scholarly journals A Substrate-Independent TR-FRET Histone Deacetylase Inhibitor Assay

2011 ◽  
Vol 16 (10) ◽  
pp. 1247-1253 ◽  
Author(s):  
Bryan D. Marks ◽  
Stephen A. Fakhoury ◽  
William J. Frazee ◽  
Hildegard C. Eliason ◽  
Steven M. Riddle
Keyword(s):  
Expression System ◽  
Hdac Inhibitors ◽  
Clear Understanding ◽  
Targeted Therapeutics ◽  
Epitope Tag ◽  
Enzyme Preparations ◽  
Deacetylase Inhibitor ◽  
Class Iib ◽  
Substrate Independent ◽  
Target Effects

Developing molecularly targeted therapeutics with minimal off-target effects is facilitated by an understanding of compound selectivity. However, for HDAC inhibitors, a clear understanding of specificity has been challenging. In particular, it has been suggested that use of nonspecific substrates and the presence of multiple HDAC activities in enzyme preparations may complicate interpretation of inhibitor experiments. To overcome these and other potential limitations of activity-based HDAC assays, the authors have developed an assay format based on measurement of the binding affinity of inhibitors rather than measurement of enzyme activity. One advantage of this format is that it does not require use of a substrate and thus ameliorates concerns about lack of specificity of existing substrates. This assay is based on an Alexa Fluor® 647-labeled HDAC inhibitor or “tracer,” which binds with a high affinity to Class I and Class IIb HDACs. Unlike activity assays, which can be affected by the presence of residual untagged endogenous HDACs from the host expression system, the signal in this format is dependent on the presence of an epitope tag on the specific HDAC of interest. The authors demonstrate the utility of this method by determining the potencies of commonly used inhibitors for six human HDACs.

scholarly journals HDAC6 Modulates Signaling Pathways Relevant to Synaptic Biology and Neuronal Differentiation in Human Stem-Cell-Derived Neurons

2019 ◽  
Vol 20 (7) ◽  
pp. 1605 ◽  
Author(s):  
Jonathan Iaconelli ◽  
Lucius Xuan ◽  
Rakesh Karmacharya
Keyword(s):  
Nervous System ◽  
Histone Deacetylase ◽  
Hdac Inhibitors ◽  
Neuronal Cultures ◽  
Histone Deacetylase 6 ◽  
Disease Biology ◽  
Lysine Residues ◽  
Class Iib ◽  
Human Neuronal Cells ◽  
Class I Hdacs

Recent studies show that histone deacetylase 6 (HDAC6) has important roles in the human brain, especially in the context of a number of nervous system disorders. Animal models of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders show that HDAC6 modulates important biological processes relevant to disease biology. Pan-selective histone deacetylase (HDAC) inhibitors had been studied in animal behavioral assays and shown to induce synaptogenesis in rodent neuronal cultures. While most studies of HDACs in the nervous system have focused on class I HDACs located in the nucleus (e.g., HDACs 1,2,3), recent findings in rodent models suggest that the cytoplasmic class IIb HDAC, HDAC6, plays an important role in regulating mood-related behaviors. Human studies suggest a significant role for synaptic dysfunction in the prefrontal cortex (PFC) and hippocampus in depression. Studies of HDAC inhibitors (HDACi) in human neuronal cells show that HDAC6 inhibitors (HDAC6i) increase the acetylation of specific lysine residues in proteins involved in synaptogenesis. This has led to the hypothesis that HDAC6i may modulate synaptic biology not through effects on the acetylation of histones, but by regulating acetylation of non-histone proteins.

Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

2001 ◽  
Vol 281 (3) ◽  
pp. C982-C992 ◽  
Author(s):  
Craig Gatto ◽  
Scott M. McLoud ◽  
Jack H. Kaplan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Atpase Activity ◽  
Insect Cells ◽  
Expression System ◽  
Β Subunit ◽  
Α Subunit ◽  
Enzyme Preparations ◽  
High Five Cells

The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase α- and β-subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8–2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the α-subunit or β-subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the α-subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the β-subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the α-subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the α-β heterodimer within the endoplasmic reticulum apparently does not require a Na+pump-specific chaperone.

scholarly journals Blockade of Histone Deacetylase Inhibitor-Induced RelA/p65 Acetylation and NF-κB Activation Potentiates Apoptosis in Leukemia Cells through a Process Mediated by Oxidative Damage, XIAP Downregulation, and c-Jun N-Terminal Kinase 1 Activation

2005 ◽  
Vol 25 (13) ◽  
pp. 5429-5444 ◽  
Author(s):  
Yun Dai ◽  
Mohamed Rahmani ◽  
Paul Dent ◽  
Steven Grant
Keyword(s):  
Oxidative Damage ◽  
Nuclear Translocation ◽  
Hdac Inhibitors ◽  
Histone Acetyltransferases ◽  
Leukemia Cells ◽  
Ros Generation ◽  
Suberoylanilide Hydroxamic Acid ◽  
Oxygen Species ◽  
Antiapoptotic Proteins ◽  
Deacetylase Inhibitor

ABSTRACT NF-κB activation is reciprocally regulated by RelA/p65 acetylation and deacetylation, which are mediated by histone acetyltransferases (HATs) and deacetylases (HDACs). Here we demonstrate that in leukemia cells, NF-κB activation by the HDAC inhibitors (HDACIs) MS-275 and suberoylanilide hydroxamic acid was associated with hyperacetylation and nuclear translocation of RelA/p65. The latter events, as well as the association of RelA/p65 with IκBα, were strikingly diminished by either coadministration of the IκBα phosphorylation inhibitor Bay 11-7082 (Bay) or transfection with an IκBα superrepressor. Inhibition of NF-κB by pharmacological inhibitors or genetic strategies markedly potentiated apoptosis induced by HDACIs, and this was accompanied by enhanced reactive oxygen species (ROS) generation, downregulation of Mn-superoxide dismutase and XIAP, and c-Jun N-terminal kinase 1 (JNK1) activation. Conversely, N-acetyl l-cysteine blocked apoptosis induced by Bay/HDACIs by abrogating ROS generation. Inhibition of JNK1 activation attenuated Bay/HDACI lethality without affecting NF-κB inactivation and ROS generation. Finally, XIAP overexpression dramatically protected cells against the Bay/HDACI regimen but failed to prevent ROS production and JNK1 activation. Together, these data suggest that HDACIs promote the accumulation of acetylated RelA/p65 in the nucleus, leading to NF-κB activation. Moreover, interference with these events by either pharmacological or genetic means leads to a dramatic increase in HDACI-mediated lethality through enhanced oxidative damage, downregulation of NF-κB-dependent antiapoptotic proteins, and stress-related JNK1 activation.

scholarly journals A potent trifluoromethyl ketone histone deacetylase inhibitor exhibits class-dependent mechanism of action

MedChemComm ◽  
2016 ◽  
Vol 7 (3) ◽  
pp. 464-470 ◽  
Author(s):  
Andreas S. Madsen ◽  
Christian A. Olsen
Keyword(s):  
Histone Deacetylase ◽  
Mechanism Of Action ◽  
Histone Deacetylase Inhibitor ◽  
Hdac Inhibitors ◽  
Zinc Binding ◽  
Kinetic Evaluation ◽  
Deacetylase Inhibitor ◽  
Dependent Mechanism

Kinetic evaluation of HDAC inhibitors containing different zinc-binding chemotypes demonstrates that trifluoromethyl ketone-containing compounds can inhibit individual HDAC isozymes via differential mechanisms.

scholarly journals Clinical Evaluation of Combined Epigenetic Therapies on the Induction of Fetal Hemoglobin in Patients with Hematologic Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 960-960
Author(s):  
Katharine Rose Press ◽  
Jeffrey Keefer ◽  
Steven D. Gore ◽  
Hetty E. Carraway ◽  
Sarah Sakoian ◽  
...  
Keyword(s):  
Histone Deacetylase ◽  
Hdac Inhibitors ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Methyl Transferase ◽  
Disease Response ◽  
Deacetylase Inhibitor ◽  
Hbf Induction ◽  
Hbf Level

Fetal hemoglobin induction with hydroxyurea (HU) is a mainstay of therapy for β-hemoglobinopathies, especially sickle cell disease (SCD). A high level of fetal hemoglobin (HbF) has a direct relationship with acute clinical status in SCD patients including pain crises, acute chest syndrome, and death. However, not all patients benefit from HU, and more effective HbF induction strategies are needed. DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors have been shown in vitro to induce HbF production through epigenetic modification of the β-globin gene cluster. Azacitidine (AZA) is a DNMT already used in some SCD patients resistant to HbF modulation with HU. Entinostat (MS-275) is an orally available histone deacetylase inhibitor with a long half-life and established antitumor activity in preclinical models. Recent studies suggest that drugs, which act with different molecular and epigenetic mechanisms, have synergistic effects on induction of fetal hemoglobin (Fard et al. IJHOSCR 2013). In this study, we evaluated the effects of a combination of AZA and MS-275 on HbF levels. This was preformed as a correlative study of a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We sequentially measured the level of HbF the peripheral blood in 33 patients receiving different doses of AZA (range: 30mg/m2 to 50mg/m2 per day for 10 doses) and MS-275 (range: 2 to 8 mg/m2 orally on days 3 and 10). Patients completed a minimum of four 28-day cycles of combined therapy. HbF levels were measured in peripheral blood at baseline, at day 15 or 16 and day 29 or 30 of cycle 1, and after cycles 2, 4, and if applicable 6. Azacitidine dose positively correlated with HbF fold increase (mean of 1.1, 2.3, and 2.1 for doses of 30, 40, and 50 mg respectively, p=0.07) while MS-275 dose had a slightly negative correlation with HbF level (mean of 3.0, 1.8, and 1.3 for doses of 2, 4, and 6mg respectively, p=0.13). There was no correlation between baseline HbF and HbF fold increase after exposure to treatment (p=NS) and no correlation between baseline HbF levels and clinical disease response (p=0.19). Interestingly, we demonstrated a correlation between HbF fold increase and clinical disease response: median fold increase of 3.5 for patients achieving hematologic normalization (complete response, partial response, or trilineage hematological improvement) versus 1.4 in non-responders (p=0.006). The positive correlation between AZA dose and HbF increase is consistent with prior work showing that this drug induces HbF production. The correlation between clinical response and HbF induction could reflect a greater susceptibility to AZA potentially related to differing methylomes. Alternatively, it may also represent a known increase in HbF in the setting of stress erythropoiesis. The slight inverse correlation between MS-275 and HbF level was surprising, as other HDAC inhibitors are known to induce HbF in vitro. However, these results are in line with the methylation data found in the more recent randomized phase 2 trial of AZA +/- MS-275 (E1905 trial) that showed a potential pharmacodynamic antagonism of the combination (Prebet et al. J Clin Oncol. 2014). Overall, this work supports the use of AZA as a clinical inducer of HbF. It also shows the importance of trialing various combinations of HbF inducers, as not all drugs work synergistically and some may even be antagonistic in combination. Disclosures Off Label Use: Azacitidine (AZA) is a DNA methyl transferase (DNMT) inhibitor. Entinostat (MS-275) is an orally available histone deacetylase inhibitor. Both drugs were used in a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). . Keefer:MAST therapeutics: Employment. Gore:Celgene: Consultancy, Honoraria, Research Funding. Prebet:CELGENE: Research Funding.

Construction of Recombinant Producers of Enzyme Preparations for Feed Production with an Expression System Based on Penicillium verruculosum Fungus

2020 ◽  
Vol 56 (8) ◽  
pp. 875-880 ◽  
Author(s):  
A. P. Sinitsyn ◽  
O. G. Korotkova ◽  
E. A. Rubtsova ◽  
O. A. Sinitsyna ◽  
E. G. Kondrat’eva ◽  
...  
Keyword(s):  
Expression System ◽  
Penicillium Verruculosum ◽  
Enzyme Preparations ◽  
Feed Production

scholarly journals Integron activity accelerates the evolution of antibiotic resistance

2020 ◽  
Author(s):  
Célia Souque ◽  
José A. Escudero ◽  
R.Craig MacLean
Keyword(s):  
Antibiotic Resistance ◽  
Experimental Evolution ◽  
Expression System ◽  
Genomic Integrity ◽  
Resistance Evolution ◽  
On Demand ◽  
Accelerated Evolution ◽  
Gentamicin Resistance ◽  
The Impact ◽  
Target Effects

AbstractMobile integrons are widespread genetic platforms that allow bacteria to modulate the expression of antibiotic resistance cassettes by shuffling their position from a common promoter. Antibiotic stress induces the expression of an integrase that excises and integrates cassettes, and this unique recombination and expression system is thought to allow bacteria to ‘evolve on demand’ in response to antibiotic pressure. To test this hypothesis, we inserted a custom three cassette integron into P. aeruginosa, and used experimental evolution to measure the impact of integrase activity on adaptation to gentamicin. Crucially, integrase activity accelerated evolution by increasing the expression of a gentamicin resistance cassette through duplications and by eliminating redundant cassettes. Importantly, we found no evidence of deleterious off-target effects of integrase activity. In summary, integrons accelerate resistance evolution by rapidly generating combinatorial variation in cassette composition while maintaining genomic integrity.

Class IIa HDAC Inhibitors Reduce HDAC1 Activity by off-Target Effects Which Reduce GATA1 Expression In Human Erythroblasts Expanded Ex-Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4780-4780
Author(s):  
Elena Masselli ◽  
Lilian Varricchio ◽  
Barbara Ghinassi ◽  
Carolyn Whitsett ◽  
Patricia A. Shi ◽  
...  
Keyword(s):  
Indirect Effects ◽  
Caspase 3 ◽  
Hdac Inhibitors ◽  
Class Ii ◽  
Class I ◽  
Hdac Activity ◽  
Erk Phosphorylation ◽  
Erythroid Maturation ◽  
Target Effects

Abstract Abstract 4780 Histone deacetylation maintains chromatin in a condensed configuration preventing gene expression in eukaryotic cells. The deacetylation reaction is catalyzed by enzymes of the histone deacetylase (HDAC) superfamily, which perform their functions as multiprotein complexes including at least 2 HDAC isoforms, DNA docking factors (transcription factors and methyl-binding proteins) and protein kinases (PKC and Erk). The well established role of HDACs in gene silencing has suggested studies to identify HDAC inhibitors (HDACi) that, by re-activating γ-globin expression, might treat the anemia due to insufficient β-globin expression (Cao et al Blood 103:701, 2004). Over the years several HDACi have been documented to induce γ-globin expression in human erythroid cultures, adult baboons, and β-thalassemia and sickle cell patients. Among those, Class I HDACi, and in particular those that inhibit HDAC3, appear to be more potent as γ-globin gene activators (Mankidy et al, Blood 108:3179, 2006). We have recently identified two new HDACi (compound 9 and 24) which both improved maturation and reactivated γ-globin expression in β°-thalassemic erythroblasts in vitro (Mai et al Mol Pharmacol 72:111, 2007). Compound 24 inhibits both class I (HDAC1 ID50 =0.2 μ M) and class IIa (HDAC4 ID50=0.6 μ M) HDAC. Compound 9 is a class IIa specific inhibitor (HDAC4 ID50=20 μ M) and does not affect HDAC1 activity but is a more potent γ-globin inducer than compound 24. This observation suggests that HDACi may also affect HDAC activity through indirect effects which alter overall complex activity. To clarify possible off-target effects of Class II and Class I/IIa inhibitors and their consequences for erythroid maturation, we analysed expression and activity of different HDAC isoforms during maturation of normal human erythroblasts in vitro at baseline and with treatment with compounds 9 and 24. The proteins studied included GATA1 (the major transcription regulator of erythroid maturation), p21/p27kip1, two cyclin D dependent kinase inhibitors which favor maturation, Caspase 3 (the protease which specifically cleaves GATA1) and Erk (a component of the HDAC complex). During normal erythroid maturation (without HDACi), all the HDAC isoforms were expressed at the mRNA and protein levels. Immunoprecipitation studies followed by determination of HDAC activity indicated that the activities which changed most during maturation are those of HDAC1 (class I), increased by 2-fold, and HDAC5 (class IIa), decreased by 2-fold. In addition, co-immunoprecipitation studies revealed an increase in the association between HDAC1 and GATA1 with erythroid maturation. Changes in the expression of key regulatory proteins were observed with normal erythroid maturation: activation of Caspase 3 decreased with resultant increase in GATA-1, and phosphorylation of pErk decreased while expression of p21 and p27 increased. With exposure to increasing HDACi concentrations (0.2, 2 and 6 μ M), there were class-specific, concentration-dependent alterations in protein expression: compound 9 (Class IIa inhibitor) induced Caspase 3 activation and reduced GATA1 content, while compound 24 decreased Caspase3 activation and greatly increased GATA1 content. In addition, compound 9 did not induce Erk phosphorylation and decreased p21 expression, while compound 24 did induce Erk phosphorylation and inhibited p27 expression (see figure). These results confirm the hypothesis that, in addition to class I inhibitors that directly inhibit class I HDAC, class II HDACi can also affect class I HDAC activity, through indirect effects that involve other components of the complex (repression of GATA1 expression and decrease of Erk phosphorylation). Disclosures: No relevant conflicts of interest to declare.

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