Mitomycin C is a clinically significant antineoplastic antibiotic having found wide use in combination with other agents for the treatment of patients with advanced breast cancer and to a lesser extent cervical and ovarian cancers. Information gleaned from previous studies have provided the basis of our current understanding of the mode of action of this drug in the absence of DNA. Recent investigations have also demonstrated that mitomycin C bonds to DNA in a sequence selective fashion. However, little is known of how the chemical machinery implanted in the drug is harnessed to .provide for this specificity. In this proposal, an integrated approach has been outlined for the further understanding of how the drug and receptor site function. The following issues are addressed: Do neighboring bases flanking the mitomycin guanine alkylation site play an important role in conferring site specificity for both the monoalkylation and the bisalkylation (cross-linking) process? Do specific interactions between mitomycin substituents and the DNA minor groove facilitate these transformations? Which DNA sites are most prone to alkylation? What are the functions of specific mitomycin mono- and bis-alkylated products? A series of sensitive monitoring techniques are introduced to accomplish these objectives. Four enzymatic assays (e.g., UVRABC nuclease, transcription, polymerase, lambda exo) are used with mitomycin modified genomic and synthetic DNAs to provide detailed information concerning the DNA-drug bonding events. The mode of action of this clinically significant agent will be further analyzed by high-field NMR studies and molecular modeling investigations on select mitomycin modified DNA complexes. Analysis of this data is expected to permit the determination of the key interactions that lead to the selective bonding of the drug to DNA. Knowledge gained from these collective experiments will serve as the molecular basis for the rational design of new DNA site specific mitomycin drug candidates.
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