DNA topoisomerases (top1 & top2) are important targets for anticancer therapeutics. The top2 inhibitors, etoposide and DNA intercalators (such as adriamycin and derivatives) are the most commonly used anticancer drugs today. Camptothecins are specific top1 poisons and have recently been approved by the FDA for the treatment of human carcinomas resistant to prior chemotherapy. The goals of this project are: i) to elucidate the molecular interactions between topoisomerase inhibitors and their target enzymes, ii) to elucidate the molecular pathways that respond to topoisomerase-mediated DNA damage and contribute to the selectivity of topoisomerase inhibitors in cancer cells, and iii) discover novel topoisomerase inhibitors.Goal 1: To elucidate the molecular interactions between topoisomerase inhibitors and their target enzymes, we have set up a baculovirus expression system for high expression of recombinant top1. We have used this top1 enzyme with oligonucleotides containing a single polycyclic aromatic adduct that mimics a topoisomerase inhibitor, and found that intercalation at the site of top1 cleavage mimics the effect of camptothecin. Based on molecular modeling and crystal structure data, we proposed that polycyclic aromatics intercalate in the DNA and stabilize an intermediate in which a DNA base is flipped out of the DNA duplex. A second approach to elucidate the drug binding sites has been to identify top1 mutations that selectively confer camptothecin resistance. Analysis of camptothecin-resistant human prostate carcinoma cell lines (DU145/RC.1 & 1) demonstrated that amino acid residue 364 of top1 is important for camptothecin activity (interaction with top1?). In these cells, mutation of arginine 364 to histidine confers high resistance to camptothecin both with the mutated recombinant top1 enzyme and in cells.Goal 2: To elucidate the molecular pathways that respond to topoisomerase-mediated DNA damage, we have initiated studies with a newly discovered enzyme, tyrosyl-DNA-phosphodiesterase (TDP-1) that selectively removes the tyrosyl residue bound at the 3-end of the DNA. In collaboration with Dr. Grandas (University of Barcelona) and Dr. Nash (NIH), we found that the activity of TDP-1 is optimum when the top1 peptide is short and when it is linked to a long DNA oligonucleotide. This suggests that the catalytic site of TDP-1 interacts both with the DNA and a short peptide segment. These findings underline the potential importance of top1 proteolysis prior to TDP-1 action in cells.Goal 3: We have pursued our investigations for the discovery and molecular pharmacology investigations of novel topoisomerase I inhibitors. First, in the areas of camptothecins, we have identified novel camptothecins with enhanced stability in the bloodstream and which should be useful clinical candidates. We have also discovered in collaboration with Dr. Gamcsik (Duke University) and Dr. Wall (Research Triangle Institute) new camptothecin-peptide conjugates (glutathione bound to position 7 of camptothecin) that produce remarkably stable top1 cleavage complexes. These compounds have been patented because they can be used to specifically deliver drugs to the tumor cells. Secondly, we have continued our studies on the indenoisoquinolines that we discovered in collaboration with Drs Cushman. We now have more potent top1 poisons that are being investigated for pre-clinical development. 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 indolocarbazoles using cell lines with top1 alterations that should confer drug resistance.
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