This laboratory is committed to increasing our understanding of the molecular basis of action of new antitumor antibiotics that interact with DNA. Experience for almost 35 years has shown that this basic approach leads to the discovery of novel mechanisms of DNA damage and repair what have important implications for cancer cell toxicity and mutagenesis. A major aspect of this endeavor is the development of these agents as useful biochemical tools in the study of nucleic acid structure and function. This work has led to the identification of a new and expanding chemical and mechanistic class of potent antitumor agents with neocarzinostatin as the prototype, having the unprecedented enediyne structure, that are converted into diradical DNA damaging agents. It is proposed to: (1) investigate unusual nucleic acid structures, such as bulges, hairpins, mismatches, DNA- RNA hybrids, as targets for neocarzinostatin chromophore (NCS-Chrom); (2) elucidate the structure of the noncovalent, sequence-specific complex formed between drug and DNA by 1H NMR and X-ray crystallography, (3) characterize the unprecedented NCS-Chrom-DNA covalent adduct formed on the deoxyribose of DNA in terms of structure and effects on transcription and replication; (4) elucidate drug activation mechanisms, not involving thiol, including the role of bulged DNA as an effector molecule; (5) study the transfer of deuterium from C-4' at the T of the target sequence AGT-ACT into the C-2 radical site of NCS-Chrom; (6) identify drug-induced DNA deoxyribose damage intermediates resulting from hydrogen atom abstraction at C-4' of the sugar and leading to the formation of 3'-phosphoglycolate ends; (7) determine the structural basis of the resistance of bistranded DNA lesions containing abasic sites to AP endonucleases; and (8) study the mechanism of action of the new and highly potent enediyne antitumor antibiotic, C-1027, and synthetic dynemicin analogues that work by free radical mechanisms. The above studies, in addition to those planned to further elucidate the mechanism whereby nitroaromatic and other radiation sensitizers substitute for dioxygen in enediyne-induced DNA oxidative damage, will hopefully provide mechanistic insights of value in the design of highly specific probes of nucleic acid structures, which may be targets for gene modification and regulation.
