DIYNENE ANTIBIOTICS AND THEIR DNA CLEAVAGE CHEMISTRY

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The neocarzinostatin chromophore, calicheamicin lambda 1 I, esperamicin A1, and dynemicin A1 are representative members of the growing class of diynene antitumor antibiotics. Each of these compounds is capable of causing single- and double-strand DNA cleavages at very low concentrations (nM) in the presence of thiols and molecular oxygen. The in vitro and in vivo activity of these compounds has given hope that, through preferential uptake or selective delivery to cancer cells, for example, by the agency of a monoclonal antibody, effective therapies might be possible. Several such strategies are currently under investigation, and at least one is in Phase II clinical trials at present. More broadly, the successful design of sequence-selective DNA cleaving agents based on a thorough understanding of effective natural products is of enormous potential utility in molecular biology. Described in this new application is a set of diverse but interlocking experimental approaches to a detailed understanding of the thiol activation, sequence recognition, binding and reaction of calicheamicin with DNA. Calicheamicin is striking among the diynene antitumor antibiotics for its high potency, its high sequence selectively and its propensity for double-strand scissions. The cleavage preferences of the drug will be surveyed for an evolutionary broad group of DNA's. These preferred sites will be examined in greater detail in synthetic oligonucleotides using a variety of structural and kinetic techniques. Atom transfer experiments will secure the identities of hydrogens lost from the DNA backbone and reveal unambiguously the orientation of the drug in the minor groove. These findings will be compared with results from hydroxyl radical footprinting experiments and careful analyses of the mobilities of the cleavage fragments on high resolution gel electrophoresis. The DNA fragments will also be characterized by chemical and spectroscopic means to establish the kinetics of the various steps of drug activation and DNA cleavage will be examined to determining the overall dynamics of the process. Finally, NMR experiments are proposed to give more refined structural information aided by molecular modeling.