DNA topoisomerases (top1 & top2) have been identified as essential targets for anticancer research. Etoposide and DNA intercalators are top2 inhibitors and are the most commonly used anticancer drugs today. Camptothecins are specific top1 poisons and have recently been introduced in the clinic. The goals of this project are: i) to elucidate the molecular interactions between topoisomerase inhibitors and their target enzymes and ii) to discover novel topoisomerase inhibitors.We have continued our investigations in the areas of camptothecins. First, we have identified novel camptothecins that are more stable in the bloodstream and which should be useful as clinical candidates. Secondly, we have characterized a human prostate carcinoma cell line and found a novel top1 mutation that confers camptothecin resistance. Interestingly, this residue is in close contact with the DNA, which is consistent with our model that camptothecins stack against the +1 base in the DNA cleavage site of the top1 cleavage complex.In the area of top2, we have determined that mutations in the alpha-4 region of top2 have profound impact on DNA cleavage activity and response to drugs. From these results, we infer that this alpha-4 helix is critical for interacting with the DNA duplex that is cleaved during the topoisomerization reaction (G strand). This same region also probably interacts with drugs, which is consistent with the binding of top2 poison at the enzyme-DNA interface.We have also been studying novel non-camptothecin top1 inhibitors. First, we have continued our studies on the indenoisoquinolines that we discovered in collaboration with Drs. Paull & Cushman. We now have more potent top1 poisons that are being investigated for pre-clinical development. Secondly, we have found that ecteinascidin 743 is a novel top1 poison. Ecteinascidin 743 is an extremely potent anticancer drug in clinical trials, which forms adducts in the DNA minor groove (guanine N2). Our recent results indicate that ecteinascidin 743 forms protein-linked DNA breaks in cells and that top1 is a target of the drug. By contrast to camptothecins, these breaks are located in specific regions of the genome and are very persistent after drug removal, which should make them highly cytotoxic. Finally, we have started investigations on indolocarbazole derivatives that are a new class of top1 inhibitors that will be introduced in the clinic soon. We are currently determining: i) the drug molecular interactions with top1 using various camptothecin-resistant top1 mutants in cells and in biochemical assays, and ii) whether top1 is the only target of indolocarbaxoles using cell lines with top1 alterations that should confer drug resistance. - breast cancer, Cell Cycle, chemotherapy, colorectal cancer, DNA binding proteins, DNA repair, Pharmacology, proteases,
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