scholarly journals Hydroxamic Acid-Modified Peptide Microarrays for Profiling Isozyme-Selective Interactions and Inhibition of Histone Deacetylases

2020 ◽  
Author(s):  
Carlos Moreno-Yruela ◽  
Adela-Eugenie Vrsanova ◽  
Hans M. Maric ◽  
Christian Adam Olsen
Keyword(s):  
Posttranslational Modifications ◽  
Histone Deacetylases ◽  
Hydroxamic Acid ◽  
Protein Complexes ◽  
Hdac Inhibitors ◽  
Effector Proteins ◽  
Peptide Sequence ◽  
Class I ◽  
Peptide Microarrays ◽  
And Function

Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled through the enzymatic action of epigenetic effector proteins and protein complexes. Further, various PTM signatures or combinations are recognized by so-called reader domains. Histone microarrays have been widely applied to investigate such histone–reader interactions. So far, however, these could not be used to directly study the fast and transient interactions of Zn<sup>2+</sup>-dependent histone deacetylase (HDAC) enzymes. Here, we describe the synthesis of hydroxamic acid-modified histone-derived peptides and their use in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. We further demonstrate their suitability to discover and map HDAC isozyme-specific substrates. Functional assays confirmed the prediction of HDAC–peptide binding requirements and the conversion of acetylated substrates in response to PTMs and mutations. Subsequent analysis of the hydroxamic acid-containing peptides identified compounds with nanomolar potency and unanticipated selectivity. Follow-up analyses confirmed a major contribution of the peptide sequence to substrate turnover, inhibitor potency, and isozyme selectivity. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme- or protein complex-selective HDAC inhibitors and affinity probes.

scholarly journals Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases

2020 ◽  
Author(s):  
Carlos Moreno-Yruela ◽  
Michael Bæk, ◽  
Adela-Eugenie Vrsanova ◽  
Hans M. Maric ◽  
Clemens Schulte ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Histone Deacetylases ◽  
Hydroxamic Acid ◽  
Hdac Inhibitors ◽  
Class I ◽  
Peptide Microarrays ◽  
Cellular Assays ◽  
Transient Interactions ◽  
And Function

Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn<sup>2+</sup>-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution demonstrate their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes<br>

scholarly journals Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Carlos Moreno-Yruela ◽  
Michael Bæk ◽  
Adela-Eugenie Vrsanova ◽  
Clemens Schulte ◽  
Hans M. Maric ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Histone Deacetylases ◽  
Hydroxamic Acid ◽  
Hdac Inhibitors ◽  
Class I ◽  
Peptide Microarrays ◽  
Cellular Assays ◽  
Transient Interactions ◽  
And Function

AbstractHistones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.

scholarly journals Hydroxamic acid-modified peptide microarrays for profiling isozyme-selective interactions and inhibition of histone deacetylases

2020 ◽  
Author(s):  
Carlos Moreno-Yruela ◽  
Michael Bæk, ◽  
Adela-Eugenie Vrsanova ◽  
Hans M. Maric ◽  
Clemens Schulte ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Histone Deacetylases ◽  
Hydroxamic Acid ◽  
Hdac Inhibitors ◽  
Class I ◽  
Peptide Microarrays ◽  
Cellular Assays ◽  
Transient Interactions ◽  
And Function

Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn<sup>2+</sup>-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution demonstrate their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes<br>

Suppression of monosodium urate crystal-induced cytokine production by butyrate is mediated by the inhibition of class I histone deacetylases

2015 ◽  
Vol 75 (3) ◽  
pp. 593-600 ◽  
Author(s):  
Maartje C P Cleophas ◽  
Tania O Crişan ◽  
Heidi Lemmers ◽  
Helga Toenhake-Dijkstra ◽  
Gianluca Fossati ◽  
...  
Keyword(s):  
Hdac Inhibitor ◽  
Histone Deacetylases ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Hdac Inhibitors ◽  
Class I ◽  
Acute Gout ◽  
Monosodium Urate ◽  
Healthy Donors ◽  
Anti Inflammatory

ObjectivesAcute gouty arthritis is caused by endogenously formed monosodium urate (MSU) crystals, which are potent activators of the NLRP3 inflammasome. However, to induce the release of active interleukin (IL)-1β, an additional stimulus is needed. Saturated long-chain free fatty acids (FFAs) can provide such a signal and stimulate transcription of pro-IL-1β. In contrast, the short-chain fatty acid butyrate possesses anti-inflammatory effects. One of the mechanisms involved is inhibition of histone deacetylases (HDACs). Here, we explored the effects of butyrate on MSU+FFA-induced cytokine production and its inhibition of specific HDACs.MethodsFreshly isolated peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with MSU and palmitic acid (C16.0) in the presence or absence of butyrate or a synthetic HDAC inhibitor. Cytokine responses were measured with ELISA and quantitative PCR. HDAC activity was measured with fluorimetric assays.ResultsButyrate decreased C16.0+MSU-induced production of IL-1β, IL-6, IL-8 and IL-1β mRNA in PBMCs from healthy donors. Similar results were obtained in PBMCs isolated from patients with gout. Butyrate specifically inhibited class I HDACs. The HDAC inhibitor, panobinostat and the potent HDAC inhibitor, ITF-B, also decreased ex vivo C16.0+MSU-induced IL-1β production.ConclusionsIn agreement with the reported low inhibitory potency of butyrate, a high concentration was needed for cytokine suppression, whereas synthetic HDAC inhibitors showed potent anti-inflammatory effects at nanomolar concentrations. These novel HDAC inhibitors could be effective in the treatment of acute gout. Moreover, the use of specific HDAC inhibitors could even improve the efficacy and reduce any potential adverse effects.

scholarly journals A Limited Group of Class I Histone Deacetylases Acts To Repress Human Immunodeficiency Virus Type 1 Expression

2009 ◽  
Vol 83 (10) ◽  
pp. 4749-4756 ◽  
Author(s):  
Kara S. Keedy ◽  
Nancie M. Archin ◽  
Adam T. Gates ◽  
Amy Espeseth ◽  
Daria J. Hazuda ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Histone Deacetylases ◽  
Hdac Inhibitors ◽  
Class I ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Silencing of the integrated human immunodeficiency virus type 1 (HIV-1) genome in resting CD4+ T cells is a significant contributor to the persistence of infection, allowing the virus to evade both immune detection and pharmaceutical attack. Nonselective histone deacetylase (HDAC) inhibitors are capable of inducing expression of quiescent HIV-1 in latently infected cells. However, potent global HDAC inhibition can induce host toxicity. To determine the specific HDACs that regulate HIV-1 transcription, we evaluated HDAC1 to HDAC11 RNA expression and protein expression and compartmentalization in the resting CD4+ T cells of HIV-1-positive, aviremic patients. HDAC1, -3, and -7 had the highest mRNA expression levels in these cells. Although all HDACs were detected in resting CD4+ T cells by Western blot analysis, HDAC5, -8, and -11 were primarily sequestered in the cytoplasm. Using chromatin immunoprecipitation assays, we detected HDAC1, -2, and -3 at the HIV-1 promoter in Jurkat J89GFP cells. Targeted inhibition of HDACs by small interfering RNA demonstrated that HDAC2 and HDAC3 contribute to repression of HIV-1 long terminal repeat expression in the HeLa P4/R5 cell line model of latency. Together, these results suggest that HDAC inhibitors specific for a limited number of class I HDACs may offer a targeted approach to the disruption of persistent HIV-1 infection.

scholarly journals Synthesis, Molecular Docking and Biological Characterization of Pyrazine Linked 2-Aminobenzamides as New Class I Selective Histone Deacetylase (HDAC) Inhibitors with Anti-Leukemic Activity

2021 ◽  
Vol 23 (1) ◽  
pp. 369
Author(s):  
Hany S. Ibrahim ◽  
Mohamed Abdelsalam ◽  
Yanira Zeyn ◽  
Matthes Zessin ◽  
Al-Hassan M. Mustafa ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Cancer Cells ◽  
Histone Deacetylases ◽  
Hdac Inhibitors ◽  
Md Simulations ◽  
Docking Studies ◽  
Class I ◽  
Hek293 Cells ◽  
Human Acute Myeloid Leukemia

Class I histone deacetylases (HDACs) are key regulators of cell proliferation and they are frequently dysregulated in cancer cells. We report here the synthesis of a novel series of class-I selective HDAC inhibitors (HDACi) containing a 2-aminobenzamide moiety as a zinc-binding group connected with a central (piperazin-1-yl)pyrazine or (piperazin-1-yl)pyrimidine moiety. Some of the compounds were additionally substituted with an aromatic capping group. Compounds were tested in vitro against human HDAC1, 2, 3, and 8 enzymes and compared to reference class I HDACi (Entinostat (MS-275), Mocetinostat, CI994 and RGFP-966). The most promising compounds were found to be highly selective against HDAC1, 2 and 3 over the remaining HDAC subtypes from other classes. Molecular docking studies and MD simulations were performed to rationalize the in vitro data and to deduce a complete structure activity relationship (SAR) analysis of this novel series of class-I HDACi. The most potent compounds, including 19f, which blocks HDAC1, HDAC2, and HDAC3, as well as the selective HDAC1/HDAC2 inhibitors 21a and 29b, were selected for further cellular testing against human acute myeloid leukemia (AML) and erythroleukemic cancer (HEL) cells, taking into consideration their low toxicity against human embryonic HEK293 cells. We found that 19f is superior to the clinically tested class-I HDACi Entinostat (MS-275). Thus, 19f is a new and specific HDACi with the potential to eliminate blood cancer cells of various origins.

scholarly journals Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Oxana Schmidt ◽  
Nadja Nehls ◽  
Carolin Prexler ◽  
Kristina von Heyking ◽  
Tanja Groll ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Microarray Analysis ◽  
Ewing Sarcoma ◽  
Histone Deacetylases ◽  
Hdac Inhibitors ◽  
Class I ◽  
Specific Expression ◽  
Regulatory Complex ◽  
Xenograft Mice

Abstract Background Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). Here histone deacetylases (HDAC) class I were investigated. Methods Their role was determined using different inhibitors including TSA, Romidepsin, Entinostat and PCI-34051 as well as CRISPR/Cas9 class I HDAC knockouts and HDAC RNAi. To analyze resulting changes microarray analysis, qRT-PCR, western blotting, Co-IP, proliferation, apoptosis, differentiation, invasion assays and xenograft-mouse models were used. Results Class I HDACs are constitutively expressed in EwS. Patients with high levels of individual class I HDAC expression show decreased overall survival. CRISPR/Cas9 class I HDAC knockout of individual HDACs such as HDAC1 and HDAC2 inhibited invasiveness, and blocked local tumor growth in xenograft mice. Microarray analysis demonstrated that treatment with individual HDAC inhibitors (HDACi) blocked an EWS-FLI1 specific expression profile, while Entinostat in addition suppressed metastasis relevant genes. EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics including Doxorubicin in the presence of HDACi. Furthermore, HDACi treatment mimicked RNAi of EZH2 in EwS. Treated cells showed diminished growth capacity, but an increased endothelial as well as neuronal differentiation ability. HDACi synergizes with EED inhibitor (EEDi) in vitro and together inhibited tumor growth in xenograft mice. Co-IP experiments identified HDAC class I family members as part of a regulatory complex together with PRC2. Conclusions Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and in combination therapy, co-treatment with HDACi is an interesting new treatment opportunity for this malignant disease.

Abstract 158: Regulation of miR-21 Expression by Acetylation in Myocardial Infarction

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Ludivine Renaud ◽  
Harinath Kasiganesan ◽  
Erhe Gao ◽  
Santhosh K Mani ◽  
Jeffrey A Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myocardial Infarction ◽  
Transcription Factors ◽  
Histone Deacetylases ◽  
Hdac Inhibitors ◽  
Class I ◽  
Transcriptional Level ◽  
Sodium Calcium Exchanger ◽  
Acetylation State ◽  
Innovative Therapies

Cardiovascular diseases are one of the leading causes of morbidity and mortality in the world, underlining the need for innovative therapies and diagnosis. Recent reports have identified microRNAs (miRNAs) as central players in regulating gene expression and showed that several miRNAs are aberrantly expressed in cardiac arrhythmia, hypertrophy, fibrosis, ischemia, vascular atherosclerosis and heart failure. Gene expression is also regulated at the transcriptional level by histone deacetylases (HDACs) under basal and pathological conditions. We previously demonstrated that 1) class I and class II HDACs play an important role in the basal expression and upregulation of the sodium-calcium exchanger (Ncx1) gene in adult cardiomyocytes and pressure-overloaded ventricle and 2) treatment with class I/IIb HDAC inhibitors trichostatin (TSA) or suberoylanilide hydroxamic acid (SAHA) improved ventricular function by suppressing matrix metalloproteinases (MMPs) gene expression in myocardial infarction (MI).Therefore it is possible that protein acetylation regulates the expression of some miRNAs and we hypothesize that HDAC inhibition would attenuate the shift in expression of certain miRNAs that are aberrantly expressed post-MI. In a pilot study, ligation of the left anterior descending (LAD) coronary artery was performed to induce MI with and without SAHA treatment. Because of its misregulation and relevance in cardiac hypertrophy and MI, we examined the expression level of miR-21. qRT-PCR confirmed that miR-21 is increased by 8 fold 7 days post-MI. Interestingly, SAHA treatment significantly attenuated the abnormal expression of miR-21. To our knowledge, it is the first report of the regulation of a miRNA by HDACs in the heart. We anticipate that not only miR-21 but other miRNAs will fall under the same mechanistic control via acetylation. The miR-21 promoter contains binding sites for several transcription factors that get acetylated and we speculate that one or more HDAC mediate the expression of miR-21 by controlling the acetylation state of transcription factors interacting with the miR-21 promoter.

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