Biomedical subjects

Manfred Jung

Publications and source records attributed to Manfred Jung.

At least 19 recordsLinked to original sources

PubMed2007

Inhibitors of histone deacetylases as potential therapeutic tools for high-risk embryonal tumors of the nervous system of childhood.

The origin of malignant embryonal tumors is incompletely understood and certain risk groups remain difficult to treat. The epigenetic structure of DNA and its lesions play a role in the origin of these neoplasms. Manipulation of the epigenome may offer novel treatment options. The authors evaluated the cytotoxicity of histone deacetylase inhibitors (HDI) [MS-275, SAHA, TSA, M344, M360, D85, SW55, SW187 and valproic acid (VPA)] on 13 embryonal tumor cell lines [4 medulloblastomas, 5 neuroblastomas, 2 atypical teratoid/rhabdoid tumors (AT/RT), and 2 malignant rhabdoid tumors of the kidney (RTK)] in MTT assay. In addition, HDI effects on hyperacetylation, reexpression of growth regulatory genes and apoptosis were characterized by Western analysis, RT-PCR and annexin-V staining. All HDI inhibited cell proliferation in a time- and dose-dependent manner. VPA was least cytotoxic with GI50 values after 72 hr ranging from 53.6 to 332.9 microM, while TSA was most efficient with GI50 values after 72 hr ranging from 0.01 to 8.8 microM. M344 and M360 were also highly effective. Western blot revealed hyperacetylation of histone H4 after HDI treatment. Reactivation of several genes including the proapoptotic CASP8 was identified by RT-PCR. Annexin-V staining demonstrated a dose and time dependent induction of apoptosis. HDI inhibited the growth of medulloblastoma, neuroblastoma and rhabdoid tumors in vitro. Treatment with HDI induced the reactivation of growth regulatory genes and consequently apoptosis. Our results warrant further studies and may help in the design of new protocols geared at the treatment of high risk embryonal tumors.

Acetylation↗

PubMed2006

Adenosine mimetics as inhibitors of NAD+-dependent histone deacetylases, from kinase to sirtuin inhibition.

NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.

Acetylation↗

PubMed2006

The role of NAD+ dependent histone deacetylases (sirtuins) in ageing.

Histone deacetylases (HDACs) are enzymes that are able to deacetylate lysine side chains in histones and certain non-histone proteins which leads to altered states of conformation and activity for the proteins in question. Three classes of histone deacetylases have been recognized in humans. Class I and II are zinc-dependent amidohydrolases and eleven subtypes have been discovered (HDAC1-11). Class III enzymes depend in their catalysis on NAD(+) and subsequently, O-acetyl ADP ribose and nicotinamide are formed as a consequence of the acetyl transfer. Due to the homology to the yeast histone deacetylase Sir2p the NAD(+)-dependent deacetylases are also termed sirtuins and seven members (Sirt1-7) are known in humans. Sirtuins are found from bacteria to eukaryotes and altogether about 60 isoforms have been characterized in different organisms. Sirtuins have been implicated in the regulation of molecular mechanisms of aging. The overexpression of sirtuin enzymatic activity leads to an increase of lifespan in Saccharomyces cerevisiae and Caenorhabditis elegans that can also be reached by calorie restriction. Sirtuins have been proposed to act as sensors for glucose uptake that respond to the levels of NAD(+) but more complex ways of action have been suggested as well. This article will present the members of the human sirtuin family with their respective functions and review the existing druglike inhibitors and activators of sirtuin activity.

Aging↗

PubMed2005

SIRT1 regulates HIV transcription via Tat deacetylation.

The human immunodeficiency virus (HIV) Tat protein is acetylated by the transcriptional coactivator p300, a necessary step in Tat-mediated transactivation. We report here that Tat is deacetylated by human sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent class III protein deacetylase in vitro and in vivo. Tat and SIRT1 coimmunoprecipitate and synergistically activate the HIV promoter. Conversely, knockdown of SIRT1 via small interfering RNAs or treatment with a novel small molecule inhibitor of the SIRT1 deacetylase activity inhibit Tat-mediated transactivation of the HIV long terminal repeat. Tat transactivation is defective in SIRT1-null mouse embryonic fibroblasts and can be rescued by expression of SIRT1. These results support a model in which cycles of Tat acetylation and deacetylation regulate HIV transcription. SIRT1 recycles Tat to its unacetylated form and acts as a transcriptional coactivator during Tat transactivation.

Acetylation↗

PubMed2005

Chromatin modifications as targets for new anticancer drugs.

Chromatin proteins undergo diverse posttranslational modifications, esp. acetylation and methylation, that contribute to the control of transcriptional processes. The result of these modifications in its various states is called the histone code. This review presents an overview of those modifications of chromatin proteins that affect the side chains of lysines and arginines and define variations of the chromatin acetylome and methylome. The relevant enzymes are presented and the feasibility to influence their activity by inhibition or activation is discussed. The manipulation of these enzymes is an exciting strategy towards an increased understanding of their role in the functionality of a cell. Additionally, this may lead to new approaches for the treatment of diseases that are based on a dysregulation of transcription, especially cancer.

Acetylation↗

PubMed2005

Hydroxamide derivatives of short-chain fatty acid have erythropoietic activity and induce gamma gene expression in vivo.

OBJECTIVE: The hydroxamic acid derivatives of short-chain fatty acids, butyryl and propionyl hydroxamate, subericbishydoxamic acid, and suberoylanilide hydroxamic acid, are potent inhibitors of histone deacetylase (HDAC) and have been shown to induce fetal hemoglobin in vitro. In this study we examined whether these compounds have erythropoietic activity and can induce gamma globin gene expression in vivo. MATERIALS AND METHODS: Transgenic mice heterozygous for a deletion beta thalassemia and hemizygous for a human gamma globin transgene were treated with these compounds and hematologic responses as well as the induction of gamma gene expression were evaluated. Hematologic studies included measurement of reticulocytes, hematocrit, and the in vivo levels of BFU-E. Effects on globin gene expression were assessed by measuring F reticulocytes and the human gamma/murine alpha globin mRNA ratios by RNAse protection assay. RESULTS: Propionyl and butyryl hydroxamate increased reticulocytes by 71% and 139%, the in vivo BFU-E counts by 75% and 51%, and the in vivo gamma gene expression by 33.9% and 71% respectively. SBHA and SAHA had no erythropoietic activity in vivo. CONCLUSION: Hydroxamic acid derivatives can stimulate the in vivo erythropoiesis and fetal globin production in a thalassemic murine model. The combined effect of certain histone deacetylase inhibitors on erythropoiesis and on gamma gene expression make these compounds desirable targets for development of therapeutics for beta chain hemoglobinopathies.

Animals↗

PubMed2005

In vitro assays for the determination of histone deacetylase activity.

Histone deacetylases are important regulators of transcription and an emerging target for anticancer drugs. We present an overview over various assay formats that include radiolabelled histones, oligopeptides, and small molecules as substrates. The advantages and disadvantages of the various formats in terms of, e.g., substrate availability, throughput or subtype selectivity are discussed. Detailed procedures for various assay types that can be used for different problems, such as library screening or fluorescent inhibitor testing, are given. We present a new protocol for a simple high-throughput assay for NAD+-dependent (class III) histone deacetylases, also termed sirtuins.

Animals↗

PubMed2005

Effect of inhibitors of histone deacetylase on the induction of cell differentiation in murine and human erythroleukemia cell lines.

Histone deacetylase (HDAC) inhibitors are a novel class of promising anti-cancer agents. Little information is available on the capacity of structurally different HDAC inhibitors to induce terminal cell differentiation in different cell types in relation to enzyme inhibition and subtype selectivity. Consequently, the aim of this study was to provide a comprehensive comparison of these effects. New biarylalanine inhibitors of HDAC were synthesized and compared to a series of standard inhibitors from different laboratories. Chromatographically purified rat liver and immunoprecipitated FLAG-tagged recombinant human HDACs were used as sources of HDAC activity. Enzyme inhibition was studied using a fluorescent substrate and its conversion was monitored by high-performance liquid chromatography. The ability to induce cell differentiation was compared in murine (Friend DS-19) and human (K562) erythroleukemic cell lines, and was quantified by benzidine staining. Inhibition of cell proliferation was evaluated by cell counting. All HDAC inhibitors were identified as potent inhibitors of erythroleukemic cell proliferation. However, we observed a complex pattern of differentiation induction: structurally similar inhibitors resulted in disparate activity profiles, whereas similar profiles were detected within distinct structural classes. Among the newly synthesized biarylalanine compounds, a 3'-methoxy derivative was identified as a very effective inducer of terminal cell differentiation. We conclude that investigation of subtype selectivity of selected HDAC inhibitors does not provide a clear link between selectivity and the observed cellular activity profile. The predictive value of in vitro HDAC inhibition assays for identifying anti-proliferative compounds has been emphasized.

Aminobiphenyl Compounds↗

PubMed2004

Subtype selective substrates for histone deacetylases.

To probe the steric requirements for deacylation, we synthesized lysine-derived small molecule substrates and examined structure-reactivity relationships with various histone deacetylases. Rat liver, human HeLa, and human recombinant class I and II histone deacetylases (HDACs) as well as human recombinant NAD(+)-dependent SIRT1 (class III enzyme) were used in these studies. A benzyloxycarbonyl substituent on the alpha-amino group yielded the highest conversion rates. Replacing the epsilon-acetyl group with larger lipophilic acyl substituents led to a pronounced decrease in conversion by class I and II enzymes; the class III enzyme displayed a greater tolerance. Incubations with recombinant FLAG-tagged human HDACs 1, 3, and 6 showed a distinct subtype selectivity among small molecule substrates. The subtype selectivity of HDAC inhibitors could be predicted with these substrates and an easily obtainable mixture of HDAC subtypes.

Animals↗

PubMed2004

Enhancement of radiation sensitivity of human squamous carcinoma cells by histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors are emerging therapeutic agents with potential for disruption of critical cellular processes in cancer cells. Transcriptional regulation, differentiation, cell cycle arrest, radiation sensitization, and apoptosis have been observed in response to exposure to HDAC inhibitors. In the present study, we observed that several potent HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, M344 (an analogue of hydroxamic acid), and the cyclic tetrapeptide, depsipeptide (FR90228), modulate cellular responses to ionizing radiation in cells of two human squamous carcinoma lines (SQ-20B and SCC-35), previously characterized as intrinsically resistant to radiation. Also exposure to IC(50) concentrations of these inhibitors, radiation sensitivities were enhanced in both cell lines. Depsipeptide exhibited the greatest effect on SQ-20B cells, decreasing D(0) values from 2.62 Gy to 1.64 Gy. M344 was the most active drug in sensitizing SCC-35 cells, decreasing D(0) values from 1.91 Gy to 1.21 Gy. The mechanisms underlying HDAC inhibitor-induced radiosensitization were further investigated by extending trichostatin A studies to assess cell cycle distributions and levels of apoptosis. Treatment of SQ-20B cells with radiosensitizing concentrations of trichostatin A resulted in cell cycle arrest in G(1) phase (>70%) and inhibition of DNA synthesis. Contrary to previous reports, induction of apoptosis was very low and caspase 3 and 9 were not activated. Taken together, these results implicate G(1) arrest and inhibition of DNA synthesis in the mechanisms underlying radiation sensitization by trichostatin A and support the use of HDAC inhibitors for targeting radioresistant cancers.

Apoptosis↗

PubMed2003

Farnesyl-O-acetylhydroquinone and geranyl-O-acetylhydroquinone suppress the proliferation of murine B16 melanoma cells, human prostate and colon adenocarcinoma cells, human lung carcinoma cells, and human leukemia cells.

Farnesyl-O-acetylhydroquinone (IC(50)=2.5 microM/l) suppressed the proliferation of murine B16F10 melanoma cells with a potency much greater than those of farnesol (IC(50)=45 microM/l) and farnesyl anthranilate (IC(50)=46 microM/l), its alcohol, and ester counterparts with proven anti-tumor activities in vivo. Geranyl-O-acetylhydroquinone (IC(50)=5.1 microM/l) also had a much-improved activity compared to geraniol (IC(50)=160 microM/l) and geranyl anthranilate (IC(50)=30 microM/l). The suppression by farnesyl-O-acetylhydroquinone was concentration- and time-dependent and was accompanied by arrest of cell cycle at G1 and G2/M phases as shown by flow cytometry. Farnesyl-O-acetylhydroquinone and lovastatin had additive impact on B16 cell proliferation. Farnesyl-O-acetylhydroquinone also suppressed the proliferations of human cancer cells HL-60, DU145, PC-3, LNCaP, Caco-2, and A549. Our results suggested that farnesyl derivatives, suppressors of tumor 3-hydroxy-3-methylglutaryl coenzyme A reductase activities, have potential as chemopreventive or chemotherapeutic agents.

Animals↗

PubMed2003

Identification of selective inhibitors of NAD+-dependent deacetylases using phenotypic screens in yeast.

Sir2 and Hst1 are NAD+-dependent deacetylases involved in transcriptional repression in yeast. The two enzymes are highly homologous yet have different sensitivity to the small-molecule inhibitor splitomicin (compound 1) (Bedalov, A., Gatbonton, T., Irvine, W. P., Gottschling, D. E., and Simon, J. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 15113-15118). We have now defined a critical amino acid residue within a small helical module of Hst1 that confers relative resistance to splitomicin. Parallel cell-based screens of 100 splitomicin analogues led to the identification of compounds that exhibit a higher degree of selectivity toward Sir2 or Hst1. A series of compounds based on a splitomicin derivative, dehydrosplitomicin (compound 2), effectively phenocopied a yeast strain that lacked Hst1 deacetylase while having no effect on the silencing activities of Sir2. In addition, we identified a compound with improved selectivity for Sir2. Selectivity was affirmed using whole-genome DNA microarray analysis. This study underscores the power of phenotypic screens in the development and characterization of selective inhibitors of enzyme functions.

Amino Acid Sequence↗

PubMed2003

Induction of human gamma globin gene expression by histone deacetylase inhibitors.

We investigated the induction of human gamma globin gene activity by 3 classes of histone deacetylase inhibitors: amide analogues of trichostatin A, hydroxamic acid analogues of trapoxin, and scriptaid and its analogues. The screening consisted of measuring the effects of these compounds on gamma and beta human gene promoter activity by using cultures of GM979 cells stably transfected with a construct containing a gamma promoter linked to firefly luciferase and a beta promoter linked to renilla luciferase. Compounds belonging to all 3 classes induced gamma gene promoter activity in the screening assay in low micromolar concentrations. Histone deacetylase (HDAC) inhibitors increased acetylation of histone H4 and induced the expression of endogenous murine embryonic genes. They also increased the levels of gamma mRNA and the frequency of fetal hemoglobin-containing erythroblasts in erythroid burst-forming unit (BFUe) cultures from healthy adult individuals. Compounds that displayed very similar degrees of inhibition of the HDAC activity in an HDAC enzymatic assay differed strikingly on their effects on gamma gene promoter activity, raising the possibility of selectivity of HDACs that interact with the gamma globin gene chromatin.

Amides↗

PubMed2003

Nonisotopic substrate for assaying both human zinc and NAD+-dependent histone deacetylases.

Histone deacetylases (HDACs) are involved in the regulation of transcription and their inhibitors are a promising class of new anticancer drugs. We have previously reported Boc(Ac)Lys-AMC, also termed MAL, as a fluorescent substrate for HDACs. Now we present a modification of MAL called Z-MAL that is characterized by an increased rate of conversion by histone deacetylases of classes I and II and the recently discovered sirtuins (histone deacetylases class III). MAL and Z-MAL are the first nonradioactive substrates for class III enzymes. The new substrate Z-MAL allows for shorter assay times in inhibitor screening and is applicable to diverse sources of deacetylase activity even with completely different catalytic mechanisms. Interestingly, MAL shows some relative preference toward class II, indicating that subtype selectivity in small-molecule HDAC substrates might be obtained.

Animals↗

PubMed2003

Hydroxamide derivatives of short-chain fatty acids are potent inducers of human fetal globin gene expression.

OBJECTIVE: To examine whether hydroxamic acids are inducers of fetal hemoglobin expression, we tested the effects on gamma gene expression of butyric and propionic hydroxamic acids and of two other hydroxamic acids (SBHA and SAHA), which are potent inhibitors of histone deacetylase (HDAC). We also investigated whether there is a correlation between HDAC inhibitory activity of the compounds and their ability to induce gamma-globin gene expression. MATERIALS AND METHODS: Effects on gamma-globin expression were assessed by two methods: 1) a screening assay in which specific gamma-globin gene inducers are recognized by their ability to increase gamma firefly luciferase activity significantly more than beta-renilla luciferase activity; and 2) measurements of gamma-globin mRNA and the frequency of fetal hemoglobin-positive erythroblasts in cultures of burst-forming unit erythroid (BFU-E) from normal individuals. HDAC in vitro activity was measured with a partially purified rat liver HDAC and a fluorogenic substrate. RESULTS: All compounds tested increased gamma firefly luciferase activity, gamma/gamma+beta mRNA ratios, and percentage of fetal hemoglobin-containing erythroblasts in BFU-E cultures, in a dose-dependent fashion. Butyryl-hydroxamic acid 100 microM increased the gamma/gamma+beta mRNA ratios by 5.8-fold and the frequency of fetal hemoglobin-containing erythroblasts by 4.1-fold. Propionyl-hydroxamic acid 150 microM increased the gamma/gamma+beta ratios by 6.3-fold and the fetal hemoglobin-containing erythroblasts by 3.9-fold. SBHA induced gamma-globin gene expression at very low concentrations, 5 to 20 microM in the luciferase system and 2 to 8 microM in BFU-E cultures; SAHA at 1 to 7.5 microM in the luciferase system and 1 to 2.5 microM in the BFU-E cultures. HDAC in vitro inhibition was observed in the millimolar range for propionate and butyrate. IC(50) determinations led to values of 384 microM for propionyl-hydroxamate, 47 microM for butyryl-hydroxamate, 0.93 microM for SBHA, and 0.26 microM for SAHA. CONCLUCION: Our data indicate that hydroxamic acid-based HDAC inhibitors are potent gamma-globin gene inducers and that the concentration range of their effects on gamma gene expression can be correlated roughly with their HDAC inhibitory potencies.

Animals↗

PubMed2003

A homogeneous nonisotopic histone deacetylase activity assay.

Histone deacetylases (HDACs) are important regulators of transcription, and their inhibitors are a promising class of anticancer agents. The methods for the determination of HDAC activity and its inhibition that are currently available suffer from various drawbacks, such as animal testing, radioactive substrates, or limited throughput. Therefore, a fast nonisotopic method for the measurement of HDAC activity is highly desirable. The authors present such an assay that relies on the fluorescent HDAC substrate developed previously in their group. After incubation of the substrate with the enzyme, a derivatization leads to efficient fluorescence quenching in the deacetylated metabolite. Thus, only the fluorescence emitted by the remaining substrate is detected, which allows for a convenient detection of HDAC activity in a homogeneous format that can be performed on multiwell plate readers. This procedure, called HDASH (histone deacetylase assay-homogeneous), should be a valuable tool in transcriptional research and especially drug discovery.

Animals↗

PubMed2003

Trichostatin A and structurally related histone deacetylase inhibitors induce 5-lipoxygenase promoter activity.

5-Lipoxygenase (5-LO) mRNA expression in Mono Mac 6 cells is induced by the histone deacetylase inhibitor trichostatin A (TsA). In order to study the effects of TsA and several structurally related compounds such as MD85, D237 and M232 on 5-LO promoter activity, we have analyzed the response of a 5-lipoxygenase (5-LO) promoter luciferase reporter gene construct to histone deacetylase inhibitors in transiently transfected Mono Mac 6 and HeLa cells. We show that the activity of 5-LO promoter constructs comprising the sequences -778 to and of several successive deletions of the 5-LO promoter is strongly increased upon TsA treatment. The data suggest a significant involvement of histone deacetylases in the regulation of 5-LO gene transcription. The basal activity of the 5-LO promoter strongly depends on the presence of multiple Sp1-binding sites (GC-boxes), five of which are positioned in tandem. Deletion of the five tandemized GC-boxes in the 5-LO reporter gene construct revealed that the induction of 5-LO promoter activity by TsA seems to be independent of these GC-boxes. Methylation of 5-LO reporter gene constructs by M.Hpall reduced 5-LO promoter activity but did not prevent induction of promoter activity by TsA, although the activated reporter gene activities were lower compared to the unmethylated plasmid, indicating the dominance of methylation over TsA-sensitive histone deacetylation in silencing of the 5-LO gene. The structure-activity data obtained for histone deacetylase inhibitors suggest that this assay system might serve as a cellular screening tool for the development of HDAC inhibitors.

Animals↗

PubMed2002

Structure-activity relationships on phenylalanine-containing inhibitors of histone deacetylase: in vitro enzyme inhibition, induction of differentiation, and inhibition of proliferation in Friend leukemic cells.

Inhibitors of histone deacetylases (HDACs) are a new class of anticancer agents that affect gene regulation. We had previously reported the first simple synthetic HDAC inhibitors with in vitro activity at submicromolar concentrations. Here, we present structure-activity data on modifications of a phenylalanine-containing lead compound including amino acid amides as well as variations of the amino acid part. The compounds were tested for inhibition of maize HD-2, rat liver HDAC, and for the induction of terminal cell differentiation and inhibition of proliferation in Friend leukemic cells. In the amide series, in vitro inhibition was potentiated up to 15-fold, but the potential to induce cell differentiation decreased. Interestingly, an HDAC class selectivity was indicated among some of these amides. In the amino acid methyl ester series, a biphenylalanine derivative was identified as a good enzyme inhibitor, which blocks proliferation in the submicromolar range and is also a potent inducer of terminal cell differentiation.

Acetylation↗