DNA topoisomerases (top1 and 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 determine the response to topoisomerase inhibitors in cancer cells, iii) discover novel topoisomerase inhibitors, and iv) elucidate the function of mitochondrial topoisomerase I.Goal 1: Our recent studies demonstrate that topoisomerase inhibitors are a paradigm for drug discovery as these drugs alter the binding of topoisomerases to DNA by binding at the enzyme-DNA interface when the topoisomerases form their transient DNA cleavage complex intermediates. We refer to this type of inhibition as """"""""interfacial inhibition"""""""" and propose this type of inhibition to be one of Nature's paradigms for drug discovery. This concept has profound implication for the discovery of inhibitors of macromolecular complexes that stabilize protein complexes (novel approach) rather than screening only for drugs that prevent the formation or dissociate protein complexes (past and current approach). We have determined the structures of several topoisomerase I-DNA complexes with single point mutations resulting in camptothecin resistance. These studies provide a rationale for the drug-resistance mutations. They also provide evidence for the validity of the enzyme-DNA structures to be used for molecular docking and rational drug discovery. To further elucidate the molecular interactions between topoisomerase inhibitors and their target enzyme-DNA complexes, 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 found that acetaldehyde adducts, which form readily during alcohol consumption can enhance camptothecin-induced topoisomerase I-DNA complexes.Goal 2: Enhanced drug efflux is a common resistance mechanism to therapy. Camptothecins are transported by the half transporter ABCG2, otherwise named BCRP or MXR. We have evaluated the implication of ABCG2 in the activity of camptothecins. We found the new camptothecin derivatives, the homocamptothecins to be more active than the camptothecins presently in clinical trials in ABCG2-overexpressing cell lines. We have also looked at differential expression of the ABC transporter in camptothecin-resistant cells. 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. We have demonstrated 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. Using recombinant Tdp-1 and modified oligonucleotides, we are looking for TDP-1 inhibitors to block the repair pathways downstream from topoisomerase I-mediated DNA damage in order to selectively enhance the activity of camptothecins in checkpoint-deficient cells.Goal 3: We have pursued our investigations for the discovery and molecular pharmacology investigations of novel topoisomerase I inhibitors. The indenoisoquinolines were discovered in collaboration with Dr. Cushman. The indenoisoquinolines have several potential advantages over camptothecins: 1/ they are chemically stable; 2/ they trap topoisomerase I cleavage complexes at specific genomic sites that differ from those trapped by camptothecins; 3/ their cellular half-life is much longer than camptothecins with cleavage complexes that are more stable than those trapped by camptothecins. 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 looking for mitochondrial DNA damage that may be induced by mitochondrial topoisomerase I, and attempting to knock out the gene in mice.
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