Nucleic acid bulges have been implicated in a number of biological processes, including TAR (transactivation response element) RNA function in HIV AIDS and as intermediates in the unstable expansion of nucleotide repeats due to DNA strand slippage in at least 12 human neurodegenerative genetic diseases, including Huntington's disease and fragile X syndrome, and certain cancers, especially colon. The goal is to design and synthesize spirocyclic bulge-specific, wedge-shaped molecules, based on earlier work with the enediyne antitumor antibiotic neocarzinostatin that modulate these processes. These agents have been found to promote DNA strand slippage synthesis in simple model systems; their structures will be modified to optimize this process and then derivatized to generate alkylating and cleaving species that interfere with the expansion. Mechanistic studies will be undertaken to determine the molecular and structural basis for this action, and the model systems will be modified to more closely resemble the disease situation. Mammalian cell culture systems, developed as Huntington's disease models, will be used to test the effect of these agents. The use of small synthetic molecules to modify gene replication and expression represents a novel way of treating """"""""gain-of-function"""""""" genetic diseases. Solution structures of relevant drug/nucleic acid complexes will be elucidated by high resolution NMR spectroscopy. Selection from RNA diversity libraries (SELEX) will be used to identify the structural parameters for optimal RNA bulge binding. Enediyne antibiotics have been used recently in the treatment of relapsed acute myelogenous leukemia. One of the most potent members of this family of agents, C-1027, forms interstrand crosslinks involving the deoxyribose moieties of opposite DNA strands, especially under anaerobic conditions. This lesion, postulated to account for cell killing in oxygen-poor centers of solid cancers, will be isolated and its unique chemical structure determined. The conformational changes induced by this lesion in duplex DNA will be analyzed by NMR. These studies will provide insight into its action and offer a basis for the design of more effective drugs. ? ?
