Dual Inhibitor Of Topoisomerase Research Articles

Abstract B37: Selective tumor localization of CRLX101, a novel nanoparticle-drug conjugate

Abstract CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin conjugated to a biocompatible copolymer of cyclodextrin and polyethylene glycol (PEG), is currently being evaluated clinically in multiple treatment-refractory solid tumors. CRLX101 is a dual inhibitor of topoisomerase 1 (TOPO 1) and hypoxia-inducible factor 1α (HIF-1α) and has been shown pre-clinically to be active in many different tumor types. It has a long circulation half-life and is believed to release camptothecin in a slow and prolonged fashion in the tumor. Camptothecin is an active anti-tumor agent preclinically but was not developed clinically due to its poor tolerability in patients. The development of CRLX101, which has not demonstrated significant toxicity in over 250 patients dosed to date, offers a unique opportunity to improve cancer treatment in a meaningful way. We have previously published that in gastric cancer patients treated with CRLX101, camptothecin fluorescence can be visualized in tumor tissue but not adjoining normal tissue. Based on these data we hypothesized that CRLX101 utilizes the enhanced permeability and retention (EPR) effect to accumulate selectively in tumors. In this study, we sought to mechanistically dissect the process of CRLX101 entry and accumulation into tumor cells using multiple methods, both in vitro and in xenograft tumors in vivo. Using confocal microscopy, we can detect camptothecin fluorescence in CRLX101- treated tumor cells in culture as well as in tumor tissue from mice treated with CRLX101. We can co-localize this camptothecin with intact nanoparticles using an anti-PEG antibody that specifically detects the PEG loops in the NDCs. We can also demonstrate that macropinocytosis plays a role in the manner by which tumor cells take up CRLX101. Using an anti-CD31 antibody, we can visualize the distance traversed by CRLX101 from the tumor vasculature over time. We have developed novel analytical methods to precisely quantify both released and CRLX101-conjugated camptothecin over time in CRLX101 treated tumor cells in vitro, as well as in tumor tissue from mice treated with CRLX101 in vivo. Using cell viability assays, we can correlate the kinetics of camptothecin released inside tumor cells to the degree of tumor cell kill. We believe that these data are an important step forward in understanding the precise mechanism(s) underlying selective delivery of CRLX101 into tumor tissue. Citation Format: Christian G. Peters, Douglas Lazarus, Donna Brown, Ningning Zhang, Roy Case, Ellen Rohde, Scott Eliasof, Lata Jayaraman. Selective tumor localization of CRLX101, a novel nanoparticle-drug conjugate. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B37.

The dual topoisomerase inhibitor A35 preferentially and specially targets topoisomerase 2α by enhancing pre-strand and post-strand cleavage and inhibiting DNA religation.

DNA topoisomerases play a key role in tumor proliferation. Chemotherapeutics targeting topoisomerases have been widely used in clinical oncology, but resistance and side effects, particularly cardiotoxicity, usually limit their application. Clinical data show that a decrease in topoisomerase (top) levels is the primary factor responsible for resistance, but in cells there is compensatory effect between the levels of top1 and top2α. Here, we validated cyclizing-berberine A35, which is a dual top inhibitor and preferentially targets top2α. The impact on the top2α catalytic cycle indicated that A35 could intercalate into DNA but did not interfere with DNA-top binding and top2α ATPase activity. A35 could facilitate DNA-top2α cleavage complex formation by enhancing pre-strand and post-strand cleavage and inhibiting religation, suggesting this compound can be a topoisomerase poison and had a district mechanism from other topoisomerase inhibitors. TARDIS and comet assays showed that A35 could induce cell DNA breakage and DNA-top complexes but had no effect on the cardiac toxicity inducer top2β. Silencing top1 augmented DNA break and silencing top2α decreased DNA break. Further validation in H9c2 cardiac cells showed A35 did not disturb cell proliferation and mitochondrial membrane potency. Additionally, an assay with nude mice further demonstrated A35 did not damage the heart. Our work identifies A35 as a novel skeleton compound dually inhibits topoisomerases, and predominantly and specially targets top2α by interfering with all cleavage steps and its no cardiac toxicity was verified by cardiac cells and mice heart. A35 could be a novel and effective targeting topoisomerase agent.

Design, synthesis and biological evaluation of novel 7-alkylamino substituted benzo[a]phenazin derivatives as dual topoisomerase I/II inhibitors

A novel series of benzo[a]phenazin derivatives bearing alkylamino side chains were designed, synthesized and evaluated for their topoisomerases inhibitory activity as well as cytotoxicity against four human cancer cell lines (HL-60, K-562, HeLa, and A549). These compounds were found to be dual inhibitors of topoisomerase (Topo) I and Topo II, and exhibited excellent antiproliferative activity, in particular against HL-60 cells with submicromolar IC50 values. Further mechanistic studies showed that this class of compounds acted as Topo I poisons by stabilizing the Topo I-DNA cleavage complexes and Topo II catalytic inhibitors by inhibiting the ATPase activity of hTopo II. Molecular docking studies revealed the binding modes of these compounds for Topo I and Topo II.

ChemInform Abstract: Synthesis and Biological Evaluation of Benzo[a]phenazine Derivatives as a Dual Inhibitor of Topoisomerase I and II.

AbstractMost of the title compounds show good antiproliferative activity against four cancer cell lines tested.

The discovery and optimization of novel dual inhibitors of topoisomerase ii and histone deacetylase

A novel class of podophyllotoxin derivatives have been designed and synthesized based on the synergistic antitumor effects of topoisomerase II and histone deacetylase inhibitors. Their inhibitory activities towards histone deacetylases and Topo II and their cytotoxicities in cancer cell lines were evaluated. The aromatic capping group connection, linker length and zinc-binding group were systematically varied and preliminary conclusions regarding structure–activity relationships are discussed. Among all of the synthesized hybrid compounds, compound 24d showed the most potent HDAC inhibitory activity at a low nanomolar level and exhibited powerful antiproliferative activity towards HCT116 colon carcinoma cells at a low micromolar level. Further exploration of this series led to the discovery of potent dual inhibitor 32, which exhibited the strongest in vitro cytotoxic activity.

Correction: STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G2Arrest in the Absence of DNA Damage

The third author's name was spelled incorrectly. The correct name is: N.R. Thimmegowda. The correct citation is: Kim S-O, Sakchaisri K, Thimmegowda NR, Soung NK, Jang J-H, et al. (2013) STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G2 Arrest in the Absence of DNA Damage. PLoS ONE 8(1): e53908. doi:10.1371/journal.pone.0053908

STK295900, a dual inhibitor of topoisomerase 1 and 2, induces G(2) arrest in the absence of DNA damage.

STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G2 phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent.

Synthesis and biological evaluation of benzo[a]phenazine derivatives as a dual inhibitor of topoisomerase I and II

Topoisomerases (Topo I and Topo II) are very important players in DNA replication, repair, and transcription, and are a promising class of antitumor target. In present study, a series of benzo[a]phenazine derivatives with alkylamino side chains at C-5 were designed, synthesized, and their biological activities were evaluated. Most of derivatives showed good antiproliferative activity with a range of IC50 values of 1-10 μM on the four cancer cell lines HeLa, A549, MCF-7, and HL-60. Topoisomerase-mediated DNA relaxation assay results showed that derivatives could effectively inhibit the activity of both Topo I and Topo II, and the structure-activity relationship studies indicated the importance of introducing an alkylamino side chain. Further mechanism studies revealed that the compounds could stabilize the Topo I-DNA cleavage complexes and inhibit the ATPase activity of hTopo II, indicating that they are a rare class of dual topoisomerase inhibitors by acting as Topo I poisons and Topo II catalytic inhibitors. Moreover, flow cytometric analysis and caspase-3/7 activation assay showed that this class of compounds could induce apoptosis of HL-60 cells.

Abstract A169: Identification of compounds for treatment of solid tumors using spheroid screening

Abstract A primary aim of cancer therapy is to irradicate solid tumor masses. Anticancer drugs are nevertheless not systematically developed with this aim in mind - screening and mechanistic studies are usually performed using cells grown under 2-D monolayer conditions. We have developed a procedure to screen for compounds that induce apoptosis of 3-D multicellular tumor spheroids. The chemical properties of spheroid screening hits differed significantly from conventional monolayer screening hits with regard to compound hydrophobicity and polar surface area. The spheroid screen resulted in hits which were more effective than standard chemotherapeutical agents in inducing wide-spread apoptosis of tumor spheroids. Hypotheses regarding the mechanisms of action for the selected compounds were generated using the Connectivity Map (CMAP; a compendium of gene expression signatures from drug treated cell lines). One of the hits in the spheroid screen was the alkaloid thaspine from the South American tree Croton lechleri which we show to be a dual topoisomerase inhibitor. Thaspine is insensitive to overexpression of drug efflux transporters and induces apoptosis of tumor cells in vivo. Counter-screening for compounds that do not induce apoptosis of normal epithelial cells resulted in a limited set of agents, most of which were found to be novel microtubuli inhibitors. The results show that interesting anti-cancer drugs can be identified using spheroid screening. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A169.

Identification of a Novel Topoisomerase Inhibitor Effective in Cells Overexpressing Drug Efflux Transporters

BackgroundNatural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology.Method and FindingsA library of natural products (NCI Natural Product set) was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine), an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo.ConclusionsThe alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

Novel tetra-acridine derivatives as dual inhibitors of topoisomerase II and the human proteasome

Acridine derivatives, such as amsacrine, represent a well known class of multi-targeted anti-cancer agents that generally interfere with DNA synthesis and inhibit topoisomerase II. But in addition, these tricyclic molecules often display secondary effects on other biochemical pathways including protein metabolism. In order to identify novel anti-cancer drugs, we evaluated the mechanism of action of a novel series of bis- and tetra-acridines. As expected, these molecules were found to interact with DNA and inhibit the topoisomerase II-mediated DNA decatenation. Interestingly when tested on human tumour cells either sensitive (HL-60) or resistant (HL-60/MX2) to topoisomerase II inhibitors, these molecules proved equicytotoxic against the two cell lines, suggesting that they do not only rely on topoisomerase II inhibition to exert their cytotoxic effects. In order to identify alternative targets, we tested the capacity of acridines 1– 9 to inhibit the proteasome machinery. Four tetra-acridines inhibited the proteasome in vitro, with IC 50 values up to 40 times lower than that of the reference proteasome inhibitor lactacystin. Moreover, unlike peptide aldehydes used as reference inhibitors for the proteasome, these new acridine compounds demonstrated a good selectivity towards the proteasome, when tested against four unrelated proteases. A cellular assay based on the degradation of a proteasome protein substrate indicated that at least two of the tetra-acridines maintained this proteasome inhibition activity in a cellular context. This is the first report of tetra-acridines that demonstrate dual topoisomerase II and proteasome inhibition properties. This new dual activity could represent a novel anti-cancer approach to circumvent certain forms of tumour resistance.

Induction of genotoxic and cytotoxic damage by aclarubicin, a dual topoisomerase inhibitor

The anthracycline aclarubicin (ACLA) is an intercalative antibiotic and antineoplastic agent that efficiently binds to DNA, leading to a secondary inhibition of the catalytic activity of topoisomerase II (topo II) on DNA. Besides this activity, ACLA has been reported to exert a concomitant poisoning effect on topo I, in a fashion similar to that of the antitumor drug camptothecin and its derivatives. As a consequence of this dual (topo II catalytic inhibiting/topo I poisoning) activity of ACLA, the picture is somewhat confusing with regards to DNA damage and cytotoxicity. We studied the capacity of ACLA to induce catalytic inhibition of topo II as well as cytotoxic effects and DNA damage in cultured Chinese hamster V79 cells and their radiosensitive counterparts irs-2. The ultimate purpose was to find out whether differences could be observed between the two cell lines in their response to ACLA, as has been widely reported for radiosensitive cells treated with topo poisons. Our results seem to agree with the view that the radiosensitive irs-2 cells appear as hypersensitive ACLA as compared with radiation repair-proficient V79 cells. The recovery after ACLA treatment was also followed-up, and the irs-2 mutant was found to be less proficient than V79 to repair DNA strand breaks induced by ACLA.

Ex vivo effects of the dual topoisomerase inhibitor tafluposide (F 11782) on cells isolated from fresh tumor samples taken from patients with cancer

Tafluposide (F 11782), a novel epipodophylloid with a unique mechanism of interaction with both topoisomerase I and II, has shown outstanding antitumor activity in vivo against a panel of experimental human tumor xenografts. The aim of this study was to evaluate its cytotoxicity against fresh tumor cells taken from patients. Cells derived from bone marrow, peripheral blood, malignant effusions or solid biopsies from 84 patients with either hematological or solid tumors were exposed continuously to 0.8-100 nuM tafluposide for 48 h, 96 h or 7 days. Cell survival was measured using an MTT assay or the ATP assay and LC(50) values (drug concentration required for 50% cell kill) were calculated. Tafluposide showed significant cytotoxicity against cells derived from either hematological or solid tumors, with a marked inter-patient variation. There was no significant difference between the effect of tafluposide in samples from untreated or previously treated patients (p>0.05 for all cancer types). Whilst tafluposide appeared to show weak (p<0.05) cross-resistance with the topoisomerase II inhibitor etoposide in acute myeloid leukemia (AML), there did not appear to be any correlation with the effect of the topoisomerase I inhibitor topotecan (p>0.05) in either hematological or solid malignancies. True synergism was identified when combining tafluposide with cisplatin in ovarian cancer [combination index (CI)=0.14, 0.79] and with etoposide in AML (CI=0.49, 0.63 and 0.78). Our results suggest that tafluposide is a strong candidate for inclusion in clinical trials, particularly in hematological malignancies.

Oxa-aza-benzo[de]anthracenes: design, synthesis and evaluation of a structurally new class of dual topoisomerase inhibitors

Oxa-aza-benzo[de]anthracenes: design, synthesis and evaluation of a structurally new class of dual topoisomerase inhibitors

A Dual Topoisomerase Inhibitor, TAS‐103, Induces Apoptosis in Human Cancer Cells

TAS‐103 (6‐[[2‐(dimethylamino)ethyl]amino]‐3‐hydroxy‐7H‐indeno[2,1‐c]quinolin‐7‐one dihydrochloride), a dual topoisomerase (topo) inhibitor, was developed as an anticancer agent by targeting topo I and topo II and has previously been shown to be effective against lung tumors. In this study, we investigated the cytotoxic activity of TAS‐103 in various human cancer cell lines (including gastric, colon, squamous, lung, and breast cancer cells) and the induction of apoptosis by TAS‐103. We next established stable transfectants of Bcl‐2 in the gastric cancer cell line AZ521 and found that Bcl‐2 blocked TAS‐103‐induced apoptosis. In addition, we demonstrated that the activities of ICE‐like and CPP32‐like proteases are involved in the signal transduction pathway of TAS‐103‐induced apoptosis. In summary, TAS‐103 is a novel type of anticancer agent with a unique mechanism and could be useful as a lead compound for development of new drugs.

Combination effects of TAS-103, a novel dual topoisomerase I and II inhibitor, with other anticancer agents on human small cell lung cancer cells.

TAS-103 [6-((2-(dimethylamino) ethyl)amino)-3-hydroxy-7H-iindeno(2,1-c)quinolin-7-one dihydrochloride] is a newly synthesized dual inhibitor of topoisomerase I and II. Since anticancer drugs are used in combination with other drugs for effective chemotherapy, we investigated the cytotoxic effect of TAS-103 in combination with other conventional anticancer agents, such as cisplatin, vindesine, doxorubicin, 5-fluorouracil, and the antitopoisomerase inhibitors SN-38 and etoposide in vitro. Inhibition of the growth of the human small-cell lung cancer cell line SBC-3 was evaluated using the tetrazolium dye (MTT) assay. Drug interactions were evaluated by isobologram analysis and the determination of combination indices supplemented by a three-dimensional model. Simultaneous use of TAS-103 and cisplatin had a supraadditive effect, but combinations of TAS-103 with other drugs had an additive or marginally subadditive effect. Three-dimensional model analysis added more information about the synergistic concentration ranges of two drugs (cisplatin 200-400 nM and TAS-103 7 10 nM). Sequential use of TAS-103 and cisplatin had only an additive effect. These results suggest that the concomitant use of TAS-103 and cisplatin has a greater cytotoxic effect on cancer cells than single drug use, and may provide a beneficial effect in the treatment of small-cell lung cancer.

Novel selective inhibitors for human topoisomerase I, BM2419-1 and -2 derived from saintopin.

Compounds BM2419-1 and -2 were isolated from a culture broth of a fungus Paecilomyces sp. BM2419. It was shown that these novel compounds were artifacts derived from saintopin, a dual inhibitor of topoisomerase I and II by independent processes. In the human topoisomerase I inhibition assay using the recombinant Saccharomyces cerevisiae, BM2419-1 and -2 inhibited selectively the yeast growth dependent on human topoisomerase I induction with IC50 values of 0.3 ng/ml and 6.0 ng/ml, respectively.