In vitro assays for the determination of histone deacetylase activity

Heltweg, Birgit; Trapp, Johannes; Jung, Manfred

doi:10.1016/j.ymeth.2005.03.003

Cited by 111 publications

(139 citation statements)

References 20 publications

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“…29 Under assay conditions, which were used for the human isoform, smSirt2 showed less conversion of the substrate ZMAL. In the course of a buffer optimization, we identified Triton X-100 to have beneficial effects on smSirt2 activity (Fig.…”

Section: Homogeneous Fluorescence-based Assay For Smsirt2mentioning

confidence: 97%

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Fluorescence-Based Screening Assays for the NAD+-Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni

et al. 2015

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Sirtuins are NAD + -dependent histone deacetylases (HDACs) that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Five sirtuin isoforms are encoded in the genome of the parasitic pathogen Schistosoma mansoni. During its life cycle, S. mansoni undergoes drastic changes in phenotype that are associated with epigenetic modifications. Previous work showed strong effects of hSirt2 inhibitors on both worm life span and reproduction. Thus, we postulate smSirt2 as a new antiparasite target. We report both the optimization of a homogeneous fluorescence-based assay and the development of a new heterogeneous fluorescence-based assay to determine smSirt2 activity. The homogeneous assay uses a coumarin-labeled acetyl lysine derivative, and the heterogeneous version is using a biotinylated and fluorescence-labeled oligopeptide. Magnetic streptavidin-coated beads allow higher substrate loading per well than streptavidin-coated microtiter plates and make it possible to screen for inhibitors of either smSirt2 or its human isoform (hSirt2) for selectivity studies. We also present hits from a pilot screen with inhibitors showing an IC 50 lower than 50 µM. Binding of the hits to their targets is rationalized by docking studies using a homology model of smSirt2.

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Section: Homogeneous Fluorescence-based Assay For Smsirt2mentioning

confidence: 97%

“…29 The assay was performed in black 384-well nonbinding plates (Greiner Bio-One, Monroe, NC) with a reaction volume of 20 µL per well. All tests were performed at least in duplicate using Z-(Ac)Lys-AMC (ZMAL) as a substrate.…”

Section: Homogeneous Fluorescence-based Assay For Smsirt2mentioning

confidence: 99%

Fluorescence-Based Screening Assays for the NAD+-Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni

et al. 2015

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“…The inhibitory activity against hSirt1, hSirt2 and hSirt3 was determined by a histone deacetylase assay, previously established [69], with further details provided in the …”

Section: In Vitro Assaymentioning

confidence: 99%

Identification of Bichalcones as Sirtuin Inhibitors by Virtual Screening and <em>in Vitro </em>Testing

Karaman

Alhalabi

Swyter

et al. 2018

Preprint

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Abstract:Sirtuins are nicotinamide adenine dinucleotide (NAD + )-dependent class III histone deacetylases and have been linked to the pathogenesis of numerous diseases such as HIV, metabolic disorders, neurodegeneration and cancer. Docking of the virtual pan-African natural products library (p-ANAPL), followed by in vitro testing, resulted in the identification of two inhibitors of sirtuin 1, 2 and 3 (sirt1-3). Two bichalcones; rhuschalcone IV (8) and rhuschalcone I (9), previously isolated from the medicinal plant Rhus pyroides, were shown to be active in the in vitro assay, with rhuschalcone I showing the best activity against sirt1, having an IC50 = 40.8 µM. Based on the docking experiments, suggestions for improving the biological activities of the newly identified hit compounds have been provided.

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“…11,12 In this protocol, a derivative of acetyl lysine is deacetylated by the enzyme(s) and the product formation is measured by a subsequent detection step. This involves coupling with a protease, usually trypsin, which leads to liberation of the fluorophore AMC with a concomitant shift in fluorescence wavelength.…”

Section: Trypsin Assaymentioning

confidence: 99%

Cellular analysis of the action of epigenetic drugs and probes

et al. 2017

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Small molecule drugs and probes are important tools in drug discovery, pharmacology, and cell biology. This is of course also true for epigenetic inhibitors. Important examples for the use of established epigenetic inhibitors are the study of the mechanistic role of a certain target in a cellular setting or the modulation of a certain phenotype in an approach that aims toward therapeutic application. Alternatively, cellular testing may aim at the validation of a new epigenetic inhibitor in drug discovery approaches. Cellular and eventually animal models provide powerful tools for these different approaches but certain caveats have to be recognized and taken into account. This involves both the selectivity of the pharmacological tool as well as the specificity and the robustness of the cellular system. In this article, we present an overview of different methods that are used to profile and screen for epigenetic agents and comment on their limitations. We describe not only diverse successful case studies of screening approaches using different assay formats, but also some problematic cases, critically discussing selected applications of these systems.

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In vitro assays for the determination of histone deacetylase activity

Cited by 111 publications

References 20 publications

Fluorescence-Based Screening Assays for the NAD+-Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni

Fluorescence-Based Screening Assays for the NAD+-Dependent Histone Deacetylase smSirt2 from Schistosoma mansoni

Identification of Bichalcones as Sirtuin Inhibitors by Virtual Screening and <em>in Vitro </em>Testing

Cellular analysis of the action of epigenetic drugs and probes

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