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.

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.

abstract: The effects of specific histone deacetylase inhibitors (HDACi) on transgene expression in combination with a novel polymer as a delivery vehicle are investigated in this research. Polymer vectors, although safer than viruses, are notorious for low levels of gene expression. In this investigation, the use of an emerging chemotherapeutic anti-cancer drug molecule, HDACi, was used to enhance the polymer-mediated gene expression. HDACi are capable of inhibiting deacetylation activities of histones and other non-histone proteins in the cytoplasm and nucleus, as well as increase transcriptional activities necessary for gene expression. In a prior study, a parallel synthesis and screening of polymers yielded a lead cationic polymer with high DNA-binding properties, and even more attractive, high transgene expressions. Previous studies showed the use of this polymer in conjunction with cytoplasmic HDACi significantly enhanced gene expression in PC3-PSMA prostate cancer cells. This led to the basis for the investigation presented in this thesis, but to use nuclear HDACi to potentially achieve similar results. The HDACi, HDACi_A, was a previously discovered lead drug that had potential to significantly enhance luciferase expression in PC3-PSMA cells. The results of this study found that the 20:1 polymer:plasmid DNA weight ratio was effective with 1 uM and 2 uM HDACI_A concentrations, showing up to a 9-fold enhancement. This enhancement suggested that HDACi_A was effectively aiding transfection. While not an astounding enhancement, it is still interesting enough to investigate further. Cell viabilities need to be determined to supplement the results. / Dissertation/Thesis / M.S. Bioengineering 2012

No description available.

Breast cancer is one of the leading causes of death in women cancer cases worldwide. Cancer is the result of environmental and genetic factors that contribute to alterations in cellular control, proliferation, differentiation and apoptosis. Vitamin D is emerging as an important nutrient in the prevention and treatment of cancer due to its ability to modulate proliferation and apoptosis in vivo and in vitro. To accomplish this, Vitamin D exerts its biological activity by binding to a specific, high-affinity intracellular vitamin D receptor (VDR). VDR expression is identified in mammary cancer cell lines, but levels are reduced compared to non-cancerous cells, which limits vitamin D-induced gene expression. Our study investigated two compounds with histone deacetylase inhibitor (HDACI) activity, trichostatin A (TSA), and sulforaphane (SFN), and how they influence the expression of vitamin D-induced gene expression. By isolating mRNA to create cDNA, we were able to run RT-PCR to analyze the overall gene expression. The genes investigated were: CYP24A1, CYP27B1, VDR and TRPV6. We found that in MCF-7 breast cancer cells, 1,25(OH)2D3 treatment alone induced the expression of VDR, CYP24A1 and CYP27B1. TRPV6 mRNA expression was not evident. TSA alone increased expression of VDR and CYP24A1, but SFN alone had no effect. Co-treatments of 1,25(OH)2D3 and TSA raised CYP24A1, but not significantly. Co-treatments with SFN seemed to decrease CYP24A1 expression, not significantly. Our findings support further study of the effects of the HDACI TSA in breast cancer, and suggest that this HDACI may be beneficial in augmenting vitamin D cellular responsiveness.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24532号 / 医科博第146号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 金子 新, 教授 上杉 志成, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

L'acétylation des lysines est une modification post-traductionnelle des protéines dont l’ajout et l’élimination sont catalysés respectivement par les histones acétyltransférases (HAT) et les désacétylases d'histones (HDAC). Cette modification joue un rôle majeur dans la régulation de processus cellulaires tels que l'expression génique, la mobilité cellulaire et le métabolisme. Il est maintenant bien établi qu'une altération de l'activité des désacétylases, entrainant ainsi une dérégulation de l'acétylome, est associée au développement tumoral. Par conséquent, les HDAC sont considérées comme des cibles prometteuses en thérapie anti-cancéreuse ce qui a conduit au développement de nombreux inhibiteurs de HDAC. Cependant, la recherche de nouvelles molécules avec un potentiel anti-cancéreux accru et moins d’effets secondaires est indispensable. Nous avons identifié cinq acides 4-hydroxybenzoïques comme nouveaux inhibiteurs de HDAC, trois inhibiteurs qui ciblent plusieurs HDAC et deux inhibiteurs spécifiques de HDAC6. Les inhibiteurs qui ciblent plusieurs HDAC induisent l'acétylation de certaines lysines des histones H3 et H4 dans les cellules de leucémie myéloïde chronique humaine K-562. Le traitement des cellules induit un arrêt de la progression du cycle cellulaire associé à la modulation de l'expression des cyclines et l'activation de la transcription du gène codant p21. Enfin, les trois composés qui inhibent plusieurs HDAC induisent une mort par apoptose qui est confirmée par l'observation du clivage et de l'activation des caspases. Les inhibiteurs spécifiques de HDAC6 induisent une hyperacétylation importante de la tubuline-α corrélée à une condensation des microtubules dans les cellules cancéreuses adhérentes de prostate (cellules PC-3 et LNCaP). Ces composés induisent une mort par apoptose des cellules cancéreuses en suspension K-562 accompagnée du clivage des caspases et de l'activation de la protéine pro-apoptotique BAX. Enfin, les molécules altèrent la fonction de la protéine chaperonne HSP90α observée par une forte diminution de l'expression de ses protéines clientes: Bcr-Abl et le récepteur aux androgènes. Par ailleurs, les cinq composés n'affectent pas la viabilité des cellules saines. L'ensemble de ce travail révèle que les acides 4-hydroxybenzoïques sont des molécules prometteuses pour le développement de nouveaux composés ayant des propriétés anti-tumorales intéressantes / Lysine acetylation is a post-translational modification characterized by addition and removal acetyl group by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. This modification plays a crucial role in multiple cellular processes including gene expression, cell motility and metabolism. It is now well established that disruption of deacetylase activity, leading to a pathological acetylation profile, is associated to cancer development. Consequently, HDACs are considered as promising targets for anticancer therapy, which led to the development of novel HDAC inhibitors. However, discovery and synthesis of new molecules is essential to increase anticancer potential and decrease adverse health effects of already known compounds. We identified five 4-hydroxybenzoic acids as new HDAC inhibitors: three pan-HDAC inhibitors and two HDAC6-specific inhibitors. Pan-HDAC inhibitors induce acetylation of selected lysines within histones H3 and H4 in human chronic myeloid leukemia K-562 cells. Treatment of cells induces cell cycle arrest associated with increased cyclin expression and the transcriptional activation of p21. Finally, these pan-HDAC inhibitors induce apoptotic cell death further confirmed by the cleavage and activation of caspases. HDAC6-specific inhibitors induce hyperacetylation of α-tubulin in correlation with microtubule condensation in adherent prostate cancer cells (PC-3 and LNCaP cells). These compounds induce apoptotic cell death in K-562 cells accompanied by caspase cleavage and the activation of the pro-apoptotic protein BAX. Furthermore, these molecules alter the chaperon function of HSP90α, which is observed through the robust decrease of the expression of its client proteins (i.e. Bcr-Abl and androgen receptor). Noteworthy, the five compounds did not affect healthy cell viability. Taken together these results revealed that 4-hydroxybenzoic acids are attractive molecules for the development of new compounds with promising anticancer properties

Friedreich’s ataxia (FRDA) is an inherited recessive disorder characterized by progressive neurological disability and heart disease. It is caused by a pathological intronic hyperexpansion of a GAA repeat in the FXN gene, encoding the essential mitochondrial protein frataxin. At the homozygous state, the GAA expansion induces a heterochromatin state with decreased histone acetylation and increased methylation, resulting in a partial deficiency of frataxin expression. This was established in cells from FRDA patients. We showed that the same chromatin changes exist in a GAA based mouse model, KIKI, generated in our laboratory. Furthermore, treatment of KIKI mice with a novel Histone Deacetylase Inhibitor (HDACi), 106, a pimelic diphenylamide that increases frataxin levels in FRDA cell culture, restored frataxin levels in the nervous system and heart of KIKI mice and induced histone hyperacetylation near the GAA repeat. As shown by microarrays, most of the differentially expressed genes in KIKI were corrected towards wild type. In an effort to improve the pharmacological profile of compound 106, we synthesized more compounds based on its structure and specificity. We characterized two of these compounds in FRDA patients’ peripheral blood lymphocytes and in the KIKI mouse model. We observed a sustained frataxin upregulation in both systems, and, by following the time course of the events, we concluded that the effects of these compounds last longer than the time of direct exposure to HDACi. Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACis for FRDA. Laboratory tools to follow disease progression and assess drug efficacy are needed in a slowly progressive neurodegenerative disease such as FRDA. We used microarrays to characterize the gene expression profile in peripheral lymphocytes from FRDA patients, carriers and controls. We identified gene expression changes in heterozygous, clinically unaffected GAA expansion carriers, suggesting that they present a biochemical phenotype, consistent with data from animal models of frataxin deficiency. We identified a subset of genes changing in patients as a result of pathological frataxin deficiency establishing robust gene expression changes in peripheral lymphocytes. These changes can be used as a biomarker to monitor disease progression and potentially assess drug efficacy. To this end, we used he same methodology to characterize the gene expression profiles in peripheral lymphocytes after treatment with pimelic diphenylamide HDACi. This treatment had relevant effects on gene expression on peripheral patients’ blood lymphocytes. It increased frataxin levels in a dose-dependent manner, and partially rescued the gene expression phenotype associated with frataxin deficiency in the tested cell model, thus providing the first application of a biomarker gene set in FRDA. / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Introduction AP2α is a 52 kDa retinoic acid inducible and developmentally regulated activator of transcription, which binds to the DNA in a sequence-specific manner. Transcription factor AP-2α was isolated from HeLa cells by affinity chromatography using specific binding sites with in SV40 and human metallothionein promoters. Further screening of HeLa cDNA library with oligonucleotide probes predicted partial peptide sequence which led to the isolation of AP-2α cDNA and subsequently it was mapped to chromosome 6 near HLA locus. A differentially spliced version of AP-2α, which lacks most of the C-terminus, encodes a dominant negative protein (AP-2B). Subsequent studies led to the identification of four more isoforms: AP-2β, AP-2γ, AP-2δ and AP-2ε. AP-2 family members can form homo or hetero dimers among themselves through the unique C-terminal helix span helix motif and bind DNA through basic domain lies N-terminus of DNA binding domain. Several evidences suggest that AP-2α can act as a tumor suppressor gene. It has been shown that AP-2α can activate growth suppressor genes like p21WAF1/CIP1. Transforming viral oncogenes like adenovirus E1A and SV40 large T antigen have been shown to alter AP-2α function. In addition, reduced expression of AP-2α has been reported in human breast, ovary, colon, skin, brain and prostate cancers. Further, supporting evidences suggest that more invasiveness and tumorogenicity was observed when dominant negative mutant of AP-2α was expressed in melanoma cells. In this work, we have carried out a systematic study to find the various signal transduction pathways which regulate AP-2 activity as well as we attempted to demonstrate the importance of DNA binding domain in the growth inhibitory functions of AP-2α. HDAC inhibitors (HDIs) activate AP-2 activity through spleen tyrosine kinase (Syk) In the literature, ample evidences are available that genotoxic drugs such as adriamycin, induce tumor suppressors like p53 and p73. In this study, we have screened pharmacological drugs which damage DNA and specific inhibitors of various signal transduction pathways for their ability to activate AP-2 activity. AP-2 specific reporter, 3Χ-AP2-CAT was used in this study to measure the AP-2 activity. Of all the compounds studied, we found that Histone Deacetylase Inhibitors (HDIs) efficiently activated AP-2 activity and was found to be specific as they failed to activate 3X-AP2 mut CAT, which contains mutated AP-2 binding sites as well as pGL tk Luc, which contains thymidine kinase minimal promoter and no AP-2 binding sites. To understand the mechanism of HDI-mediated of AP-2 activation, AP-2 isoforms and its coactivators transcript and protein levels were analyzed. We found significant change in transcript levels of the some of the molecules tested. While the endogenous protein levels of various AP-2 isoforms were undetectable, we found stabilization of AP-2α protein expressed from exogenous source in cells treated with HDIs. HDI stabilized AP-2α was found to be functionally active as it showed increased sequence-specific DNA-binding as well as increased apoptosis. While HDIs known for their ability to modulate the gene activities by chromatin remodeling, it is also known that they alter various signal transduction pathways. In an effort to find pathway(s) by which HDIs activate AP-2 activity, we found that HDIs failed to activate AP-2 reporter in the presence of staurosporine suggesting the involvement a staurosporine sensitive pathway(s) in this process. Stauosporine is a non-specific kinase inhibitor of different signaling pathways. Further studies using different pathway specific inhibitors identified that spleen tyrosine kinase (Syk) is essential for HDIs mediated activation of AP-2 activity. Syk is a non receptor tyrosine kinase which is known to be activated in stress conditions. Syk is considered to be a tumor suppressor since Syk over expression leads to growth suppression of breast cancer cells and is also inactivated in a subset of breast cancers. These results suggest that HDI mediated activation of AP-2 involves AP-2α stabilization through Syk pathway. Regulation of AP-2 by MAP kinase pathway Cell growth, differentiation, and apoptosis are mediated by the activation of mitogenactivated protein kinase (MAPK) pathways. These kinases constitute MAP kinase cascades mainly regulated through phosphorylation status. In mammalian cells, at least four MAPKs, namely, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), p38 and ERK5/big MAP kinase have been identified. The ERKs are usually activated by mitogenic stimuli which in turn increase the proliferation and survival. Over expression of any activator of this signaling cascade lead to the unregulated proliferation of cells. In many cancers, ERK pathways are known to be up regulated. In this study, we found that MEK (MEK is the immediate upstream regulator of ERK) inhibitors - PD98059 and U0126 activate 3X-AP2-CAT suggesting that AP-2 activity is repressed by activated MAP kinase pathway. MEK inhibitor mediated activation was found to be specific because they failed to activate transcription from pGL tk Luc which contains thymidine kinase minimal promoter and no AP-2 binding sites. To understand the mechanism of MEK inhibitor-mediated of AP-2 activation, AP-2 isoforms and its coactivators transcript and protein levels were analyzed. We found significant change in transcript levels of the some of the molecules tested. The endogenous protein levels of various AP-2 isoforms were undetectable. When AP-2α was exogenously expressed, while no change in protein levels and DNA-binding ability was seen, we found evidence for appearance of post-ranslationally modified AP-2α protein in U0126 treated cells. We also found CITED2 (CBP/p300-interacting transactivator 2, co-activator of AP-2α) transcript levels were up regulated in UO126 treated cells. Post translational modifications of AP-2α and increased and increased CITED2 levels may be responsible for MEK inhibitor mediated AP-2 activation. Thus we conclude that ERK pathway, which is an oncogenic MAP kinase pathway, inhibits AP-2 activity thereby suggesting the importance of down regulation of AP-2 activity during transformation. Essential role of DNA-binding domain of AP-2α for its growth inhibitory functions Transcription factor AP-2α has three distinct domains, N-terminal transactivation domain (52-108 aa), C-terminal DNA binding domain (204-408 aa) and dimerization domain (277-395 aa) which lies within the DNA binding domain. AP-2α exerts its effects through binding to specific DNA sequence in the promoter of its target genes leading to either repression or activation. Recent evidences suggest that AP-2α represses many genes through its competitive binding to overlapping AP-2 and other transcription factor binding sites. This suggests an important role exclusively for the DNA binding domain in AP-2α mediated functions. To address the importance of DNA binding domain for AP-2α mediated apoptosis, we have tested the ability different deletion/point mutants of AP-2α with varying DNA binding and transactivation capability to perform growth suppressor function and ability to induce apoptosis. Replication-deficient recombinant adenoviruses expressing different mutants were used in this study. We found that an intact DNA-binding domain alone even in the absence of activation domain is sufficient for AP-2α to inhibit colony formation and to induce significant levels of apoptosis. These results suggest an important role for DNA binding domain growth inhibitory functions of AP-2α and thereby implying the importance of transcriptional repression in AP-2α functions.