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1.
Artigo em Inglês | MEDLINE | ID: mdl-36029930

RESUMO

Deterioration of inhibitory synapse may be an essential neurological basis underlying abnormal social behaviours. Manipulations that regulate GABAergic transmission are associated with improved behavioural phenotypes in sociability. The synaptic protein, Ephrin-B2 (EB2), plays an important role in the maintenance and reconfiguration of inhibitory synapses in the medial prefrontal cortex (mPFC). However, the inhibitory cell-type specific role of EB2 in the pathophysiology and treatment of social deficits remains unknown. As expected, we revealed that tdTomato-expressing cells were only found in GABAergic neurons instead of excitatory neurons in transgenic EB2-vGATCre mice. This result indicated that depletion of EB2 would occur in those neurons, which further contribute to social deficits. In addition, specific over-expression of mPFC EB2 restored the defective social behaviour abnormalities. These results suggest that the effect of EB2 on social deficits is anatomically and cell-type specific. More importantly, the global upregulation of HDAC4 expression was found in EB2-vGATCre mice. Significant subcellular nuclear shuttling of HDAC4 in vGAT+ neurons was examined and quantified, suggesting a role of nuclear HDAC4 in mediating the mechanism underlying EB2 impairment in vGAT+ neurons. Treatment with LMK235 led to a remarkable rescue of social deficits, thus our data revealed a new domain for the potential utility of HDAC targeting agents to treat social deficits. In conclusion, these results not only revealed a novel molecular mechanism underlying the pathophysiology of social deficits, but also suggested a potential intervention avenue for the treatment of social deficits.


Assuntos
Efrina-B2 , Histona Desacetilases , Animais , Camundongos , Proteínas de Transporte , Efrina-B2/metabolismo , Neurônios GABAérgicos/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Camundongos Transgênicos , Mutação , Sinapses/metabolismo
2.
Methods Mol Biol ; 2589: 3-15, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255614

RESUMO

Besides the physiological role of histone deacetalylases in maintaining normal cellular integrity, the acetylation landscape is changed in cancer cells, which has been implicated as a potential target in cancer therapy. The overexpression of certain HDACs correlates with specific cancer types. Therefore, the development of specific HDAC inhibitors may extend the therapeutic strategy for cancer therapy. Here, we describe how to investigate the therapeutic potential of specific HDACi by treatment in a mouse model for B-cell lymphoma, exemplified by the HDAC6 inhibitor Marbostat-100.


Assuntos
Inibidores de Histona Desacetilases , Linfoma , Camundongos , Animais , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Histonas , Histona Desacetilases/genética , Acetilação , Linfoma/tratamento farmacológico , Modelos Animais de Doenças
3.
Methods Mol Biol ; 2589: 75-85, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255618

RESUMO

Reliable preclinical drug testing models for cancer research are urgently needed with zebrafish embryo models emerging as a powerful vertebrate model for xenotransplantation studies. Here, we describe the evaluation of toxicity, efficacy, and on-target activity of histone deacetylase (HDAC) inhibitors in a zebrafish embryo yolk sac xenotransplantation model of medulloblastoma and neuroblastoma cells. For this, we performed toxicity assays with our zebrafish drug library consisting of 28 clinically relevant targeted as well as chemotherapeutic drugs with zebrafish embryos. We further engrafted zebrafish embryos with fluorescently labeled pediatric tumor cells (SK-N-BE(2)-C, HD-MB03, or MED8A) and monitored the progression after HDAC inhibitor treatment of xenotransplanted tumors through tumor volume measurements with high-content confocal microscopy in a multi-well format. The on-target activity of HDAC inhibitors was verified through immunohistochemistry staining on paraffin-embedded early larvae. Overall, the zebrafish embryo xenotransplantation model allows for fast and cost-efficient in vivo evaluation of targeted drug toxicity, efficacy, and on-target activity in the field of precision oncology.


Assuntos
Neoplasias , Peixe-Zebra , Animais , Humanos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Xenoenxertos , Neoplasias/tratamento farmacológico , Medicina de Precisão , Modelos Animais de Doenças , Histona Desacetilases , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral
4.
Methods Mol Biol ; 2589: 51-73, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255617

RESUMO

Class I histone deacetylases (HDACs) are important regulators of cellular functions in health and disease. HDAC1, HDAC2, HDAC3, and HDAC8 are promising targets for the treatment of cancer, neurological, and immunological disorders. These enzymes have both catalytic and non-catalytic functions in the regulation of gene expression. We here describe the generation of a genetic toolbox by the CRISPR/Cas9 methodology in nearly haploid human tumor cells. This novel model system allows to discriminate between catalytic and structural functions of class I HDAC enzymes and to mimic the treatment with specific HDAC inhibitors.


Assuntos
Inibidores de Histona Desacetilases , Neoplasias , Humanos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteínas Repressoras
5.
Methods Mol Biol ; 2589: 145-155, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255623

RESUMO

Class I histone deacetylase (HDAC) enzymes are key regulators of cell proliferation and are frequently dysregulated in cancer cells. Here we describe the synthesis of a novel series of class-I selective HDAC inhibitors containing anilinobenzamide moieties as ZBG connected with a central (piperazin-1-yl)pyrazine moiety. Compounds were tested in vitro against class-I HDAC1, 2, and 3 isoforms. Some highly potent HDAC inhibitors were obtained and were tested in pancreatic cancer cells and showed promising activity. Moreover, we summarize how the growth-inhibitory effects of these compounds can be determined in murine pancreatic cancer cell lines.


Assuntos
Inibidores de Histona Desacetilases , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Inibidores de Histona Desacetilases/farmacologia , Pirazinas/farmacologia , Linhagem Celular Tumoral , Histona Desacetilases/metabolismo , Proliferação de Células , Isoformas de Proteínas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Relação Estrutura-Atividade , Histona Desacetilase 1/metabolismo
6.
Methods Mol Biol ; 2589: 129-144, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255622

RESUMO

Systemic administration of histone deacetylase inhibitors (HDACi), like valproic acid (VPA), is often associated with rapid drug metabolization and untargeted tissue distribution. This requires high-dose application that can lead to unintended side effects. Hence, drug carrier systems such as nanoparticles (NPs) are developed to circumvent these disadvantages by enhancing serum half-life as well as organ specificity.This chapter gives a summary of the biological characterization of HDACi-coupled NPs in vitro, including investigation of cellular uptake, biocompatibility, as well as intracellular drug release and activity. Suitable methods, opportunities, and challenges will be discussed to provide general guidelines for the analysis of HDACi drug carrier systems with a special focus on recently developed cellulose-based VPA-coupled NPs.


Assuntos
Inibidores de Histona Desacetilases , Nanopartículas , Inibidores de Histona Desacetilases/farmacologia , Ácido Valproico/farmacologia , Portadores de Fármacos , Celulose
7.
Methods Mol Biol ; 2589: 157-177, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255624

RESUMO

The aberrant activity of histone deacetylases (HDACs) across a broad range of cancers and other disease indications has led to the development of small-molecule inhibitors that target one or more members of the HDAC protein family. Emerging HDAC inhibitors that show promise in drug discovery programs must be assessed across a range of in vitro assays to establish an inhibitor profile for potency and cellular selectivity towards target HDAC(s) as well as preliminary absorption, distribution, metabolism, and excretion (ADME) features. Here we provide an overview of methods to determine a subset of pivotal in vitro drug-like parameters for HDAC inhibitors (HDACi). We initially describe protocols for parallel artificial membrane permeability assays (PAMPA) to evaluate the passive permeability of small molecules across lipid membranes. Subsequently, we elaborate on cytotoxicity assays using CellTiter-Blue to determine HDACi-induced cell death in healthy/diseased cellular models. We next focus on assessing the target engagement of inhibitors with the appropriate HDAC isoforms in a cellular environment via Western blotting of acetylated HDAC substrates. Finally, we provide detailed guidelines on how to assess the metabolic stability of HDACi through whole blood stability assays. Collectively, these assays provide an overview of the permeability, selectivity, and stability of the HDAC inhibitor under development.


Assuntos
Inibidores de Histona Desacetilases , Histona Desacetilases , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Isoformas de Proteínas/metabolismo , Membranas Artificiais , Lipídeos
8.
Methods Mol Biol ; 2589: 195-205, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255626

RESUMO

The ability of histone deacetylase inhibitors (HDACi) like valproic acid (VPA) as a therapeutic for inflammatory diseases or cancer has increased the interest in HDACi and their targeted transport to diseased tissues. Administration of VPA immobilized on polymeric carriers was found to be a suitable approach to circumvent drawbacks such as rapid metabolization, short serum half-life, or side effects. Polysaccharides are convenient biopolymeric carriers due to their biocompatibility and biodegradability. Furthermore, the hydroxy-, amino-, or carboxylic groups are predestinated for functionalization. The esterification of three hydroxy groups of cellulose with VPA leads to products having a high amount of VPA loading. Subsequent shaping yielded uniform nanoparticles (NPs) of around 150 nm in size capable of releasing VPA in a controlled way under physiological conditions.


Assuntos
Inibidores de Histona Desacetilases , Nanopartículas , Inibidores de Histona Desacetilases/farmacologia , Ácido Valproico/farmacologia , Celulose
9.
Methods Mol Biol ; 2589: 293-302, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255632

RESUMO

This book chapter describes a plasmid-based reporter method, first described by Bennardo et al. (2008) that we use in our laboratory for determining the activity of the repair of DNA double-strand breaks by nonhomologous end joining. This method can be used to measure the impact of epigenetic modifiers of the histone deacetylase family on this DNA repair pathway by flow cytometry.


Assuntos
Quebras de DNA de Cadeia Dupla , Inibidores de Histona Desacetilases , Inibidores de Histona Desacetilases/farmacologia , Reparo do DNA por Junção de Extremidades , Reparo do DNA , DNA , Histona Desacetilases
10.
Methods Mol Biol ; 2589: 241-252, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255629

RESUMO

Primary hepatocytes are the gold standard in pharmaco- and toxicokinetic studies during preclinical development of drug candidates. Such cells are a valuable tool to identify potential hepatotoxicity, an important adverse drug reaction. Primary hepatocytes can be obtained not only from wild-type mice but also from genetically engineered knockout mouse strains. Liver perfusion yields murine primary hepatocytes (mpH) with high vitality, expressing an array of metabolic enzymes and transporters that are impaired or even absent in established liver cell lines. Furthermore, mpH display no genetic alterations and are proficient in the DNA damage response pathway. This makes mpH a suitable model to analyze the effects of histone deacetylase inhibitors on DNA damage and cell viability. Here, we report an efficient and fast protocol for the isolation of mpH by liver perfusion. These mpH can be used for downstream applications such as the detection of the DNA damage marker γH2AX by confocal laser scanning microscopy.


Assuntos
Hepatócitos , Inibidores de Histona Desacetilases , Camundongos , Animais , Inibidores de Histona Desacetilases/farmacologia , Hepatócitos/metabolismo , Dano ao DNA , Fígado , Sobrevivência Celular
11.
Methods Mol Biol ; 2589: 207-221, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255627

RESUMO

Cyanoacrylates define a class of inhibitors which are capable to form a transient covalent bond with a cysteine flanking the binding site, thereby increasing the residence time and prolonging the inhibitory effect on the target protein under nonequilibrium conditions. Herein, we describe the synthetic access to cyanoacrylate-based HDAC4 inhibitors and the procedures for the characterization of the transient nature of the covalent bond between cyanoacrylates and thiols or cysteines in HDAC4.


Assuntos
Cianoacrilatos , Cisteína , Cisteína/metabolismo , Sítios de Ligação , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química
12.
Methods Mol Biol ; 2589: 337-344, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255635

RESUMO

Reactive oxygen species (ROS) are induced by several chemotherapeutics. In this protocol, we describe a flow cytometry-based method for the analysis of the intracellular levels of ROS in vital leukemic cells in response to the histone deacetylase inhibitor vorinostat. This measurement of ROS using the cell-permeable dye CM-H2DCFDA indicates intracellular oxidative stress.


Assuntos
Inibidores de Histona Desacetilases , Estresse Oxidativo , Inibidores de Histona Desacetilases/farmacologia , Espécies Reativas de Oxigênio , Vorinostat/farmacologia , Linhagem Celular Tumoral , Apoptose , Ácidos Hidroxâmicos/farmacologia
13.
Methods Mol Biol ; 2589: 269-291, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255631

RESUMO

Posttranslational modifications are important for protein functions and cellular signaling pathways. The acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), with the latter being grouped into four phylogenetic classes. The class III of the HDAC family, the sirtuins (SIRTs), contributes to gene expression, genomic stability, cell metabolism, and tumorigenesis. Thus, several specific SIRT inhibitors (SIRTi) have been developed to target cancer cell proliferation. Here we provide an overview of methods to study SIRT-dependent cell metabolism and mitochondrial functionality. The chapter describes metabolic flux analysis using Seahorse analyzers, methods for normalization of Seahorse data, flow cytometry and fluorescence microscopy to determine the mitochondrial membrane potential, mitochondrial content per cell and mitochondrial network structures, and Western blot analysis to measure mitochondrial proteins.


Assuntos
Sirtuínas , Sirtuínas/metabolismo , Lisina/metabolismo , Filogenia , Acetilação , Histona Desacetilases/metabolismo , Histona Acetiltransferases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Inibidores de Histona Desacetilases/farmacologia
14.
Methods Mol Biol ; 2589: 253-268, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255630

RESUMO

The endoplasmic reticulum (ER) is a multifunctional cell organelle which is important for the folding and processing of proteins. Different endogenous and exogenous factors can disturb the ER homeostasis, causing ER stress and activating the unfolded protein response (UPR) to remove misfolded proteins and aggregates. ER stress and the UPR are associated with several human diseases, such as diabetes, Alzheimer's or Parkinson's disease, and cancer. Histone deacetylase inhibitors (HDACi) are used to treat cancer and were shown to induce ER stress/to modulate the UPR, although the exact mechanism is not fully understood and needs further research. Several approaches to monitoring ER stress exist. Here we describe methods including qPCR, Western blot, transmission electron microscopy, and fluorescence microscopy to analyze changes in mRNA and protein expression levels as well as defects in ER structures after HDAC inhibitor-induced ER stress.


Assuntos
Estresse do Retículo Endoplasmático , Inibidores de Histona Desacetilases , Humanos , Inibidores de Histona Desacetilases/farmacologia , Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , RNA Mensageiro/metabolismo
15.
Methods Mol Biol ; 2589: 455-466, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255642

RESUMO

During the preclinical development of small molecule inhibitors, compounds or compound libraries are typically first screened using purified target enzymes in vitro to select candidates with high potency. In the later stages of the development, however, functional cell-based assays may provide biologically more relevant data. In this chapter, we describe a detailed protocol for determining the potency of inhibitors targeting human histone deacetylase 6 in complex cellular environments. Cells are first treated with a dilution series of tested compounds, cell lysates separated by SDS-PAGE, and electrotransferred to a blotting membrane. The inhibitor potency is then determined indirectly by quantifying the levels of acetylated tubulin as a surrogate readout.


Assuntos
Inibidores de Histona Desacetilases , Tubulina (Proteína) , Humanos , Desacetilase 6 de Histona/metabolismo , Tubulina (Proteína)/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Acetilação
16.
Methods Mol Biol ; 2589: 429-454, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255641

RESUMO

Epigenetic alterations have been identified in various tumor types. In part, these alterations are mediated via increased histone deacetylase activity. Although preclinical results of monotherapies with histone deacetylase inhibitors (HDACi) are promising, success in clinical trials is limited. Reasons for these limitations may be de novo or acquired resistance to HDAC inhibitors that could be overcome with rational combination therapies. This requires knowledge of resistance mechanism along with the involved genetic networks. One way to identify such genetic networks is the implementation of a CRISPR-based technology allowing transcriptional repression (CRISPRi) and activation (CRISPRa) at a genome-wide scale. We describe a simple approach to amplify and validate sgRNA libraries, generate a myeloid progenitor cell line expressing catalytically dead Cas9 (dCas9) fusion proteins with transcriptional effectors to repress or activate genetic regions of interest and demonstrate a complementary genome-wide HDACi resistance screening approach. Furthermore, we present bioinformatics tools for quality control and analysis of the sequencing data.


Assuntos
Redes Reguladoras de Genes , Inibidores de Histona Desacetilases , Inibidores de Histona Desacetilases/farmacologia , Proteína 9 Associada à CRISPR , Expressão Gênica , Histona Desacetilases/genética , Sistemas CRISPR-Cas
17.
Methods Mol Biol ; 2589: 467-480, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255643

RESUMO

Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains.


Assuntos
Lisina , Peixe-Zebra , Animais , Desacetilase 6 de Histona/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Domínio Catalítico , Cristalização , Lisina/metabolismo , Ubiquitinas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Acetilação
18.
Methods Mol Biol ; 2589: 481-492, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255644

RESUMO

Histone deacetylase 6 (HDAC6) is an emerging clinical target for the treatment of several hematological cancers and central nervous system disorders. HDAC6 catalyzes the deacetylation of lysine residues on substrates such as tubulin, with profound implications in key cellular processes, including cellular motility and migration. This critical deacetylation activity occurs at the catalytic domain 2 (CD2) of HDAC6, and small molecule inhibitors of HDAC6 are designed to target CD2. We briefly highlight previously reported strategies for recombinant bacterial expression and purification of the HDAC6 CD2. We aim to discuss competition assays that have been used to evaluate the potency of potential HDAC6 inhibitors against CD2 via displacement of pre-bound fluorescent HDAC-probes. Moreover, we elaborate on previous protocols that have been employed in inhibitor screening and present an HDAC6-selective probe that also enables rapid and reliable high-throughput screening of new chemical entities designed to target the HDAC6 CD2.


Assuntos
Inibidores de Histona Desacetilases , Tubulina (Proteína) , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Desacetilase 6 de Histona/metabolismo , Tubulina (Proteína)/metabolismo , Lisina/metabolismo , Acetilação , Polarização de Fluorescência
19.
Methods Mol Biol ; 2588: 279-293, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36418694

RESUMO

Histone acetylation and deacetylation of DNA-associated proteins have been shown to alter the architecture of chromatin, affecting gene expression and controlling a wide range of biological events. These events are balanced by two sets of cellular enzymes, histone-deacetylases (HDACs) and histone acetyl-transferases (HATs). Pharmacological inhibition of histone-deacetylases (HDACs) using HDAC-inhibitors (HDACis) has been shown to promote dental pulp cell reparative processes with therapeutic implications in various fields including regenerative dentistry. To date, pan-HDACi have generally been used rather than isoform-specific HDACi targeting, despite the fact that HDAC-specific inhibitors have been developed to target HDACs in several tissues. To identify potential therapeutic targets in the tooth, the expression and distribution of HDAC-isoforms need to be analyzed. This chapter focuses on techniques to analyze expression, location, and distribution of individual HDAC-isoforms under mineralizing conditions using both histology and cell biology, along with a description of basic techniques for culturing and mineralization of rodent dental pulp cells.


Assuntos
Polpa Dentária , Histonas , Acetilação , Processamento de Proteína Pós-Traducional , Histona Desacetilases/genética , Inibidores de Histona Desacetilases/farmacologia
20.
Methods Mol Biol ; 2588: 353-367, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36418697

RESUMO

Recently, the central role of microRNAs (miRNAs) and epigenetic modifiers in biological and pathological processes, such as stem cell differentiation and inflammation, has stimulated interest. In particular, their influence in dental pulp stem cell (DPSC) differentiation has been highlighted as an exciting avenue of research in the field of Regenerative Endodontics. Although specific miRNAs have been shown to be altered in expression during dental pulp mineralization and repair processes, their interaction with epigenetic modifiers, such as histone deacetylase inhibitors (HDACi) or DNA methyltransferase inhibitors (DNMTi), has not been explored. Currently available next-generation sequencing (NGS) technologies offer the potential to explore in detail the miRNA expression profile of cells, and to investigate the effects of pharmacological inhibitors such as epigenetic modifiers on this profile. This chapter describes the experimental methods required to induce mineralization of DPCs in the presence and absence of epigenetic modifiers and analyze the resulting miRNA expression profiles using RNA sequencing (RNAseq), with a focus on bioinformatic analysis.


Assuntos
Polpa Dentária , MicroRNAs , MicroRNAs/genética , Inibidores de Histona Desacetilases/farmacologia , Diferenciação Celular/genética , Técnicas de Cultura de Células
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