DNA topoisomerases (top1 & top2) are the targets for some of the most effective 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, iii) discover novel topoisomerase inhibitors, and iv) elucidate the function of mitochondrial topoisomerase I. Goal 1: To elucidate the molecular interactions between topoisomerase inhibitors and their target enzyme-DNA complexes, and between topoisomerases and damaged DNA, we have studied topoisomerase-mediated cleavage of oligonucleotides containing site-specific modification, such as a single polycyclic aromatic adduct that mimics a topoisomerase inhibitor. We found that intercalation at the sites of topoisomerase cleavage mimics the effect of camptothecin with topoisomerase I and of intercalating anticancer drugs in the case of topoisomerase II. We have also directly determined the structural effects of DNA modifications (oxative lesions and cytosine arabinoside incorporation) that trap topoisomerase I by co-crystallization studies. 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. Goal 2: To elucidate the molecular pathways that respond to topoisomerase-mediated DNA damage, we have continued our studies with the 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. We have also demonstrated for the first time that Tdp1 is associated with XRCC1, the scaffolding protein in the BER (Break-Induced Repair) pathway, and that cells deficient for XRCC1 are selectively hypersensitive to camptothecin. We have also continued using microarray technology to elucidate the cellular pathways that determine cellular response and more importantly, cellular killing or resistance to topoisomerase inhibitors. We found that the transcriptional response to camptothecin affects RNAs that drive cell cycle arrest or apoptosis, depending on the dose of drug and survival outcome. We have also found that the effector pathways for programmed cell death (apoptosis) are markedly attenuated in prostate cancer cells that are resistant to camptothecins, while some cell growth/apoptosis pathways are activated. This finding led to the """"""""permissive apoptosis model"""""""", i.e. that turning off effector apoptosis genes allows the upregulation of growth activator genes, which otherwise would induce apoptosis. 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. We have also studied the cellular pharmacology of homocamptothecins, a novel series of inhibitors that differ from camptothecin by the presence of a stable seven-membered ring instead of the six-membered ring of camptothecins, which leads to their inactivation by conversion to carboxylates. Secondly, we have continued our studies on the indenoisoquinolines that we discovered in collaboration with Drs Cushman. We recently obtained co-crystals of one of the indolocarbazoles bound to the topoisomerase I-DNA complex. We now have more potent top1 poisons that are being investigated for pre-clinical development. Goal 4: We discovered human mitochondrial topoisomerase I, a specific enzyme encoded by a nuclear gene. We have now found the presence of homologs in all vertebrate genomes sequenced: mouse, rat, chicken, and zebra fish. However, the gene is absent in non-vertebrate including the Ciona intestinalis, yeast and plants. We are currently attempting to knock out the gene in mice.
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