Inhibition Of Topo Research Articles

Modification of 3-arylisoquinolines into 3,4-diarylisoquinolines and assessment of their cytotoxicity and topoisomerase inhibition.

Inspired by the initial success of the monoarylisoquinolines and the quest to identify more potent and selective anticancer agents with topoisomerase (topo) inhibitory activity, series of diarylisoquinolines (3,4-diarylisoquinolones and 3,4-diarylisoquinolinamines) were designed and synthesized. Synthesis of these compounds primarily involved lithiated toluamide-benzonitrile cycloaddition, Suzuki coupling, and nucleophilic aromatic substitution reactions. Eight of the derivatives were selectively toxic against human ductal breast epithelial tumor cells (T47D), human prostate cancer cells (DU145), and human colorectal adenocarcinoma cells (HCT-15), but had no effect on normal human breast epithelial cells (MCF10A). The topo inhibitory activities of the diarylisoquinoline compounds were relatively dependent upon their chemical structure. 3,4-Diarylisoquinolones generally did not inhibit topo I and only showed moderate inhibition of topo II. In contrast, several 3,4-diarylisoquinolinamines showed superior topo I inhibitory activity. Isoquinolinamine derivatives had greater affinity for topo I than for topo II. Topo inhibition by 3,4-diarylisoquinolines was further supported by docking models showing intercalative and/or H-bond interactions between these compounds and the DNA/topo(s). An analysis of the correlation between the cytotoxicity and topo inhibition of these compounds indicated that the primary biological target of derivatives with potent cytotoxicity was topo, which in turn establishes diaryl-substituted isoquinolines as a novel class of potential anticancer drugs.

Synthesis and bioactivity of novel xanthone and thioxanthone l-rhamnopyranosides

Xanthone l-rhamnopyranoside derivative 11 was discovered as one of novel topo-I inhibitors.

A New-Class Antibacterial-Almost. Lessons in Drug Discovery and Development: A Critical Analysis of More than 50 Years of Effort toward ATPase Inhibitors of DNA Gyrase and Topoisomerase IV.

The introduction into clinical practice of an ATPase inhibitor of bacterial DNA gyrase and topoisomerase IV (topo IV) would represent a new-class agent for the treatment of resistant bacterial infections. Novobiocin, the only historical member of this class, established the clinical proof of concept for this novel mechanism during the late 1950s, but its use declined rapidly and it was eventually withdrawn from the market. Despite significant and prolonged effort across the biopharmaceutical industry to develop other agents of this class, novobiocin remains the only ATPase inhibitor of gyrase and topo IV ever to progress beyond Phase I. In this review, we analyze the historical attempts to discover and develop agents within this class and highlight factors that might have hindered those efforts. Within the last 15 years, however, our technical understanding of the molecular details of the inhibition of the gyrase and topo IV ATPases, the factors governing resistance development to such inhibitors, and our knowledge of the physical properties required for robust clinical drug candidates have all matured to the point wherein the industry may now address this mechanism of action with greater confidence. The antibacterial spectrum within this class has recently been extended to begin to include serious Gram negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. In spite of this recent technical progress, adverse economics associated with antibacterial R&D over the last 20 years has diminished industry's ability to commit the resources and perseverance needed to bring new-class agents to launch. Consequently, a number of recent efforts in the ATPase class have been derailed by organizational rather than scientific factors. Nevertheless, within this context we discuss the unique opportunity for the development of ATPase inhibitors of gyrase and topo IV as new-class antibacterial agents with broad spectrum potential.

Abstract C50: Identification of secondary targets to enhance anthracycline-based therapy

Abstract Breast cancer is the most common cancer in women worldwide. African-American women (AA) tend to have more aggressive breast cancers that present more frequently as estrogen receptor, progesterone receptor (PR) and HER2 negative which are referred to as “triple negative” breast cancers (TNBC). TNBC tumors carry worse prognoses and are harder to manage because they lack the molecular targets required for therapies. In patients suffering from TNBC cytotoxic agents such as anthracyclines are some of the few therapeutic alternatives. Anthracyclines are among the most common chemotherapeutic agents used to-date. Although effective, resistance to this agent usually develops in the course of treatment and serious dose-related toxicity, namely chronic cardio-toxicity, limits their use. To increase our knowledge of the pathways that protect cells from anthracyclines we carried out a genome-wide genetic screening in the eukaryotic model organism S. cerevisiae to identify gene defects that generate cells hypersensitive to doxorubicin. A total of 71 deletion strains displayed varying levels of sensitivity to anthracyclines. Major pathways were represented by multiple genes, including homologous recombination/DNA repair, the heat-shock response, chromatin remodeling and aminoacid metabolism. To further investigate the mechanisms that render cells hypersensitive to anthracyclines when genes of these pathways are inactivated, we determined the sensitivity of several mutants to a series of cytotoxic agents. Our findings indicate that inactivation of the heat-shock protein 40 encoded by the genes YDJ1 and ZUO1, as well as the heat-shock protein 70 encoded by the gene SSZ1, render cells highly sensitive to the toxicity of doxorubicin and cisplatin. In addition, deletion of chromatin remodeling factor SNF2 resulted in sensitivity to doxorubicin of 100,000-fold higher than wild type cells and it is also sensitive to daunorubicin, cisplatin, as well as to DNA alkylating agents, topoI inhibitor campthotecin or replication blocks from hydroxyurea. Interestingly, mutations of the HOM6 gene increases the accumulation of an aldehyde intermediate that synergizes with anthracyclines. Taken together, our data indicates that inactivation of any of these pathways can be targeted to hypersensitize cancer cells to cytotoxic agents to enhance chemotherapeutic efficacy in TNBC. This project was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 2 G12 RR003020 and 8 G12 MD007582-28. Citation Format: Hernan Flores-Rozas, Tryphon Mazu, Miles D. Freeman, Jana S. Miles. Identification of secondary targets to enhance anthracycline-based therapy. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C50. doi:10.1158/1538-7755.DISP13-C50

Synthesis and evaluation of 18F-labeled ATP competitive inhibitors of topoisomerase II as probes for imaging topoisomerase II expression

Type II topoisomerase (Topo-II) is an ATP-dependent enzyme that is essential in the transcription, replication, and chromosome segregation processes and, as such, represents an attractive target for cancer therapy. Numerous studies indicate that the response to treatment with Topo-II inhibitors is highly dependent on both the levels and the activity of the enzyme. Consequently, a non-invasive assay to measure tumoral Topo-II levels has the potential to differentiate responders from non-responders. With the ultimate goal of developing a radiofluorinated tracer for positron emission tomography (PET) imaging, we have designed, synthesized, and evaluated a set of fluorinated compounds based on the structure of the ATP-competitive Topo-II inhibitor QAP1. Compounds 18 and 19b showed inhibition of Topo-II in in vitro assays and exhibited moderate, Topo-II level dependent cytotoxicity in SK-BR-3 and MCF-7 cell lines. Based on these results, 18F-labeled analogs of these two compounds were synthesized and evaluated as PET probes for imaging Topo-II overexpression in mice bearing SK-BR-3 xenografts. [18F]-18 and [18F]-19b were synthesized from their corresponding protected tosylated derivatives by fluorination and subsequent deprotection. Small animal PET imaging studies indicated that both compounds do not accumulate in tumors and exhibit poor pharmacokinetics, clearing from the blood pool very rapidly and getting metabolized over. The insights gained from the current study will surely aid in the design and construction of future generations of PET agents for the non-invasive delineation of Topo-II expression.

Zbh-1205: a Novel Camptothecin Derivate Revealed Potent Antitumor Activities Mainly Through Cell Apoptosis Pathway

ABSTRACT Aim: The aim of this study is to characterize the antitumor activity of a novel camptothecin derivate ZBH-1205 and compare with other Topo I inhibitors CTP-11 and SN38. Methods: MTT assay was employed to assess cell growth inhibition with treatment of ZBH-1025, CPT-11 or SN38. A panel of 11 human tumor cell lines was used in the assay and normal human diploid 293 cell line was also included. DNA relaxation assays were used to detected the formation of single-strand breaks induced by Topo-1 in the presence of ZBH-1205. Induction of cell apoptosis by ZBH-1205, CPT-11 and SN38 was analyzed by flow cytometer using FITC Annexin V/PI double staining method. DNA content assay was used to detect cell cycle profile. The levels of pro- and active-Caspase 3 and PARP was examined by Western blot and flowcytometry. The in vivo tumor growth inhibition of Topo I drugs was evaluated in human colon cancer xenografts. Results: ZBH-1205 retains Topo-I poisoning activities in DNA relaxation assays, as compared with CPT-11 and SN38. IC50 values for ZBH-1205 were consistently lower in cell growth assays for 11 tumor cell lines(IC50 values of ZBH-1205: 0.0009mM–2.5671mM). ZBH-1205 induced apoptosis in SW1116 and K562 cells in a dose and time dependent manner. Such abilities were greater than that of CPT-11 and SN38 especially at lower concentrations. Annexin-V+/PI- cells became detectable starting from ∼18h (or before) of drugs exposure. The induction of cell cycle arrest remains the same among three Topo I drugs. Consistent with this, there was a time and dose dependent decrease in pro-caspase-3 and PARP expression, and a significant increase in active-caspase-3. In tumor xenografts, a stronger tumor growth inhibition was observed with ZBH-1205 treatment than those of other two Topo I drugs. Conclusions: The results of current study clearly shows that ZBH-1205 is more active than SN38 and CPT-11. Also, different from CPT-11 and SN38, its antitumor mechanisms were more involved in apoptosis-related signaling pathway. Notably, ZBH-1205 can significantly induce tumor cell apoptosis at lower concentrations, which enables the drug to be more efficient and overcome the toxic effects of CPT-11 in vivo. These findings open a new route for improving the pharmacological properties of CPT-11 derivatives and support the development of novel series of CPT-11 as anticancer agents in the clinic. Disclosure: All authors have declared no conflicts of interest.

Molecular docking studies of curcumin natural derivatives with DNA topoisomerase I and II-DNA complexes.

DNA topoisomerase I (topo I) and II (topo II) are essential enzymes that solve the topological problems of DNA by allowing DNA strands or double helices to pass through each other during cellular processes such as replication, transcription, recombination, and chromatin remodeling. Their critical roles make topoisomerases an attractive drug target against cancer. The present molecular docking study provides insights into the inhibition of topo I and II by curcumin natural derivatives. The binding modes suggested that curcumin natural derivatives docked at the site of DNA cleavage parallel to the axis of DNA base pairing. Cyclocurcumin and curcumin sulphate were predicted to be the most potent inhibitors amongst all the curcumin natural derivatives docked. The binding modes of cyclocurcumin and curcumin sulphate were similar to known inhibitors of topo I and II. Residues like Arg364, Asn722 and base A113 (when docked to topo I-DNA complex) and residues Asp479, Gln778 and base T9 (when docked to topo II-DNA complex) seem to play important role in the binding of curcumin natural derivatives at the site of DNA cleavage.

Organometallic ruthenium(II) scorpionate as topo IIα inhibitor; in vitro binding studies with DNA, HPLC analysis and its anticancer activity

New organoruthenium [(η6-arene)RuII(L)Cl]Cl (1: arene = p-cymene, L = L1; 2: arene = p-cymene, L = L2; 3: arene = benzene, L = L1; 4: arene = benzene; L = L2; L1 = bis(3,5-dimethylpyrazolyl)parabenzoic acid, L2 = bis(3,5-dimethylpyrazolyl)metabenzoic acid) have been synthesized and characterized by analytical and spectroscopic methods. The molecular structure of [(η6-p-cymene)RuCl(L1)]Cl (1) was determined by single crystal X-ray diffraction studies. Preliminary in vitro binding studies of 1–4 with CT DNA were carried out by employing various biophysical techniques which revealed their avid DNA binding via non-covalent binding mode viz; partial intercalation of the η6-arene group as well as electrostatic surface interaction through one oxygen atom of the phosphate backbone of the DNA helix; however, complexes 1 and 3 display higher binding propensity in comparison to 2 and 4, as quantified by Kb. The interaction was further analysed by HPLC technique. The results of the cleavage experiments of pBR322 DNA with 1 and 3 displayed significantly good cleavage at 20–40 μM, following the oxidative pathway. These findings have revealed that the hydrophobic arene, and the chloride leaving group have important roles in the novel mechanism of recognition of DNA by (η6-arene)ruthenium(II) complexes, and will aid the design of more effective anticancer complexes, as well as new site-specific DNA reagents. Furthermore, the anticancer activity of the complexes 1 and 3 on 15 cell lines of different histological origin has been studied. It has been observed that 1 exhibits higher cytotoxicity than 3, and the cells undergo apoptotic cell death.

Structural determinants of the catalytic inhibition of human topoisomerase IIα by salicylate analogs and salicylate-based drugs

We previously identified salicylate as a novel catalytic inhibitor of human DNA topoisomerase II (topo II; EC 5.99.1.3) that preferentially targets the alpha isoform by interfering with topo II-mediated DNA cleavage. Many pharmaceuticals and compounds found in foods are salicylate-based. We have now investigated whether these are also catalytic inhibitors of topo II and the structural determinants modulating these effects. We have determined that a number of hydroxylated benzoic acids attenuate doxorubicin-induced DNA damage signaling mediated by the ATM protein kinase and inhibit topo II decatenation activity in vitro with varying potencies. Based on the chemical structures of these and other derivatives, we identified unique properties influencing topo II inhibition, including the importance of substitutions at the 2′- and 5′-positions. We extended our findings to a number of salicylate-based pharmaceuticals including sulfasalazine and diflunisal and found that both were effective at attenuating doxorubicin-induced DNA damage signaling, topo II DNA decatenation and they blocked stabilization of doxorubicin-induced topo II cleavable complexes in cells. In a manner similar to salicylate, we determined that these agents inhibit topo II-mediated DNA cleavage. This was accompanied by a concomitant decrease in topo II-mediated ATP-hydrolysis. Taken together, these findings reveal a novel function for the broader class of salicylate-related compounds and highlight the need for additional studies into whether they may impact the efficacy of chemotherapy regimens that include topo II poisons.

A pilot study of supraphysiologic testosterone (T) and oral etoposide (E) in men with castrate-resistant prostate cancer (CRPC).

45 Background: Prostate cancer (PC) cells become resistant to chronic castration via an adaptive increase in androgen receptor (AR) expression, a liability that can be exploited therapeutically. Mechanistically, supraphysiologic androgens can induce PC cell death through topoisomerase 2 (topo2) mediated double-strand DNA breaks and disruption of DNA relicensing due to persistence of AR at origins of replication during the cell cycle. Methods: We evaluated parenteral T in combination with the topo2 inhibitor E in men with CRPC and low metastatic burden (≤5 bone and <10 soft tissue metastases). To rapidly cycle from supraphysiologic (>1500 ng/dL) to near castrate T levels, men received intramuscular T cypionate 400 mg on day (D) 1 and oral E 100 mg D 1-14 of a 28 D cycle. After 3 cycles, men with declining PSA could continue T alone every 28 D. The primary endpoint was PSA response, defined as a PSA below baseline, after cycle 3. Secondary endpoints included objective response rates and safety. Results: Sixteen men enrolled of which 14 completed 3 cycles of T+E and were evaluable for response. 7/14 went on the T-only expansion stage. After 3 cycles, 6/14 men had a PSA response. 7 (50%) had a PSA response at any timepoint [4 (29%) had PSA declines ≥50%] (table). In men with RECIST-evaluable disease, 2 had progressive and 3 had stable disease (SD), 4 had partial (PR) and 1 had a complete response (CR). Median response duration was 248 D (range, 80 to 359 D). Post T, 10/12 men had a PSA decline to subsequent androgen ablative therapies. Most adverse events (AEs) were ≤grade 2 and considered effects of E. One man died from neutropenic sepsis before completing the 1st cycle. AEs possibly related to T included lower extremity edema (n=1), priapism (n=1) and asymptomatic pulmonary embolism (n=2). No one developed new pain on T. Conclusions: T with or without E demonstrated preliminary efficacy in patients with CRPC as manifested by PSA and objective responses. This regimen is generally safe. Cyclic T-based therapies warrant further study. Clinical trial information: NCT01084759. [Table: see text]

Heterobimetallic o-vanillin functionalized complexes: In vitro DNA binding validation, cleavage activity and molecular docking studies of CuII–Sn2IV analogs

The heterobimetallic chemical entities 1–4 of o-vanillin functionalized Schiff base have been synthesized and characterized by elemental analysis and spectroscopic methods viz., UV–vis, IR, ESI–mass, NMR (in 2 and 4) and EPR (in 1 and 3). The NiII–Sn2IV analogs were synthesized only for structural elucidation by NMR spectroscopy. To evaluate the biological preference with the molecular target DNA, interaction of the CuII–Sn2IV entities 1 and 3 with CT DNA has been explored by employing various biophysical methods revealing the electrostatic mode of binding via oxygen of sugar–phosphate backbone of DNA helix. The Kb values of 1 and 3 were found to be 2.31 × 104 and 3.67 × 104 M−1, respectively suggesting the greater binding propensity of 3. Furthermore, site of action was ascertained by the interaction studies of 1 and 3 with 5′-AMP employing UV–vis titrations, 1H and 31P NMR studies which implicates the preferential selectivity of these complexes to N1 of adenosine moiety. Moreover, the antimicrobial activities of 1 and 3, revealed 3 as a good antimicrobial agent. The cleavage activity of 3 was evaluated by agarose gel electrophoresis assay with pBR322 DNA, revealing the involvement of singlet oxygen species via oxidative cleavage pathway. Additionally, 3 exhibited significant inhibitory effects on the catalytic activity of Topo I at a very low concentration, 15 μM, suggesting that 3 is an efficient catalytic inhibitor of human Topo I. The computer-aided molecular docking techniques were carried out to ascertain the mode of action toward the molecular target DNA and Topo I for 1 and 3.

Targeting The Nuclear Export Signal In Multiple Myeloma

IntroductionIn multiple myeloma (MM), de novo drug resistance to topoisomerase (topo) II poisons occurs at high cell densities due to trafficking of topo IIα from the nucleus to the cytoplasm where it is no longer in contact with the DNA and thus unable to induce cell death (Turner et al. 2009, Cancer Res, 69, 6899-905, Engel et al. 2004, Exp Cell Res, 295, 421-31). We have previously demonstrated that topo IIα is exported from the nucleus of human myeloma cells by a CRM1-dependent mechanism ( Valkov et al 2000, Br J Haematol, 108, 331-45, Engel et al. 2004, Exp Cell Res, 295, 421-31) and we have also identified the nuclear export signals (NES) for topo IIα at amino acids 1017-28 (site A) and 1054-66 (site B) ( Turner et al. 2004, J Cell Sci, 117, 3061-71). Blocking nuclear export with a CRM1 inhibitor or by siRNA has been shown to sensitize drug-resistant MM cells to topo II poisons ((Turner et al. 2009, Cancer Res, 69, 6899-905, Turner et al 2013, J. of Cancer, In press). Even though the active NES site (site A) of topo IIα conforms to the hydrophobic amino acid motif for an NES, the amino acid sequence does not occur in any other human protein. In addition, this NES is in a pocket formed by the three-dimensional structure of the topo IIα protein. This specificity of the NES could lead to the development of drugs that may exclusively block the NES of topo IIα and not affect the CRM 1-dependent export of other nuclear proteins. MethodsIn this study, our aims were to; 1) generate an atomic homology model of human topo IIα to identify lead compounds targeting the NES of topo IIα (nuclear export signal inhibitors, NESi), 2) determine if the compounds bind specifically to topo IIα and inhibit nuclear export by immunofluorescence microscopy, nuclear-cytoplasmic fractionation, immunoprecipitation, proximity ligation assay, and Biacore kinetic/affinity assay, and 3) test the anticancer activity of these compounds in multiple myeloma and normal cells with and without a topo IIα inhibitor. Results and ConclusionsWe generated an atomic homology model of human topo IIα to provide the basis for in silico molecular docking screenings of 139,735 small molecules (MW < 500) to identify lead compounds or NESi targeting the NES of topo IIα. The highest docking score NESi compounds (46 total) were assayed for their ability to induce apoptosis in drug-resistant human H929 myeloma cells when used in combination with the topo II inhibitor doxorubicin. We isolated four lead compounds that induced apoptosis (caspase 3 cleavage) when used with doxorubicin and that inhibited nuclear export of topo IIα, as shown by immuno-fluorescence microscopy and nuclear-cytoplasmic fractionation. Immunoprecipitation, proximity ligation assay and Biacore kinetic/affinity assay showed that the most potent lead compound, NCI-9138, inhibited binding of topo IIα to the export receptor CRM1. Inhibition was specific to topo IIα because p53 trafficking was unaffected and the inhibitor did not affect topo IIα protein expression or topo IIα function (decatenation). The NESi were found to sensitize drug-resistant human myeloma cells to doxorubicin but did not affect normal fibroblast cell lines (Flow2000 or WI-38) or human PBMCs. These topo IIα-specific nuclear export inhibitors may potentially lead to a new approach in circumventing drug resistance in multiple myeloma. Disclosures:No relevant conflicts of interest to declare.

Salicylate, a Catalytic Inhibitor of Topoisomerase II, Inhibits DNA Cleavage and Is Selective for the α Isoform

Topoisomerase II (topo II) is a ubiquitous enzyme that is essential for cell survival through its role in regulating DNA topology and chromatid separation. Topo II can be poisoned by common chemotherapeutics (such as doxorubicin and etoposide), leading to the accumulation of cytotoxic enzyme-linked DNA double-stranded breaks. In contrast, nonbreak-inducing topo II catalytic inhibitors have also been described and have more limited use in clinical chemotherapy. These agents, however, may alter the efficacy of regimens incorporating topo II poisons. We previously identified salicylate, the primary metabolite of aspirin, as a novel catalytic inhibitor of topo II. We have now determined the mechanism by which salicylate inhibits topo II. As catalytic inhibitors can act at a number of steps in the topo II catalytic cycle, we used multiple independent, biochemical approaches to interrogate the catalytic cycle. Furthermore, as mammalian cells express two isoforms of topo II (α and β), we examined whether salicylate was isoform selective. Our results demonstrate that salicylate is unable to intercalate DNA, and does not prevent enzyme-DNA interaction, nor does it promote stabilization of topo IIα in closed clamps on DNA. Although salicylate decreased topo IIα ATPase activity in a dose-dependent noncompetitive manner, this was secondary to salicylate-mediated inhibition of DNA cleavage. Surprisingly, comparison of salicylate's effects using purified human topo IIα and topo IIβ revealed that salicylate selectively inhibits the α isoform. These findings provide a definitive mechanism for salicylate-mediated inhibition of topo IIα and provide support for further studies determining the basis for its isoform selectivity.

Abstract B87: Preclinical investigation of the potentiation in antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, in combination with irinotecan (CPT-11) in colorectal cancer.

Abstract Background: Irinotecan (CPT-11) is a topoisomeraseI (TopoI) inhibitor, which is widely used for the treatment of metastatic colorectal cancer. CPT-11 inhibits DNA religation by TopoI, thus it stabilizes DNA-TopoI complexes and subsequently induces cytotoxicity. TAS-102, a novel oral antitumor agent, consisting of trifluorothymidine(FTD) and thymidine phosphorylase inhibitor, has reported promising clinical results in metastatic colorectal cancer. Several reports indicated that aberrant nucleoside incorporation into DNA, such as with cytarabine or gemcitabine, enhanced DNA-TopoI complexes. FTD, the effector of TAS-102, is a nucleoside which is efficiently incorporated and retained into DNA. Here, we evaluated the potency of TAS-102 in combination with CPT-11 in vitro and in vivo Materials and Methods: The combinational effect of FTD and SN-38 which is the active metabolite of CPT-11 was evaluated with several treatment schedules. Various colorectal cancer cell lines (SW48, SW620, LS174T and HCT116) were used in this assay. In one set, cells were washed after a 24 hour pre-exposure to FTD followed by a 48 hour exposure to SN-38 (FTD pre-exposure schedule); in the other set, cells were washed after a 6 hour pre-exposure to SN-38 followed by a 66 hour exposure to FTD (SN-38 pre-exposure schedule). A combinational intensity was estimated by using a combination index (CI) which indicates synergism (CI&amp;lt;0.8), additivity (0.8&amp;lt;CI&amp;lt;1.2), or antagonism (CI&amp;gt;1.2). Additionally, the antitumor effect of TAS-102 in combination with CPT-11 was investigated in a human colorectal cancer xenograft model. TAS-102 was orally administered for 14 days consecutively and CPT-11 was intravenously administered once a week to mice. Results: The combination of SN-38 and FTD showed a synergistic effect (SW48, SW620 and LS174T) or an additive effect (HCT116) in both the FTD pre-exposure schedule as well as the SN-38 pre-exposure schedule. Also, the enhancement of antitumor effect of TAS-102 in combination with CPT-11 was observed in vivo. Furthermore, these cell lines had various genotypes as regards KRAS, PI3CA and other DNA repair enzymes frequently detected in colorectal cancer, indicating that the combination's efficacy seemed to be independent of genetic status. Conclusion: TAS-102/CPT-11 represents a novel combination strategy for the treatment of advanced colorectal cancer. A synergistic effect was observed in both schedules tested, suggesting that various underlying mechanisms may exist. One potential mechanism is that FTD uptake or incorporation into DNA is increased by TopoI inhibition, and that FTD incorporation then further stabilizes the CPT-11 induced cleavable complex. However, the precise mechanism needs to be elucidated in the future. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B87. Citation Format: Keiji Ishida, Nozomu Tanaka, Sayaka Tsukioka, Kokoro Eshima, Akio Fujioka, Keisuke Yamamura, Satoko Ito, Kenichi Matsuo, Teruhiro Utsugi. Preclinical investigation of the potentiation in antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, in combination with irinotecan (CPT-11) in colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B87.

Discovery of indeno[1,2- $$c$$ c ]quinoline derivatives as dual topoisomerases I/II inhibitors: Part 3

(E) -6-Hydroxy-9-methoxy-6-(piperazin-1-yl)-11H -indeno[1,2-c ]quinolin-11-one O-2-(pyrrolidin-1-yl)ethyl oxime (2c) was identified as a potential dual topo I/II inhibitor in our previous paper. In continuation for the search of more potent compounds, we describe herein the preparation of certain indeno[1,2-c ]quinoline derivatives and evaluation of their antiproliferation, DNA binding affinity, and topoisomerases (topo I and topo II) inhibitory activities. Among them, (E) -9-[3-(dimethylamino)propoxy]-11H -indeno[1,2-c ]quinolin-11-one O-3-(dimethylamino)propyl oxime (11b) and its analog 11c exhibited approximately equal activity to the lead compound 2c against the growth of HeLa and A549 cancer cells. Both compounds 11b and 11c were more active than 2c in the inhibition of topo I and topo II. However, none of them exhibited significant DNA binding affinity while 2c was a very strong DNA binding agent. Compound 11b exhibited a high oral bioavailability of 39.8 % while the oral bioavailability of 2c and 11c was only 10.9 and 8.6 %, respectively. The in vivo anti-tumor evaluation of 11b in nude mice bearing subcutaneous breast cancer tumors revealed that treatment with low (10 mg/kg) or high (30 mg/kg) doses of 11b dramatically diminished tumor growth. Therefore, compound 11b is identified as a potential non-DNA intercalating dual topo I/II inhibitor.

Cell‐cycle specific association of transcription factors and RNA polymerase ii with the human β‐globin gene locus

The human β-globin genes are regulated by a locus control region (LCR) and are expressed at extremely high levels in erythroid cells. How transcriptional fidelity of highly expressed genes is regulated and maintained during the cell cycle is not completely understood. Here, we analyzed the association of transcription factor USF, the co-activator CBP, topoisomerase I (Topo I), basal transcription factor TFIIB, and RNA polymerase II (Pol II) with the β-globin gene locus at specific cell-cycle stages. The data demonstrate that while association of Pol II with globin locus associated chromatin decreased in mitotically arrested cells, it remained bound at lower levels at the γ-globin gene promoter. During early S-phase, association of CBP, USF, and Pol II with the globin gene locus decreased. The re-association of CBP and USF2 with the LCR preceded re-association of Pol II, suggesting that these proteins together mediate recruitment of Pol II to the β-globin gene locus during S-phase. Finally, we analyzed the association of Topo I with the globin gene locus during late S-phase. In general, Topo I association correlated with the binding of Pol II. Inhibition of Topo I activity reduced Pol II binding at the LCR and intergenic regions but not at the γ-globin gene promoter. The data demonstrate dynamic associations of transcription factors with the globin gene locus during the cell cycle and support previous results showing that specific components of transcription complexes remain associated with highly transcribed genes during mitosis.

Abstract 4653: DNA-PKcs dependent phosphorylation of topoI-S10 determines its ubiquitination by BRCA1/BARD1, rate of topoI degradation and CPT response.

Abstract Abstract: The age-adjusted mortality rate for cancer is about the same in the 21st century as it was 50 years ago. In the past decade, our understanding of oncogenesis at molecular level has increased greatly. This has resulted in the development of several targeted drugs and many more are in the pipeline. However, the lack of predictive biomarkers to stratify the patient population has restricted the benefits of these new developments. Camptothecins (CPTs) target topoisomeraseI (topoI) and represent a highly potent class of anticancer drugs. Two topoI inhibitors, topotecan and irrinotecan (CPT analogues), are in clinical use and several other topoI inhibitors are in different phases of development. SCLC, colon, ovarian, breast and cervical carcinomas are treated with topoI inhibitors. However, only 13% to 32% of patients respond to these drugs. TopoI mutations are rare in the patient population, and CPTs are not substrates for MDR transporters, therefore the rate of proteolytic degradation of topoI is a potential mechanism explaining CPT resistance. A differential rate of topoI degradation by the ubiquitin-proteosomal pathway (UPP) has been linked to CPT response, but the mechanism is not well understood. Using a functional proteomics approach, we have isolated a topoI interacting complex and have determined that: i) Ku70/Ku80/DNA-PKcs associates with topoI, and DNA-PKcs specifically phosphorylates topoI at S10, ii) S10-phosphorylated topoI is ubiquitinated by the BRCA1/BARD1 heterodimer, K-48 ubiquitin linkage indicates polyubiquitination and proteosomal degradation of topoI in response to CPT, iii) topoI S10 phosphorylation level determines the rate of topoI ubiquitination and proteosomal degradation in triple negative breast cancer (TNBC) and colon cancer cells. Higher levels of topoI pS10 ensures rapid degradation of topoI by the UPP and confers resistance to CPT. These cell based findings have further been tested in retrospective clinical studies. We have raised a monoclonal phosphospecific antibody against topoI-pS10 and shown its specificity in IF and IHC assays. In a retrospective study, we have performed IHC of FFPE tissues from metastatic CRC patients treated with FOLFIRI (combination therapy, 5FU, Leucovorin and Irinotecan). IHC analysis was performed in ten responders and ten non-responders tissues by anti topoI-pS10. Our preliminary data indicates that non-responders have very high level of topoI-pS10; in contrast the responders were negative for IHC staining. A larger retrospective study is underway to develop this predictive biomarker. Citation Format: Ankur K. Shah, Vibhu Sachdev, Julian Taylor Parker, Yevnegyia Malinkovich, Yara Hamade Tohme, Yiheng Hu, Jeffrey D. Parvin, Ajit K. Bharti. DNA-PKcs dependent phosphorylation of topoI-S10 determines its ubiquitination by BRCA1/BARD1, rate of topoI degradation and CPT response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4653. doi:10.1158/1538-7445.AM2013-4653 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 5656: Sustained delivery of CZ48, a lactone-stabilized camptothecin, by nanosuspensions.

Abstract Background: CZ48, a prodrug of camptothecin (CPT), shows promising antitumor activity as a topoisomerase I (Topo-I) inhibitor after its biotransformation to CPT in vivo. Studies establish that a prolonged exposure of Topo-I inhibitors with low concentrations is more efficacious in cancer treatment than a short term exposure with high concentrations, due to the S-phase cell cycle specific mechanism. However, poor solubility and the effective delivery challenges have been the obstacles for clinical applications of CZ48 to patients. Objectives: The research was to achieve the following objectives: (1) to prepare by wet media milling method and characterize CZ48 nanosuspensions (NSs); (2) to determine the pharmacokinetics (PK) of CZ48 and CPT from CZ48 NSs in Swiss nude mouse model; (3) to demonstrate the proof-of-concept efficacy of CZ48 NS in tumor-bearing athymic mouse model. Methods: CZ48 NSs of different sizes were formulated by media milling method. The in vitro release kinetics in plasma and phosphate buffered saline (PBS) solution (pH 7.4) were characterized, respectively, using the dialysis bag diffusion technique. The PK of CZ48 and CPT were evaluated in male Swiss nu-nu mice by intravenous (i.v.) doses of 5 and 25 mg/kg for cosolvent reference and NSs, respectively. The PK parameters of CZ48 and CPT were derived by WinNonlin 3.0. The proof-of-concept efficacy was performed in the subcutaneous tumor model of Non-small Cell Lung Cancer (NSCLC) H460 cell line in the nude mouse model. ANOVA with Tukey's post-hoc and Kaplan-Meier survival analysis were used for comparisons of PK modifications and efficacy of the CZ48 from NSs using the cosolvent as reference. Results: CZ48 NSs of particle sizes of 200 nm (NS-S) and 600 nm (NS-L) were successfully formulated and extensively characterized. The in vitro sustained release of CZ48 from NSs in plasma was achieved with 0.60 ± 0.12 %/h release rate and less than 40 % total release at 48 h, compared with 6.48 ± 0.29 %/h release rate and complete release by 28 h from cosolvent. NS-S and NS-L prolonged the blood circulation of CZ48 by 8.00, and 5.58 h, respectively, compare to 0.70 h from cosolvent. More importantly, NS-S yielded CPT with the longest half-life (12.51, 5.81 vs. 0.40 h) and largest AUC (19.78, 10.95 vs. 8.29 nM*h/(mg/kg)) than NS-L and cosolvent. CZ48-loaded NS-S exhibited significant tumor growth rate suppression compared to cosolvent (0.08 ± 0.03 vs. 0.18 ± 0.06/day) with a higher tolerable dose (25 vs. 5 mg/kg), and an improved survival rate (80% vs. 10%). Conclusion: This study is the first report to demonstrate the NS as a viable pharmaceutical carrier to deliver CZ48 in a sustained manner, achieving significant tumor growth suppression at a higher tolerable dose. The study provides a formulation strategy of great potentials to overcome the delivery barriers for other anticancer drugs. We anticipate that the CZ48 NS will be a lead candidate for the future clinical trials of CZ48. Citation Format: Diana S-L Chow, Dong Dong, Beppino C. Giovanella, Xin Liu, Cheng-Hui Hsiao, Yang Teng. Sustained delivery of CZ48, a lactone-stabilized camptothecin, by nanosuspensions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5656. doi:10.1158/1538-7445.AM2013-5656

Abstract 4367: Differential contribution of chromatin remodeling complex components to the protection from cytotoxic stress.

Abstract Anthracyclines are among the most common chemotherapeutic agents used to-date. Although effective, resistance to this agent usually develops in the course of treatment and serious dose-related toxicity, namely chronic cardio-toxicity, limits its use. In patients suffering from triple negative breast cancer (TNBC) cytotoxic agents such as anthracyclines are some of the only therapeutic alternatives. To increase our knowledge of the pathways that protect cells from anthracyclines we carried out a genome-wide genetic screening in the eukaryotic model organism S. cerevisiae to identify gene defects that generate cells hypersensitive to doxorubicin. A total of 71 deletion strains displayed varying levels of sensitivity to anthracylcines. Of these, 11 genes are involved in chromatin remodeling. The most sensitive mutations corresponded to swi3, snf2 and arp8, which are components of the SWI/SNF complex. Deletion of SNF2 resulted in sensitivity to doxorubicin of 100,000-fold higher than wild type cells. Further analysis of the SNF2 defective strain revealed that it is also sensitive to daunorubicin and cisplatin, as well as to other DNA damaging agents such as alkylating agents, the topoI inhibitor campthotecin or replication blocks from hydroxyurea. Our results indicate that the SWI/SNF complex plays a critical role in protecting cells from exposure to anthracyclines and other DNA damaging agents. Targeting these genes may provide a useful alternative to hypersensitize cancer cells to chemotherapy and provide a therapeutic alternative to TNBC. Research supported by NIH/NCRR RCMI grant #RR03020 at Florida A&amp;M University. Citation Format: Miles D. Freeman, Tryphon Mazu, Hernan Flores-Rozas. Differential contribution of chromatin remodeling complex components to the protection from cytotoxic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4367. doi:10.1158/1538-7445.AM2013-4367

Recent Advances in the Development of Catalytic Inhibitors of Human DNA Topoisomerase II&amp;#945; As Novel Anticancer Agents

DNA topoisomerases comprise an important family of enzymes that catalyse the induction of topological changes (e.g. relaxation/ supercoiling, catenation/decatenation and knotting/unknotting) in the DNA molecule. These enzymes perform their functions by creating transient either single-stranded or double-stranded breaks in the DNA molecule. Due to their ability to modulate the topology of the DNA molecule, DNA topoisomerases play vital roles in replication, transcription, chromosome separation and segregation, and thus represent an important collection of design targets for novel anticancer drugs. The aim of this review is to provide an overview of the development of catalytic inhibitors of the human topoisomerase IIα enzyme--an important member of the DNA topoisomerase family--as potential novel anticancer agents. The group of catalytic topoII inhibitors is classified into four types according to their molecular mechanism of action: inhibitors that bind to the ATP binding site, inhibitors that prevent the ATP hydrolysis step and trap the enzyme in a closed clamp, inhibitors that block the DNA cleavage and inhibitors that prevent the enzyme binding to the DNA. One of the important considerations highlighted throughout this review is the structure-based perspective of inhibitor design, giving the reader a medicinal chemist's perspective on this vibrant and active field of drug design research.