Topoisomerase I is an important target for the development of new chemotherapeutic agents for the treatment of cancer in humans. Although some members of the camptothecin class of topoisomerase I inhibitors are presently in clinical use as anticancer agents, the camptothecins suffer from a number of inherent limitations, including chemical and metabolic instability due to lactone ring opening, and rapid reversibility of topoisomerase I inhibition upon drug removal. Because of these limitations, effective chemotherapy with the camptothecins requires long I.V. infusions and prolonged and continuous exposure. Recent studies in our research group, in collaboration with others, have resulted in the synthesis of a new class of topoisomerase I inhibitors, the indenoisoquinolines. A combination of enzyme inhibition studies, in vitro cytotoxicity results in human cancer cell cultures, and in vivo animal studies have provided compelling evidence that the indenoisoquinolines will overcome some of the limitations of the camptothecins. One of the main goals of the presently proposed research program will be to design and synthesize more effective indenoisoquinolines as topoisomerase I inhibitors with potential clinical application in the treatment of cancer in humans. It can be assumed that the efficacy of the indenoisoquinoline topoisomerase I inhibitors can be maximized by taking advantage of the various chemical forces that are involved in stabilization of the ternary complexes formed from DNA, the enzyme, and the inhibitors. The forces responsible for the bonding of the drug to DNA bases in the ternary complex include charge-transfer complex formation, electrostatic attraction, and London dispersion forces. In addition to these forces, the ternary complexes are also stabilized through hydrogen bonding of the indenoisoquinolines to some of the nearby amino acid residues of the enzyme. One of the main goals of this research project will be to maximize each of these various forces in an array of new drug molecules using our knowledge of the crystal structures of the drug-enzyme-DNA ternary complexes. We presently have determined the crystal structures of complexes formed from two indenoisoquinolines, and we expect that additional crystal structures will become available through future efforts. The structures of the indenoisoquinolines will also be manipulated in order to target them to cancer cells, maximize favorable solubility properties, and increase their bioavailabilities. The new compounds prepared in this study will be evaluated in a number of biochemical and biological assays. These will include analysis of DNA cleavage patterns induced by topoisomerase I in the presence of the inhibitors, analysis of the stabilities of the ternary complexes, DNA unwinding studies to detect intercalation, inhibition of topoisomerase I-mediated DNA relaxation, detection of DNA strand breaks in cellular systems, and analysis of enzyme-inhibitory activities in camptothecin resistant mutant cell lines. The cytotoxicities of the indenoisoquinolines in cancer cell cultures will be determined, and promising candidates will be investigated for anticancer activity in a variety of animal model systems. The mechanism of action of the indenoisoquinolines will be studied in detail, and the information will be used to maximize their therapeutic potential as anticancer agents.
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