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 commonly used anticancer drugs. Camptothecins are specific Top1 poisons approved for the treatment of colon and ovarian carcinomas. 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 functions of mitochondrial topoisomerase I (Top1mt). We have pursued our investigations to discover novel topoisomerase I (Top1) inhibitors to alleviate the limitations of camptothecins while retaining their potent antitumor activity. The indenoisoquinolines were discovered and pursued in collaboration with Dr. Cushman at Purdue University. The indenoisoquinolines have several potential advantages over camptothecins: 1/ they are chemically stable; 2/ they trap Top1 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 have continued to discover and characterize novel derivatives to optimize the indenoisoquinolines. As a result, three indenoisoquinolines (NSC 706744, 725776 and 724998) have been selected for clinical development by the NCI. Our goal is to make the indenoisoquinolines the first NCI-discovered drugs in the Phase 0/I pipeline using histone gamma-H2AX as a biomarker. We have provided further evidence that Top1 inhibitors are a paradigm for interfacial inhibitors. Crystal structure studies have now established that 5 different Top1 inhibitors (topotecan, natural camptothecin, an indenoisoquinoline, a norindenoisoquinoline and an indolocarbazole) all bind at the Top1-DNA interface when the Top1 forms its 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 Natures 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 Top1-DNA complexes with single point mutations resulting in camptothecin resistance. These studies provide molecular examples of structural alterations propagated from distal point mutants to enzyme active sites. They also provide evidence for the validity of the enzyme-DNA structures to be used for molecular docking and rational drug discovery, which we are applying to our indenoisoquinoline program. We have extended our studies on the induction of topoisomerase-DNA complexes by carcinogens and during apoptosis. We had previously reported that polycyclic aromatics (benzo[a]pyrene, benzo[c]phenanthrene) and formaldehyde (a bioproduct generated in humans from alcohol metabolism) were potent inducers of Top1 cleavage complexes. We have now shown that another carcinogen, 4-nitroquinoline-1-oxide (4-NQO), which is commonly used in DNA damage/repair studies also trap Top1 cleavage complexes in cells. Regarding the induction of topoisomerase cleavage complexes during apoptosis, we have demonstrated that the formation of Top1 cleavage complexes is a conserved and ubiquitous feature of apoptosis induced by a variety of anticancer drugs including Top2 inhibitors (etoposide) and tubulin inhibitors (paclitaxel, vinblastin). One of our goals is to determine the molecular determinants that account for the selectivity of topoisomerase inhibitors for cancer cells.
We aim to translate this knowledge to the clinic by developing novel therapeutic approaches that increase the selectivity of topoisomerase inhibitors for cancer tissues, and by providing molecular biomarkers to direct therapeutic choices and follow therapeutic responses to Top1 inhibitors. Our first approach is to study step by step (dissect) the molecular pathways activated by camptothecins and Top1 inhibitors. Our recent studies have implicated phosphorylation of BLM (on threonine 99) in association with phosphorylation of histone H2AX (gamma-H2AX). We are also finding phosphorylation of Chk2, which is conditional for presence of Mre11-Rad50-Nbs1 complexes. We are investigating the functional relevance of these molecular pathways in cells that are deficient for these pathways. Our second approach is to map the molecular interactions downstream from Top1 cleavage complexes using our molecular interaction map (MIM) methodology (see Report from Dr. Kurt W. Kohn). Third, we are developing (screening) new drugs against these Top1 response pathways (Chk2 and Tdp1 inhibitors). We are studying 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. The first mitochondrial topoisomerase, Top1mt, was discovered in our laboratory. Top1mt is encoded by a nuclear gene. We have found Top1mt in all vertebrate genomes sequenced: mouse, rat, chicken, and zebra fish. However, the gene is absent in non-vertebrate including yeast and plants. We have proposed that Top1mt arose by duplication of a common ancestral TOP1 gene (found today in simple chordates) during evolution of vertebrate. The other TOP1 gene encodes the previously known Top1 devoted to the nuclear genome. We have found that Top1mt is subjected to alternative splicing. We have also generated knockout mice and are studying their phenotype. To gain insight into the molecular function of Top1mt, we are developing tools to map Top1mt sites in mitochondrial DNA and looking for inhibitors of Top1mt
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