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 and have recently been approved by the FDA 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 function of mitochondrial topoisomerase I (Top1mt).We have provided further evidence that topoisomerase I (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 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 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.We have pursued our investigations to discover novel Top1 inhibitors to alleviate the limitations of camptothecins while retaining their potent antitumor activity. 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. The indenoisoquinolines have been selected for clinical development by the NCI. Four derivatives are in the development pipeline of DTP with the goal of making the indenoisoquinolines the first NCI-discovered drugs in the Phase 0 pipeline.In spite of the same induction of Top1 cleavage complexes in cancer and normal tissues, Top1 inhibitors exhibit some selectivity for cancer tissues. One of our goal is to determine the molecular determinants that account for this selectivity.
We aim to translate this knowledge to the clinic by developing novel therapeutic approaches that increase the selectivity of Top1 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.
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