duplex DNA and generate a plethora of DNA lesions. This non-specificity makes it difficult to elucidate the biological role(s) of individual DNA lesions; i.e., cytotoxicity and/or mutagenicity. This problem is even more critical in cancer chemotherapy where there is mounting evidence that mutagenic DNA adducts are formed that pose a significant increase in risk for secondary malignancies. Moreover, the role and substrates for specific DNA repair proteins remain obscure due to presence of multiple adducts.;To address, this issue we have developed a new DNA alkylating agent, methyl-lexitropsin (Me-lex) that consists of a neutral lex dipeptide (based on N-methylpyrrole carboxamide subunits) appended with an O-methylsulfonate ester functionality. Using sequencing gels, HPLC and ELISA analyses it has been demonstrated that Me-lex equilibrium binds in the DNA minor groove at A•T rich sequences and generates almost exclusively (∼99%) 3-methyladenine (3-MeA) both in vitro and in vivo. To determine the toxicity of Me-lex and role of different DNA repair enzymes in repair of 3-MeA, studies of Me-lex were performed in wild type E. coli and mutants that were defective in different DNA repair enzymes. The results demonstrate that the toxicity of Me-lex is due to unrepaired 3-MeA, and that base excision repair is the sole repair system for detoxification of 3-MeA in E. coli. Mutagenesis studies done in wild type and base excision repair deficient yeast indicate that overall Me-lex is relatively non-mutagenic. Mutagenicity of Me-lex, i.e., 3-MeA, is attributed to subsequent formation of abasic sites rather than to 3-MeA. Moreover, the DNA minor groove binding ligand netropsin inhibits DNA alkylation both in vitro and in vivo, and significantly reduces the toxicity of Me-lex in E. coli. Thus, Me-lex because of its high cytotoxicity and low mutagenicity is a model for non-mutagenic anticancer agents. These studies have been extended to the ethyl analog of Me-Lex, Et-Lex. Et-lex shows a DNA alkylation profile that is consistent with sequence-specific binding, but with a reduced level of alkylation that is consistent with the decreased reactivity of the O-ethyl sulfonate ester functionality. |
