DNA topoisomerases (top1 & top2) are the targets for some of the most effective anticancer therapeutics. The most commonly used top2 inhibitors in cancer chemotherapy are etoposide (VP-16) and DNA intercalators (such as adriamycin and derivatives). Camptothecin derivatives are specific top1 poisons recently approved by the FDA for the treatment of colon, ovarian and lung cancers. Our research is focused on: i) elucidation of the molecular interactions between topoisomerase inhibitors and their target enzymes, ii) elucidation of the molecular response pathways to topoisomerase-mediated DNA damage, which contribute to the selectivity of topoisomerase inhibitors in cancer cells, and iii) discovery of novel topoisomerase inhibitors. Goal 1: To elucidate the molecular interactions between top1 and its inhibitors, 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 needs to be at the site of the top1 cleavage to mimic the effect of camptothecin. Based on molecular modeling and crystal structure data, we proposed that polycyclic aromatics trap top1 by stabilizing an intermediate in which the DNA base immediately 3'-from the top1-mediated cleavage site (position +1) is misplaced (flipped out of the DNA duplex) and therefore cannot be religated. 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/RC0.1 & 1) demonstrated that mutation of arginine 364 to histidine in top1 confers resistance to camptothecin activity. In the crystal structure, arginine 364 is proximal to the DNA cleavage site, which is consistent with camptothecin binding at the enzyme-DNA interface. A third approach to identify top1-drug interactions has been to perform structure-activity relationship studies with camptothecin derivatives, including the novel homocamptothecins, which possess a 6-membered E-ring. We are also performing structure-activity studies with a novel top1 inhibitor family, the indenoisoquinolines, for which we hold a joined patent with Dr. Mark Cushman (Purdue University). Similarly, to elucidate the molecular interactions between top2 and its inhibitors, we have used recombinant top2 enzyme and oligonucleotides containing a single polycyclic aromatic adduct that mimics a top2 inhibitor, and found that intercalation needs to be at the site of the top2 cleavage to mimic the drug effects. Based on molecular modeling and crystal structure data, we propose that inhibitors trap top2 by binding within the top2-mediated cleavage site (between positions -1 and +1), and interfere with the religation of the -1 base. We have also studied top2 enzymes with defined mutations in the alpha-4 helix and found that such mutations have dramatic effects on drug activity. Since the alpha-4-helix is in close proximity to the potential DNA binding site in the top2 enzyme, these observations are consistent with drug intercalation with the DNA cleavage site both for the top1 and top2 inhibitors. Goal 2: We recently reported that the cytoxicity of top1 cleavage complexes results from the collisions of replication forks with the top1 cleavage complexes. These collisions produce replication-mediated DNA double-strand breaks. To elucidate the molecular pathways downstream from top1-mediated DNA damage, we have performed 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. Ongoing studies in DNA repair-deficient cells (XRCC1- and poly[ADPribose] polymerase-deficient cells) indicate deficiency of Tdp-1 activity in these cells. These findings underline the potential importance of TDP-1 for cellular response to top1 poisons. To further investigate the repair and checkpoint pathways elicited by top1-induced replication-mediated DNA double-strand breaks, we have used several new approaches. First, we have used cDNA microarrays to analyze the transcriptional response pathways to camptothecins in p53 wild-type and p53 knockout cells. 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. Secondly, we are studying chromatin responses to top1-induced replication-mediated DNA double-strand breaks. We are finding that camptothecin-induced replication-mediated double-strand breaks induce the rapid phosphorylation of histone H2X, and that phosphorylated H2X forms nuclear foci that co-localize with DNA repair and checkpoint proteins (Mre11, Rad50, Nbs1). These finding demonstrate for the first time that replication-mediated DNA double-strand breaks induce ATR- and DNA-PK-dependent phosphorylation of H2AX, and that chromatin changes appear to be associated with repair of top1 cleavage complexes. Goal 3: We have pursued our investigations for the discovery and molecular pharmacology of novel topoisomerase I inhibitors. First, in the areas of camptothecins, we have 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 Dr. Mark 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. A CRADA is being negotiated with Enzon to formulate MJ-III-65 for preclinical development.
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