Cellular Topoisomerase Research Articles

Interactions between Gepotidacin and Escherichia coli Gyrase and Topoisomerase IV: Genetic and Biochemical Evidence for Well-Balanced Dual-Targeting.

Antimicrobial resistance is a global threat to human health. Therefore, efforts have been made to develop new antibacterial agents that address this critical medical issue. Gepotidacin is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial in clinical development. Recently, phase III clinical trials for gepotidacin treatment of uncomplicated urinary tract infections caused by uropathogens, including Escherichia coli, were stopped for demonstrated efficacy. Because of the clinical promise of gepotidacin, it is important to understand how the compound interacts with its cellular targets, gyrase and topoisomerase IV, from E. coli. Consequently, we determined how gyrase and topoisomerase IV mutations in amino acid residues that are involved in gepotidacin interactions affect the susceptibility of E. coli cells to the compound and characterized the effects of gepotidacin on the activities of purified wild-type and mutant gyrase and topoisomerase IV. Gepotidacin displayed well-balanced dual-targeting of gyrase and topoisomerase IV in E. coli cells, which was reflected in a similar inhibition of the catalytic activities of these enzymes by the compound. Gepotidacin induced gyrase/topoisomerase IV-mediated single-stranded, but not double-stranded, DNA breaks. Mutations in GyrA and ParC amino acid residues that interact with gepotidacin altered the activity of the compound against the enzymes and, when present in both gyrase and topoisomerase IV, reduced the antibacterial activity of gepotidacin against this mutant strain. Our studies provide insights regarding the well-balanced dual-targeting of gyrase and topoisomerase IV by gepotidacin in E. coli.

Real-time imaging of drug-induced trapping of cellular topoisomerases and poly(ADP-ribose) polymerase 1 at the single-molecule level.

Topoisomerases (TOP1, TOP2α, and β) are nuclear enzymes crucial for virtually all aspects of DNA metabolisms. They also are the targets of important anti-tumor chemotherapeutics that act by trapping the otherwise reversible topoisomerase-DNA covalent complex intermediates (TOPccs) that are formed during their catalytic reactions, resulting in long-lived topoisomerase DNA-protein crosslinks (TOP-DPCs) that interfere with DNA transactions. The Poly(ADP-ribose) polymerase (PARP) family protein PARP1 is activated by DNA damage to recruit DNA repair proteins, and PARP inhibitors are another class of commonly used chemotherapeutics, which bind and trap PARP molecules on DNA. To date, the trapping of TOPccs and PARP by their respective inhibitors can only be measured by immune-biochemical methods in cells. Here, we developed an imaging-based approach enabling real-time monitoring of drug-induced trapping of TOPccs and PARP1 in live cells at the single-molecule level. Capitalizing on this approach, we calculated the fraction of self-fluorescence tag-labeled topoisomerases and PARP single-molecules that are trapped by their respective inhibitors in real time. This novel technique should help elucidate the molecular processes that repair TOPcc and PARP trapping and facilitate the development of novel topoisomerase and PARP inhibitor-based therapies.

Intersubunit and intrasubunit interactions driving the MukBEF ATPase

MukBEF, a structural maintenance of chromosome-like protein complex consisting of an ATPase, MukB, and two interacting subunits, MukE and MukF, functions as the bacterial condensin. It is likely that MukBEF compacts DNA via an ATP hydrolysis–dependent DNA loop–extrusion reaction similar to that demonstrated for the yeast structural maintenance of chromosome proteins condensin and cohesin. MukB also interacts with the ParC subunit of the cellular chromosomal decatenase topoisomerase IV, an interaction that is required for proper chromosome condensation and segregation in Escherichia coli, although it suppresses the MukB ATPase activity. Other structural determinants and interactions that regulate the ATPase activity of MukBEF are not clear. Here, we have investigated the MukBEF ATPase activity, identifying intersubunit and intrasubunit interactions by protein–protein crosslinking and site-specific mutagenesis. We show that interactions between the hinge of MukB and its neck region are essential for the ATPase activity, that the ParC subunit of topoisomerase IV inhibits the MukB ATPase by preventing this interaction, that MukE interaction with DNA is likely essential for viability, and that interactions between MukF and the MukB neck region are necessary for ATPase activity and viability.

Virtual Screening to Identify the Protein Network Interaction of Hypericin with Red Complex Pathogens

Introduction: Hypericin is the anthraquinone derivative and has many properties like antiviral, antifungal and antibacterial. The red complex pathogens which include Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia in association with other microbes found in the periodontal pockets, cause severe inflammation resulting in periodontitis. Novel bioactive agents from several sources have been tested against the microbial pathogens to deduce antimicrobial activity. 
 Aim: The aim of the study is to virtually screen and identify the protein network interaction of hypericin in red complex pathogens.
 Methodology: The STITCH v5.0 pipeline was primarily used to identify the drug-protein interactions. The VirulentPred and VICMPred software were used for elucidating the functional class of the proteins and virulence property. The sub cellular localization of virulent proteins was analysed with pSORTb v3.0 software. Further, the epitopes in virulent proteins were identified using BepiPred v1.0 linear epitope prediction tool.
 Results: Heat shock protein 90 of Porphyromonas gingivalis were found to involve in the cellular process and DNA topoisomerase IV subunit B, heat shock protein 90, DNA gyrase subunit A and DNA gyrase subunit B of Treponema denticola were found to be the virulent factors. The virulent proteins were located in the cytoplasm, which would further increase the potential effect of the drug to serve as antimicrobial agents. Finally, epitopes were predicted on the virulent proteins which can be specifically docked to further ascertain their interactions with the phytocompound.
 Conclusion: Hypericin with all its potential and biological benefits can be addressed, can be used as an antimicrobial agent to eradicate dental pathogens which are recalcitrant to treatment. The mode of action of hypericin is, it is targeting crucial proteins in red complex pathogens. Further in vitro studies should be performed on a wide range of pathogens to substantiate the true interactions between the drugs and the protein repertoire of pathogens.

Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA.

In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.

HU Multimerization Shift Controls Nucleoid Compcation

Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic, but depend partly upon conserved, histone-like proteins HUαα and Huαβ and their interactions that span the nano and mesoscales from protein-DNA complexes to the bacterial chromosome and nucleoid structure. Here, we determined crystal structures of these chromosome-associated proteins in complex with native duplex DNA. Distinct DNA-binding modes of HUαα-and HUαβ elucidate fundamental features of bacterial chromosome packing regulating gene transcription. By combining crystal structures with solution X-ray scattering results, we determined architectures of HU-DNA nucleoproteins in solution under near physiological conditions. These macromolecular conformations and interactions result in contraction at the cellular level based upon in vivo imaging of native unlabeled nucleoid by soft X-ray tomography upon HUβ and ectopic HUα38 expression. Structural characterization of charge-altered HUαα - DNA complex reveals an HU molecular switch suitable to condense nucleoid and reprogram noninvasive Escherichia coli into an invasive form. Collective findings suggest that shifts between networking, cooperative and non-cooperative DNA-dependent HU multimerization control DNA compaction and supercoiling independently of cellular topoisomerase activity. By integrating X-ray crystal structures, X-ray scattering, mutational tests, and X-ray imaging that span from protein-DNA complexes to the bacterial chromosome and nucleoid structure, we show that defined dynamic HU interaction networks can promote nucleoid reorganization and transcriptional regulation as efficient general microbial mechanisms to help synchronize genetic responses to cell cycle, changing environments, and pathogenesis. Hammel, M., D. Amlanjyoti, F. E. Reyes, J. H. Chen, R. Parpana, H. Y. Tang, C. A. Larabell, J. A. Tainer and S. Adhya (2016). SCIENCE Adv2(7): e1600650.

Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase I (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes.

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process.

Structure and Chromosomal Organization of Yeast Genes Regulated by Topoisomerase II.

Cellular DNA topoisomerases (topo I and topo II) are highly conserved enzymes that regulate the topology of DNA during normal genome transactions, such as DNA transcription and replication. In budding yeast, topo I is dispensable whereas topo II is essential, suggesting fundamental and exclusive roles for topo II, which might include the functions of the topo IIa and topo IIb isoforms found in mammalian cells. In this review, we discuss major findings of the structure and chromosomal organization of genes regulated by topo II in budding yeast. Experimental data was derived from short (10 min) and long term (120 min) responses to topo II inactivation in top-2 ts mutants. First, we discuss how short term responses reveal a subset of yeast genes that are regulated by topo II depending on their promoter architecture. These short term responses also uncovered topo II regulation of transcription across multi-gene clusters, plausibly by common DNA topology management. Finally, we examine the effects of deactivated topo II on the elongation of RNA transcripts. Each study provides an insight into the particular chromatin structure that interacts with the activity of topo II. These findings are of notable clinical interest as numerous anti-cancer therapies interfere with topo II activity.

N-thioalkylcarbazoles derivatives as new anti-proliferative agents: synthesis, characterisation and molecular mechanism evaluation

Synthetic or natural carbazole derivatives constitute an interesting class of heterocycles, which showed several pharmaceutical properties and occupied a promising place as antitumour tools in preclinical studies. They target several cellular key-points, e.g. DNA and Topoisomerases I and II. The most studied representative, i.e. Ellipticine, was introduced in the treatment of metastatic breast cancer. However, because of the onset of dramatic side effects, its use was almost dismissed. Many efforts were made in order to design and synthesise new carbazole derivatives with good activity and reduced side effects. The major goal of the present study was to synthesise a series of new N-thioalkylcarbazole derivatives with anti-proliferative effects. Two compounds, 5a and 5c, possess an interesting anti-proliferative activity against breast and uterine cancer cell lines without affecting non-tumoural cell lines viability. The most active compound (5c) induces cancer cells death triggering the intrinsic apoptotic pathway by inhibition of Topoisomerase II.

A comprehensive review on anticancer mechanisms of the main carotenoid of saffron, crocin.

Crocin is derived from dried stigmas of Crocus sativus L. (saffron). It has long been used to prevent and treat various diseases. Although crocin is suggested as one of the most effective cancer therapeutic constituents of saffron stigma, its exact molecular mechanisms are not fully understood. In this study, we reviewed anticancer effects of crocin and its underlying molecular mechanisms. While several mechanisms may account for the antitumour activity of crocin, alteration of expression/activity of the genes and also epigenetic changes may be considered as necessary phenomena. These alternations may lead to inhibition of cancer cells' proliferation or/and induction of apoptosis through various mechanism including inhibition of synthesis of DNA and RNA, interaction with cellular topoisomerase, suppression of the telomerase activity and active STAT3, and targeting of microtubules. Moreover, this carotenoid could reverse the epithelial-mesenchymal transition and inhibit metastasis. Knowing molecular mechanisms of antitumoral agents could guide us to choose the best chemotherapeutic compound especially for targeted therapy and also provide insights about possible side effects.

HU multimerization shift controls nucleoid compaction

Molecular mechanisms controlling functional bacterial chromosome (nucleoid) compaction and organization are surprisingly enigmatic but partly depend on conserved, histone-like proteins HUαα and HUαβ and their interactions that span the nanoscale and mesoscale from protein-DNA complexes to the bacterial chromosome and nucleoid structure. We determined the crystal structures of these chromosome-associated proteins in complex with native duplex DNA. Distinct DNA binding modes of HUαα and HUαβ elucidate fundamental features of bacterial chromosome packing that regulate gene transcription. By combining crystal structures with solution x-ray scattering results, we determined architectures of HU-DNA nucleoproteins in solution under near-physiological conditions. These macromolecular conformations and interactions result in contraction at the cellular level based on in vivo imaging of native unlabeled nucleoid by soft x-ray tomography upon HUβ and ectopic HUα38 expression. Structural characterization of charge-altered HUαα-DNA complexes reveals an HU molecular switch that is suitable for condensing nucleoid and reprogramming noninvasive Escherichia coli into an invasive form. Collective findings suggest that shifts between networking and cooperative and noncooperative DNA-dependent HU multimerization control DNA compaction and supercoiling independently of cellular topoisomerase activity. By integrating x-ray crystal structures, x-ray scattering, mutational tests, and x-ray imaging that span from protein-DNA complexes to the bacterial chromosome and nucleoid structure, we show that defined dynamic HU interaction networks can promote nucleoid reorganization and transcriptional regulation as efficient general microbial mechanisms to help synchronize genetic responses to cell cycle, changing environments, and pathogenesis.

Mesenchymal stem cells exhibit resistance to topoisomerase inhibition

BackgroundInhibition of cellular topoisomerases has been established as an effective way of treating certain cancers, albeit with often high levels of toxicity to the bone marrow. While the involvement of mesenchymal stem cells (MSCs) in bone marrow homeostasis and regeneration has been well established, the effects of topoisomerase-inhibiting anticancer agents remain largely unknown. Materials and MethodsHuman bone marrow MSCs were treated with topoisomerase I inhibitor irinotecan or topoisomerase II inhibitor etoposide, and survival and apoptosis levels were measured. The influence of topoisomerase inhibition on cellular morphology, adhesion and migration potential and the ability to differentiate was assessed. Additionally, the role of individual DNA double-strand break repair pathways in MSCs was investigated as a potential cellular mechanism of resistance to topoisomerase inhibitors. ResultsHuman bone marrow MSCs were found relatively resistant to topoisomerase I and II inhibitors and show survival levels comparable to these of differentiated fibroblasts. Treatment with irinotecan or etoposide did not significantly influence cellular adhesion, migratory ability, surface marker expression or induction of apoptosis in human MSCs. The ability to differentiate was found preserved in MSCs after exposure to high doses of irinotecan or etoposide. MSCs were able to efficiently repair DNA double-strand breaks induced by topoisomerase inhibitors both by non-homologous end joining and homologous recombination pathways. ConclusionOur data demonstrate a topoisomerase-resistant phenotype of human MSCs that may at least in part be due to the stem cells' ability to efficiently remove DNA damage caused by these anticancer agents. The observed resistance of MSCs warrants further investigation of these cells as a potential therapeutic option for treating topoisomerase inhibitor-induced bone marrow damage.

Selective Recruitment of Nuclear Factors to Productively Replicating Herpes Simplex Virus Genomes

Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources.

Topoisomerase I as a target of erlotinib and gefitinib: Efficacy of combined treatments with camptothecin

Topoisomerases are essential nuclear enzymes that work to resolve topological problems that normally occur during DNA metabolism. Their involvement in crucial DNA associated-processes, such as replication, transcription and repair, mark them as a target of chemotherapeutic drugs such as camptothecins (CPTs). Therefore, finding other agents that may alter their activity is of great importance. Previous data showed that certain tyrosine kinase antagonists, tyrphostins, inhibit the catalytic activity of the cellular topoisomeraseI (topoI). We examined the effect of clinically used tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, on topoI in breast and prostate cancer cells. While erlotinib and gefitinib inhibit cellular topoI in treated cells without affecting the levels of the enzyme protein, in vitro assays show that erlotinib, but not gefitinib, inhibits the DNA relaxation activity of purified topoI. Erlotinib was found to reduce the DNA-binding ability of topoI, however, the reduction in topoI activity in gefitinib-treated cells is probably due to post-translational modifications of the enzyme protein. A combined treatment of either erlotinib or gefitinib with CPT increased the effect of CPT on the activity of cellular topoI, which supports the increased anticancer effect observed in MCF7 cells. These results suggest that topoI is a novel target of erlotinib and a combination of TKIs with topoI inhibitors may be an effective treatment for breast cancer.

Mycoplasma fermentans inhibits the activity of cellular DNA topoisomerase I by activation of PARP1 and alters the efficacy of its anti-cancer inhibitor.

To understand the effects of the interaction between Mycoplasma and cells on the host cellular function, it is important to elucidate the influences of infection of cells with Mycoplasma on nuclear enzymes such as DNA Topoisomerase type I (Topo I). Human Topo I participates in DNA transaction processes and is the target of anti-cancer drugs, the camptothecins (CPTs). Here we investigated the mechanism by which infection of human tumor cells with Mycoplasma fermentans affects the activity and expression of cellular Topo I, and the anti-cancer efficacy of CPT. Human cancer cells were infected or treated with live or sonicated M. fermentans and the activity and expression of Topo I was determined. M. fermentans significantly reduced (by 80%) Topo I activity in the infected/treated tumor cells without affecting the level of Topo I protein. We demonstrate that this reduction in enzyme activity resulted from ADP-ribosylation of the Topo I protein by Poly-ADP-ribose polymerase (PARP-1). In addition, pERK was activated as a result of the induction of the MAPK signal transduction pathway by M. fermentans. Since PARP-1 was shown to be activated by pERK, we concluded that M. fermentans modified the cellular Topo I activity by activation of PARP-I via the induction of the MAPK signal transduction pathway. Moreover, the infection of tumor cells with M. fermentans diminished the inhibitory effect of CPT. The results of this study suggest that modification of Topo I activity by M. fermentans may alter cellular gene expression and the response of tumor cells to Topo I inhibitors, influencing the anti-cancer capacity of Topo I antagonists.

Relationship of cellular topoisomerase IIα inhibition to cytotoxicity and published genotoxicity of fluoroquinolone antibiotics in V79 cells

Fluoroquinolone (FQ) antibiotics are bacteriocidal through inhibition of the bacterial gyrase and at sufficient concentrations in vitro, they can inhibit the homologous eukaryotic topoisomerase (TOPO) II enzyme. FQ exert a variety of genotoxic effects in mammalian systems through mechanisms not yet established, but which are postulated to involve inhibition of TOPO II enzymes. To assess the relationship of inhibition of cell nuclear TOPO II to cytotoxicity and reported genotoxicity, two FQ, clinafloxacin (CLFX) and lomefloxacin (LOFX), having available genotoxicity data showing substantial differences with CLFX being more potent than LOFX, were selected for study. The relative inhibitory activities of these FQ on nuclear TOPO IIα in cultured Chinese hamster lung fibroblasts (V79 cells) over dose ranges and at equimolar concentrations were assessed by measuring nuclear stabilized cleavage complexes of TOPO IIα–DNA. Cytotoxicity was measured by relative cell counts.Both FQ inhibited V79 cell nuclear TOPO IIα. The lowest-observed-adverse-effect levels for TOPO IIα inhibition were 55μM for CLFX, and 516μM for LOFX. The no-observed-adverse-effect-levels were 41μM for CLFX, and 258μM for LOFX. At equimolar concentrations (175μM), CLFX was more potent than LOFX. Likewise, CLFX was more cytotoxic than LOFX.Thus, the two FQ, inhibited TOPO IIα in intact V79 cells, differed in their potencies and exhibited no-observed-adverse-effect levels. These findings are in concordance with published genotoxicity data and observed cytotoxicity.

Inhibitory effects of myricetin on mammalian DNA polymerase, topoisomerase and human cancer cell proliferation

In this study, the inhibitory activities against mammalian DNA polymerases (pols) of 16 major bioflavonoids were investigated. Myricetin (3,3′,4′,5,5′,7-hexahydroxyflavone) was the most potent inhibitor of pols amongst the compounds tested, with IC50 values of 21.3–40.9μM. This compound did not affect the activities of plant (cauliflower) pol α or prokaryotic pols. Myricetin also inhibited human DNA topoisomerase II (topo II) activity with an IC50 value of 27.5μM, but did not inhibit the activities of other DNA metabolic enzymes tested. Myricetin also did not influence the direct binding to double stranded DNA as determined by thermal transition analysis. It was found to prevent the proliferation of human colon HCT116 carcinoma cells with an LD50 of 28.2μM, halt the cell cycle in G2/M phase, and induce apoptosis. These results suggest that the decrease of proliferation may be a result of the inhibition of cellular topoisomerase (topo) II rather than pols.

The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II

Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258–DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.

Identification of Cytotoxic Drugs That Selectively Target Tumor Cells with MYC Overexpression

Expression of MYC is deregulated in a wide range of human cancers, and is often associated with aggressive disease and poorly differentiated tumor cells. Identification of compounds with selectivity for cells overexpressing MYC would hence be beneficial for the treatment of these tumors. For this purpose we used cell lines with conditional MYCN or c-MYC expression, to screen a library of 80 conventional cytotoxic compounds for their ability to reduce tumor cell viability and/or growth in a MYC dependent way. We found that 25% of the studied compounds induced apoptosis and/or inhibited proliferation in a MYC-specific manner. The activities of the majority of these were enhanced both by c-MYC or MYCN over-expression. Interestingly, these compounds were acting on distinct cellular targets, including microtubules (paclitaxel, podophyllotoxin, vinblastine) and topoisomerases (10-hydroxycamptothecin, camptothecin, daunorubicin, doxorubicin, etoposide) as well as DNA, RNA and protein synthesis and turnover (anisomycin, aphidicholin, gliotoxin, MG132, methotrexate, mitomycin C). Our data indicate that MYC overexpression sensitizes cells to disruption of specific pathways and that in most cases c-MYC and MYCN overexpression have similar effects on the responses to cytotoxic compounds. Treatment of the cells with topoisomerase I inhibitors led to down-regulation of MYC protein levels, while doxorubicin and the small molecule MYRA-A was found to disrupt MYC-Max interaction. We conclude that the MYC pathway is only targeted by a subset of conventional cytotoxic drugs currently used in the clinic. Elucidating the mechanisms underlying their specificity towards MYC may be of importance for optimizing treatment of tumors with MYC deregulation. Our data also underscores that MYC is an attractive target for novel therapies and that cellular screenings of chemical libraries can be a powerful tool for identifying compounds with a desired biological activity.

Abstract 3261: Novel hemisynthetic derivatives of Amaryllidaceae alkaloids exert antitumor activity on xenograft melanoma models

Abstract Alkaloids pancratistatin and narciclasine, isolated from Amaryllidaceae, have been shown to present antitumor activity on preclinical models, including melanoma. Despite extensive investigations, pancratistatin or narciclasine analogues did not succeed to provide sufficient therapeutic benefit, [1] mostly hampered by poor water solubility and systemic toxicity [1]. To overcome those limitations, we developed a chemistry program aimed at improving both their physicochemical and pharmacological properties. A literature survey suggests that any structural modification of this highly oxigenated isocarbostiryl specific scaffold would lead to a loss of activity except on position 1, difficult to achieve but interesting to explore. A series of 40 novel 1-aminopancratistatin derivatives was synthesised starting from large quantities of narciclasine extracted from Narcissus bulbs. These novel compounds exhibit an improved pharmaceutical profile facing standard solubility, plasmatic and microsomal stability assays. Moreover, a subset of narciclasine derivatives demonstrated a significant enhancement of the antiproliferative effect with IC50 reaching the nM range, consistent with a major induction of pro-apoptotic signals, including mitochondrial depolarisation. To explore the mode of action of these alkaloids derivatives, we tested their capacity to inhibit protein synthesis, compared their pharmacological profile with 80 anticancer compounds on 16 tumor cell lines (oncoprofile.16) and explored their impact on major cellular functions such as cellular integrity, cell cycle, topoisomerases inhibition, DNA damage and synthesis. We demonstrated thus that inhibition of DNA synthesis is a key component of their mechanism of action. Based on the pharmacological properties, a structure-activity card can be built to select different categories of pancrastistatin derivatives depending on their ability to inhibit preferentially protein synthesis or/and DNA synthesis. Interestingly, the dimethylaminomethyl benzamide derivative F98604 exhibited a significant antiproliferative activity on the murine B16 and human A375 melanoma xenograft models associated with an increased therapeutic index as compared with the natural compound narciclasine. In conclusion, modulation of position 1 on the narciclasine pharmacophore overcomes the solubility issue and is a key position to modify their pharmacological profiles, this opening new perspectives as anticancer agents active against melanoma. 1. A. Kornienko, A. Evidente Chem. Rev. 2008, 108, 1982 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3261. doi:10.1158/1538-7445.AM2011-3261