A N-acetilglicosamina 6-fosfato desacetilase (EC 3.5.1.25), uma enzima envolvida na via catabólica de açucares aminados da Escherichia coli, foi cristalizada pela técnica de difusão de vapor utilizando-se fosfato como agente precipitante. Experimentos de difração de raios-X mostraram que os cristais pertencem ao sistema cristalino ortorrômbico com grupo espacial P21212. Os parâmetros de cela são a=82,09(2) Å, b=114,50(1) Å e c=80,17(1) Å. Os cristais difrataram a uma resolução máxima de 1,8 Å e um conjunto de dados foi coletado até 2.0 Å. O conteúdo da unidade assimétrica provavelmente é um dímero, produzindo um coeficiente de Matthews de 2,30 Å3 Da-1 / N-acetylglucosamine 6-phosphate deacetylase (EC 3.5.1.25), an enzyme involved in amino sugar catabolismo from Escherichia coli has been crystallized by the vapor diffusion technique using phosphate as precipitant. X-ray diffraction experiments show the crystals to belong to the orthorhombic crystals system with space group P21212. The unit cell parameters are a=82,09(2) Å, b=114,50(1) Å e c=80,17(1) Å. The crystals diffract to a maximum resolution of 1.8 Å and a initial dataset was collected to 2.0 Å. The asymmetric unit content is likely to be a dimer, yielding a Matthews coefficient of 2.30 Å3 Da-1

Xiao, Zhangang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 118-131). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Histone deacetylases (HDACs) are a class of enzymes that play a crucial role in DNA expression by removing an acetyl group from the ɛ-N-acetyl lysine residue on histone proteins. Out of 18 isoforms of HDAC enzymes which are classified into 4 classes, only 11 of them are metalloenzymes that require zinc for its catalytic activity. HDACs are considered promising target for drug development in cancer and other parasitic diseases due to their role in gene expression. Histone deacetylase inhibitors (HDACi) can cause cell cycle arrest, and induce differentiation or apotosis. While HDACi shows promising antitumor effects, their mechanism of action and selectivity against cancer cells have not been adequately defined yet. In addition, low oral bioavailability, short half-life time, bone marrow toxicity, and cardiotoxicity limit their use in clinic. Therefore, there is considerable interest in developing compounds with selectivity and specificity towards individual family members of HDACs. The prototypical pharmacophore for HDAC inhibitors consist of a metal-binding moiety that coordinates to the catalytic metal ion within the HDAC active site, a capping group that interacts with the residues at the entrance of the active site and a linker that appropriately positions the metal-binding moiety and capping group for interactions in the active site. It has been shown that modification of cap, cap linking moiety, linker or zinc binding group (ZBG) shows promises of superior potency and isoform selectivity. My thesis research involves manipulating different aspects of the pharmacophoric model to yield not only more potent, selective, and effective drugs but also to help understand the biology of HDAC isoforms. In addition, I was successful in extending studies on HDAC isoforms to other zinc metalloenzymes such as leishmanolysin (gp63) and spliceosome associated zinc-metalloenzymes to understand biology of these zinc metalloenzymes by developing potent and selective small molecule inhibitors. This will aid in improvement of existing therapeutics for treatment of cancer, leishmania, malaria and other genetic disorders.

Histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated a wealth of biological effects, including anti-proliferative, anti-inflammatory, anti-parasitic, and cognition-enhancing activities. The recent FDA approvals of the inhibitors SAHA and FK-228 have validated HDACi clinical use in cutaneous T cell lymphoma, while numerous clinical trials are currently ongoing using HDACi against a variety of disease states. While the future of the HDAC field looks increasingly promising, there are lingering issues hindering broader use. Recent data point to dysregulation of specific HDAC isoforms in many disease states. However, most current HDACi are pan-inhibitors, lacking the specificity to target individual isoforms. Adding to this, there are currently 18 identified HDAC isoforms, and most lack a defined crystal structure, further complicating the task of designing isoform-specific inhibitors. Most importantly, HDACi have demonstrated a lack of efficacy against solid tumors in the clinic, a major obstacle to broader use in cancer therapy. Several of these issues could more fully be addressed through specific targeting of HDACi, and could bring HDACi into wider and more efficacious pharmaceutical use. Targeting the specific tissue or organelle where HDAC dysregulation occurs could confer greater efficacy in vivo. To this end, we have created four classes of compounds: (1) aryltriazolyl HDACi that potently inhibit HDAC activity and prostate cancer cell growth, (2) dual-targeted inhibitors of Topoisomerase II and HDAC and (3) dual-targeted inhibitors of Topoisomerase I and HDAC, both of which have potent inhibition against both target enzymes as well as cancer cell lines, and finally (4) macrocyclic HDACi that potently inhibit the growth of lung cancer cell lines and preferentially target lung tissue in vivo.

Das Pankreaskarzinom bleibt trotz verbesserter Diagnose- und Therapiemöglichkeiten weiterhin eine Krankheit mit einer sehr schlechten Prognose und Lebenserwartung nach Diagnosestellung. Eine innovative Therapiemöglichkeit stellt eine Gruppe von Histondeacetylase-Inhibitoren dar, die einen direkten Einfluss auf die Regulation der Genexpression in Tumorzellen haben. Das Ziel der vorliegenden Arbeit bestand darin, die Wirkung des HDAC-Inhibitors 4-Phenylbutyrat auf Pankreaskarzinomzellen in-vitro und vor allem in-vivo zu untersuchen. Neben dem Einfluss auf die Zellproliferation in-vitro und in-vivo wurde in-vivo im subkutanen und orthotopen Tumormodell der Einfluss auf Tumorwachstum, Zellproliferation, Nekroseausbreitung, Regulation des Connexin 43 und Histonacetylierung im Tumorgewebe untersucht. Die Untersuchungen zeigen, dass 4-PB durch seinen hemmenden Effekt auf das Wachstum von Xenografttumoren und auf die Proliferation von Pankreastumorzellen sowie durch seine fördernde Wirkung auf die Expression von Connexin 43, Acetylierung von H4 und Bildung eine Pseudokapsel, ein potentiell wirksames Medikament bei der experimentellen Behandlung des Pankreaskarzinoms ist.

Type-1 diabetes is caused by the autoimmune destruction of insulin-producing beta cells in the pancreas. Beta-cell apoptosis involves a complex set of signaling cascades initiated by \(interleukin-1\beta (IL-1\beta)\), \(interferon-\gamma (IFN-\gamma)\), and \(tumor necrosis factor-\alpha (TNF-\alpha)\). \(IL-1\beta\) and \(TNF-\alpha\) induce \(NF\kappa B\) expression, while \(IFN-\gamma\) induces STAT1 activation. These cytokines lead to a decrease of beta-cell function. The goal of this thesis is to identify small-molecule suppressors of cytokine-induced beta-cell apoptosis using high-throughput screening approach. Using the rat INS-1E beta-cell line, I developed an assay to measure cellular viability after 48 hours of cytokine treatment. I screened 29,760 compounds for their ability to suppress the negative effects of the cytokines. I identified several compounds to be suppressors of beta-cell apoptosis. These efforts led to the discovery of \(GSK-3\beta\) and HDAC3 as novel targets for suppressing beta-cell apoptosis. I also followed up on BRD0608, a novel suppressor that increased ATP levels and decreased caspase activity in the presence of cytokines. To follow up this compound, 35 analogs related to BRD0476 were synthesized using solid-phase synthesis and tested for their protective effects in the presence of cytokines. A structurally related analog, BRD0476, was found to be more potent and active in human islets, decreasing caspase activation and increasing insulin secretion after a 6-day treatment. I performed gene-expression profiling of INS-1E cells treated with the cytokine cocktail in the absence or presence of \(10\mu M\) BRD0476. Gene-set enrichment analysis revealed that the gene sets most significantly changed by BRD0476 involved cellular responses to \(IFN-\gamma\). I therefore assessed the effects of BRD0476 on STAT1 transcriptional activity. Cytokine treatment increased the reporter-gene luciferase activity, while co-treatment with BRD0476 reduced this activity significantly. To identify the intracellular target(s) of BRD0476, I collaborated with the Proteomics Platform in Broad Institute using SILAC (stable isotope labeling by amino acids in cell culture). SILAC is a mass spectrometry-based method to identify proteins that bind a small molecule attached to a bead. Deubiquitinase USP9X was pulled down by BRD0476. Knock-down of USP9X by siRNA phenocopied the protective effects of BRD0476. Binding assays were performed to identify interactions between BRD0476 and USP9X. / Chemistry and Chemical Biology

No description available.

Histone deacetylase (HDAC) inhibition is an emerging novel therapeutic strategy in cancer therapy. HDAC inhibitors (HDACi) have shown ability to block angiogenesis and cell cycling, as well as initiate differentiation and apoptosis. In fact, suberoylanilide hydroxamic acid (SAHA) is the first in the class of HDACi approved by the FDA for the treatment of cutaneous T cell lymphoma. On the other hand, there is a sustained interest in the use of gold nanoparticles (AuNPs) for various cancer diagnostic and therapeutic applications - bioimaging, drug delivery, and binary therapy techniques such as photodynamic and photothermal therapies. This interest in AuNPs is facilitated by favorable attributes such as ease of fabrication, bioconjugation and biocompatibility, and unique optical and electronic properties. However, HDACi- and AuNPs- based antitumor agents are plagued with problems common to all chemotherapeutic agents such as lack of selectivity, which often results in systemic toxicity. Therefore, availability of a methodology to selectively deliver AuNPs and HDACi to cancer cells will significantly improve their therapeutic indices and lead to the identification of novel agents for use in diagnostic imaging and targeted cancer therapy applications.

General toxicity evaluation and risk assessment for human exposure is essential when developing new pharmaceuticals and chemicals. Developmental toxicology is an important part of this risk assessment which consumes large resources and many laboratory animals. The prediction of developmental toxicity could potentially be assessed in vitro using embryo-derived pluripotent stem cells for lead characterization and optimization. This thesis explored the potential of short-time assays with pluripotent stem cells of embryonic origin using toxicogenomics. Three established pluripotent stem cell lines; P19 mouse embryonal carcinoma (EC) cells, R1 mouse embryonic stem (mES) cells, and SA002 human embryonic stem (hES) cells were used in the studies. Valproic acid (VPA), an antiepileptic drug which can cause the neural tube defects spina bifida in human and exencephaly in mouse, was used together with microarrays to investigate the global transcriptional response in pluripotent stem cells using short-time exposures (1.5 - 24 h). In addition to VPA, three closely related VPA analogs were tested, one of which was not teratogenic in mice. These analogs also differed in their ability to inhibit histone deacetylase (HDAC) allowing this potential mechanism of VPA teratogenicity to be investigated. The results in EC cells indicated a large number of genes to be putative VPA targets, many of which are known to be involved in neural tube morphogenesis. When compared with data generated in mouse embryos, a number of genes emerged as candidate in vitro markers of VPA-induced teratogenicity. VPA and its teratogenic HDAC inhibiting analog induced major and often overlapping deregulation of genes in mES cells and hES cells. On the other hand, the two non-HDAC inhibiting analogs (one teratogenic and one not) had only minor effects on gene expression. This indicated that HDAC inhibition is likely to be the major mechanism of gene deregulation induced by VPA. In addition, a comparison between human and mouse ES cells revealed an overlap of deregulated genes as well as species specific deregulated genes.

Inflammation involves a complex interplay of immunological responses in reaction to an infection from invading pathogens and to tissue injury. However, if the inflammatory stimulus is not eliminated or if there is an aberrant response, chronic inflammation and disease can result. Most inflammatory diseases are targeted by drugs that provide only symptomatic relief by reducing the associated pain and swelling, without combating the underlying cause and progression of the disease. The purpose of this thesis was to investigate potential drug targets in different sub-cellular compartments to learn more about inflammatory pathways and the consequences of inhibiting them using different classes of small-molecule agents. Chapter 1 is a brief overview of inflammation, outlining differences between acute and chronic inflammation in relation to normal and aberrant immune responses. Major drug classes that are currently in use are briefly discussed, and some potential new inflammatory drug targets in the nucleus, cytoplasm, plasma membrane and serum that are to be investigated in the thesis are described. Chapter 2 addresses histone deacetylase (HDAC) enzymes that modulate gene transcription, and investigates the regulatory potential of HDACs and their inhibitors in macrophages. Mainly murine macrophages were investigated, but preliminary data on human macrophages is also presented. Four kinds of small-molecule inhibitors of HDAC enzymes are described in relation to their capacity to exhibit pro- or anti- inflammatory gene expression. Broad-spectrum inhibitors, those that do not discriminate between different isoforms of HDACs, were found to induce expression of a number of pro-inflammatory mediators. This was also the case for compounds that selectively inhibit class-I HDACs. On the other hand, compounds that are known to selectively inhibit class-II HDACs did not regulate pro-inflammatory genes and could be classified as anti-inflammatory based on their profiles of gene expression. An inhibitor of class-III HDACs, the sirtuins, was also briefly investigated for anti-inflammatory gene expression. This separation of the pro- and anti-inflammatory responses suggested that specific inhibitors of class II HDACs might be promising for treating inflammatory conditions. Chapters 3 and 4 discuss a group of extracellular serum proteins that are produced through the complement pathway. Complement proteins circulate in plasma while some are formed on membranes through serine protease action, and effect both innate and adaptive immune responses. Chapter 3 deals with the alternative pathway (AP) of complement activation, in particular with the protease Factor B which plays an important role as part of a protein complex (C3 convertase) in the initiation and propagation of complement. Factor B is an inactive zymogen at pH 7, but is catalytically active at alkaline pH. Substrate-based inhibitors were developed for Factor B using a chromogenic assay previously optimised in our lab. Over 60 peptide aldehydes were evaluated, with two potent inhibitors of Factor B studied further, and were found to inhibit C3 convertase by blocking cleavage of the native substrate C3 as measured by SDS-PAGE. Chapter 4 examined the properties of a homologue of Factor B found in the classical pathway (CP), the protease C2. C2 was found herein to be an inactive zymogen at pH 7. Under alkaline conditions C2 cleaved both C3 and short chromogenic peptide substrates. Substrates of 7- or 8-residues, corresponding to those in the native substrate C3, were optimally processed. C2 was inhibited by inhibitors identified in Chapter 3, which also inhibited formation of the different C3 convertase formed through the CP, as well as preventing formation of the terminal membrane attack complex (MAC) as measured by an immobilized ELISA and haemolysis assay. The selectivity of small-molecule peptidic inhibitors for different serine proteases was investigated. Chapter 5 introduces two other unrelated inflammatory proteins which have been incompletely studied herein. Protease-activated receptor 2 (PAR-2) is a cell surface protein that spans the membrane like other G-protein coupled receptors. PAR-2 has been implicated in inflammatory and proliferative diseased states, though its precise roles are still unclear. Here, PAR-2 mRNA expression was measured for different human cancer and immune cells. In macrophages, PAR-2 mRNA expression was inducible by HDACi, indicating that PAR-2 is negatively regulated by HDAC enzymes. The receptor was present and functional on the surface of HDACi-treated macrophages, permitting evaluation of PAR-2 agonists and antagonists. Caspase-1 is a cytosolic cysteine protease responsible for cleaving proIL-1β and proIL-18 into their active forms that are then secreted from cells as pro-inflammatory cytokines. Using monocytic cells, novel inhibitors of caspase-1 were developed and found to dose-dependently decrease levels of IL-1β released by the cell. Inhibition of caspase-1 may be useful for modulating circulating levels of the inflammatory cytokine IL-1β. The investigation of different prospective inflammatory targets in different locations of the cell has provided a lateral overview of inflammatory mechanisms and drug intervention strategies. Specific inhibitors have also been identified for these targets; HDACs in the nucleus, caspase-1 in the cytosol, PAR-2 and MAC on the cell surface, C2 and Factor B and C3 convertase in plasma. This unconventional study of the inflammatory network provides a different viewpoint of inflammatory proteins as drug targets.