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Design and in Vitro Characterization of Tricyclic Benzodiazepine Derivatives as Potent and Selective Antileukemic Agents

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Abstract

Currently available chemotherapeutic treatments for blood cancers (leukemia) usually have strong side effects. More selective, efficient and less toxic anticancer agents are needed. We synthesized seven, new, optically pure (12aS)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12-(2H,11H)-dione derivatives and examined their cytotoxicity towards eight cancer cell lines, including urinary bladder (TCC-SUP, UM-UC-3, KU-19-9), colon (LoVo), and breast (MCF-7, MDA-MB-231) cancer representatives, as well as two leukemic cell lines (MV-4-11, CCRF-CEM) and normal murine fibroblasts (Balb/3T3) as a reference cell line. Three of the seven newly-obtained compounds ((12aS)-8-bromo-2-(3-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione, (12aS)-8,9-dimethoxy-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione and (12aS)-8-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione, showed enhanced activity and selectivity toward the leukemic MV-4-11 cell line when compared to our previously reported compounds, with IC50 values in the range of 2.9-5.6 µM. Additionally, (12aS)-9-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione exhibited a strong cytotoxic effect against the leukemic CCRF-CEM (IC50 = 6.1 µM) and MV-4-11 (IC50 = 11.0 µM) cell lines, a moderate cytotoxic effect toward other tumor lines (IC50 = 31.8-55.0 µM) and very weak cytotoxic effect toward the Balb/3T3 reference cell line. Selected compounds were further evaluated for their potential to induce apoptotic cell death in MV-4-11 cells by measuring caspase-3 activity. We also obtained the crystal structure of three products and investigated the effect of 22 derivatives of 1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12-(2H,11H)-dione on the activity of the cancer-associated enzyme autotaxin. All compounds proved to be weak inhibitors of autotaxin, although some (R) and (S) enantiomers had Ki values of 10-19 µM. The obtained results showed that the tested compounds exhibited a selective anti-leukemic effect, which appeared not to be related directly to autotaxin. Molecular targets responsible for this effect remain to be identified. The newly obtained compounds can be used in the search for new, selective anticancer therapies.

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Preprint
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AutoDock Vina, a new program for molecular docking and virtual screening, is presented. AutoDock Vina achieves an approximately two orders of magnitude speed-up compared with the molecular docking software previously developed in our lab (AutoDock 4), while also significantly improving the accuracy of the binding mode predictions, judging by our tests on the training set used in AutoDock 4 development. Further speed-up is achieved from parallelism, by using multithreading on multicore machines. AutoDock Vina automatically calculates the grid maps and clusters the results in a way transparent to the user.
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Introduction: Autotaxin (ATX) is a lysophospholipase D enzyme that hydrolyzes lysophosphatidylcholine to lysophosphatidic acid (LPA) and choline. LPA is a bioactive lipid mediator that activates several transduction pathways, and is involved in migration, proliferation and survival of various cells. Thus, ATX is an attractive medicinal target. Areas covered: The aim of this review is to summarize ATX inhibitors, reported in patents from 2006 up to now, describing their discovery and biological evaluation. Expert opinion: ATX has been implicated in various pathological conditions, such as cancer, chronic inflammation, neuropathic pain, fibrotic diseases, etc. Although there is an intensive effort on the discovery of potent and selective ATX inhibitors in order to identify novel medicinal agents, up to now, no ATX inhibitor has reached clinical trials. However, the use of ATX inhibitors seems an attractive strategy for the development of novel medicinal agents, for example anticancer therapeutics.
Article
Autotaxin (ATX) has been reported to act as a motility and growth factor in a variety of cancer cells. The ATX protein acts as a secreted lysophospholipase D (lysoPLD) by converting lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which signals via G-protein coupled receptors and has important functions in cell migration and proliferation. The current study demonstrates that ATX expression is specifically upregulated and functionally active in acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) mutation of the FLT3 receptor gene. Moreover, ATX expression was also found in normal human CD34+ progenitor cells and selected myeloid and lymphoid subpopulations. Enforced expression of mutant FLT3-ITD by retroviral vector transduction increased ATX mRNA in selected cell lines, whereas inhibition of FLT3-ITD signaling by sublethal doses of PKC412 or SU5614 led to a significant down-regulation of ATX mRNA and protein levels. In the presence of LPC, ATX expression significantly increased proliferation. LPA induced proliferation, regardless of ATX expression and induced chemotaxis in all tested human leukemic cell lines and human CD34+ progenitors. LPC increased chemotaxis only in cells with high expression of endogenous ATX by at least 80% demonstrating the autocrine action of ATX. Inhibition of ATX using a small molecule inhibitor selectively induced killing of ATX-expressing cell lines and reduced motility in these cells. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration during hematopoiesis and that deregulation of ATX contributes to the pathogenesis of AML.
Article
A study was conducted to demonstrate chemical evolution of autotaxin (ATX) inhibitors. Alternative splicing of the ATX gene resulted in three distinct isoforms, which were differentially expressed. ATX was initially produced as a pre-proenzyme that had an N-terminal signal peptide required for secretion. This signal peptide was removed by a signal peptidase, and ATX was cleaved by proprotein convertases (PCs)-like furin. The removal of an N-terminal octapeptide in ATX by PCs was associated with an enhancement of ATX activity. The proteolytically processed ATX was secreted and consisted of several domains where it had two somatomedin B (SMB)-like domains, a central catalytic phosphodiesterase (PDE) domain, and an inactive nuclease-like domain, starting from its N-terminus. The hydrolytic activity of ATX predominantly originated from a threonine residue and two zinc ions in the ATX active site located in the PDE domain.
Article
A series of SAHA cap derivatives was designed and prepared in good-to-excellent yields that varied from 49% to 95%. These derivatives were evaluated for their antiproliferative activity in several human cancer cell lines. Antiproliferative activity was observed for concentrations varying from 0.12 to >100 microM, and a molecular modeling approach of selected SAHA derivatives, based on available structural information of human HDAC8 in complex with SAHA, was performed. Strikingly, two compounds displayed up to 10-fold improved antileukemic activity with respect to SAHA; however, these compounds displayed antiproliferative activity similar to SAHA when assayed against solid tumor-derived cell lines. A 10-fold improvement in the leukemic vs peripheral blood mononuclear cell therapeutic ratio, with no evident in vivo toxicity toward blood cells, was also observed. The herein-described compounds and method of synthesis will provide invaluable tools to investigate the molecular mechanism responsible for the reported selectively improved antileukemic activity.
Article
Autotaxin (ATX, autocrine motility factor, NPP2) has recently emerged as an attractive target for the development of anti-cancer chemotherapeutics. ATX contributes to the production of the bioactive lipid, lysophosphatidic acid (LPA), from lysophosphatidyl choline (LPC) in biological fluids including plasma, serum, and tumor cell effusates. LPA-stimulated cell proliferation, survival, motility and invasion have been demonstrated by numerous research groups. LPA receptors and ATX are upregulated in numerous cancer cell types and show expression patterns that correlate with tumor cell invasiveness. Despite considerable promise as an anti-cancer target, two complex challenges have slowed inhibitor discovery. The first of these challenges has been a lack of experimental details of the enzyme structure and its interactions with substrates or inhibitors. A second challenge has been a lack of structural diversity among initially reported inhibitors. Research reported in the last two years provides a foundation to begin addressing these challenges. Although an experimental structure of ATX is not among these recent developments, a crystal structure of the bacterial enzyme Xac. NPP is now available. This protein shares 35% identity with the central catalytic domain of ATX and provides an important starting point to begin understanding the structure of ATX. The structural diversity of known inhibitors has recently expanded to include not only phospholipid analogs, but also small molecules containing thiourea, diphenyldiazerenyl, anthracenedione and indole central cores. These two developments are essential tools for the discovery and optimization of ATX-targeted agents for evaluation as anti-cancer chemotherapeutic agents.
Article
Autotaxin (ATX) is a tumour cell motility-stimulating factor originally isolated from melanoma cell supernatants. ATX is identical to lysophospholipase D, which produces a bioactive lipid mediator, lysophosphatidic acid (LPA), from lysophosphatidylcholine. ATX is overexpressed in various malignancies, including Hodgkin lymphoma, and ATX may stimulate tumour progression via LPA production. The present study measured the serum ATX antigen levels in patients with haematological malignancies using a recently developed automated enzyme immunoassay. The serum ATX antigen levels in patients with B-cell neoplasms, especially follicular lymphoma (FL), were higher than those in healthy subjects. Serum ATX antigen levels in FL patients were associated with tumour burden and changed in parallel with the patients' clinical courses. The serum ATX antigen levels were little affected by inflammation, unlike the soluble interleukin-2 receptor and beta2-microglobulin levels. As expected, the plasma LPA levels in FL patients were correlated with the serum ATX antigen levels. Given that leukaemic tumour cells from FL patients expressed ATX, the shedding of ATX from lymphoma cells probably leads to the elevation of serum ATX antigen levels. Our results suggest that the serum ATX antigen level may be a promising and novel marker for FL.
Article
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
Article
Although patients with cancer may derive much benefit from treatment, they are at risk for developing life-threatening complications. Hypersensitivity reactions can be severe, as in the case of anaphylaxis with L-asparaginase. Cardiac toxicities consist of arrhythmias with various drugs, hemorrhagic myocarditis with cyclophosphamide and ifosfamide, cardiomyopathy with anthracyclines, and pericardial disease. Acute respiratory failure may occur as a result of ARDS caused by ATRA or cytarabine, from interstitial fibrosis, or from pulmonary veno-occlusive disease. Hemorrhagic cystitis caused by cyclophosphamide and ifosfamide can be severe and result in exsanguination if unresponsive to treatment. Disseminated intravascular coagulation and thrombotic microangiopathy can produce thrombotic or hemorrhagic complications. Gastrointestinal toxicities include significant hepatotoxicity with a variety of drugs and development of acute surgical abdomen.
Article
The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
Article
An account is given of the development of the SHELX system of computer programs from SHELX-76 to the present day. In addition to identifying useful innovations that have come into general use through their implementation in SHELX, a critical analysis is presented of the less-successful features, missed opportunities and desirable improvements for future releases of the software. An attempt is made to understand how a program originally designed for photographic intensity data, punched cards and computers over 10000 times slower than an average modern personal computer has managed to survive for so long. SHELXL is the most widely used program for small-molecule refinement and SHELXS and SHELXD are often employed for structure solution despite the availability of objectively superior programs. SHELXL also finds a niche for the refinement of macromolecules against high-resolution or twinned data; SHELXPRO acts as an interface for macromolecular applications. SHELXC, SHELXD and SHELXE are proving useful for the experimental phasing of macromolecules, especially because they are fast and robust and so are often employed in pipelines for high-throughput phasing. This paper could serve as a general literature citation when one or more of the open-source SHELX programs (and the Bruker AXS version SHELXTL) are employed in the course of a crystal-structure determination.
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Modified Cap Group Suberoylanilide Hydroxamic Acid Histone Deacetylase Inhibitor Derivatives Reveal Improved Selective Antileukemic Activity’
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(S)-2-(4-Chlorobenzoyl)-1,2,3,4-tetrahydrobenzo[e][1,2-a][1,4],12(11H,12aH)-dione – Synthesis and Crystallographic Studies’
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Molecular modelling guided design, synthesis and QSAR analysis of new small molecule non-lipid autotaxin inhibitors’
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