We have pursued our discovery and molecular pharmacology of novel topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors to alleviate the limitations of camptothecins, doxorubicin and etoposide while retaining their potent antitumor activity. The indenoisoquinolines have been discovered and pursued in collaboration with Dr. Cushman at Purdue University. We have now established that the indenoisoquinolines have several advantages over camptothecins: 1/ they are chemically stable and easy to synthesize and chemically optimize;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;4/ the Top1 cleavage complexes they produce are more stable than those trapped by camptothecins indicating a tight fit in the Top1-DNA cleavage complexes;5/ they are not substrates for the multidrug resistance efflux pumps (such as ABCB1 (Pgp), ABCG2 (Mrp/Bcrp) and ABCC1 (Mrp1). We have continued to discover and characterize novel derivatives to optimize the indenoisoquinolines. As a result, two indenoisoquinolines (NSC 725776 and 743400) have been selected for clinical development by the NCI. This drug development is a collaboration between several groups: LMP (our group and Dr. Bonner for gamma-H2AX biomarker), Clinical Oncology Branch (Dr. Doroshow and Shivaani Kummar for clinical trials), DTP and SAIC (Dr. Hollingshead, Dr. Parchment and Dr. Kinders for mouse models and pharmacodynamic biomarkers), and Purdue University (Dr. Mark Cushman for drug synthesis). Our goal is to make the indenoisoquinolines the first NCI-discovered drugs in the Phase 0/I pipeline with histone gamma-H2AX as a biomarker. We have also studied and characterized novel non-camptothecin and non-indenoisoquinoline topoisomerase inhibitors that are in clinical trials and developments. Those inhibitors belong to different chemical families: the homocamptothecins and camptothecins keto derivatives against Top1, and Dp44mT against Top2. We are currently studying the molecular and cellular pharmacology of a novel non-camptothecin Top1 inhibitors from Genzyme Co., which is just starting clinical trials. We have extended our studies on the induction of Top1-DNA complexes by carcinogens and during apoptosis. We had previously reported that polycyclic aromatics (benzo[a]pyrene, benzo[c]phenanthrene), formaldehyde (a bioproduct generated in humans from alcohol metabolism) and 4-nitroquinoline-1-oxide (4-NQO) were potent inducers of Top1 cleavage complexes. We have now shown that another carcinogen, crotonaldehyde can also trap Top1 cleavage complexes both with purified Top1 and in cells. We have also shown for the first time that crotonaldehyde adducts can form Top1-DNA adducts independently of Top1 cleavage complexes. This result is the first proof of principle that crotonaldehyde adducts can form adducts between chromatin protein (here Top1) and DNA. Regarding the induction of topoisomerase cleavage complexes during apoptosis, we have now 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). We have also shown that the formation of Top1 cleavage complexes plays an active role in the execution of apoptosis since cells with Top1 down-regulation produce abnormal chromatin condensation and delayed formation of apoptotic bodies. This finding may be important since partial (incomplete apoptosis) allows the survival of cells with carcinogenic potential and can elicit autoimmune responses. The first and still the only specific mitochondrial topoisomerase, Top1mt, was discovered in our laboratory. Top1mt is encoded by a nuclear gene present 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 vertebrates. The other TOP1 gene encodes the previously known Top1 devoted to the nuclear genome. We have generated specific antibodies for Top1mt, which enabled us to demonstrate that Top1mt is absent from nuclei and concentrated in mitochondria. We have also found that Top1mt can be trapped by camptothecin and used this finding to map the Top1mt binding sites in mitochondrial DNA (mtDNA). Mapping of Top1mt sites in the regulatory D-loop region of mtDNA in mitochondria revealed the presence of an asymmetric cluster of Top1mt sites confined to a 150-bp segment downstream from, and adjacent to, the site at which replication is prematurely terminated, generating a ≈650-base (7S DNA) product that forms the mitochondrial D-loop. Moreover, we showed that inhibition of Top1mt by camptothecin reduces formation of the 7S DNA. Our results suggest novel roles for Top1mt in regulating mtDNA replication. We have also generated Top1mt knockout mice and are presently studying their phenotype and their genotype. In collaboration with Dr. Rafa Balana, we have studied the effects of Top1 inhibitors on the leishmania donovani Top1 and analyzed the functional role of key catalytic residues. One potential outcome will be the discovery of novel antiparasite drugs potentially related to indenoisoquinolines.
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