Improved fluorogenic histone deacetylase assay for high-throughput-screening applications

Wegener, Dennis; Hildmann, Christian; Riester, Daniel; Schwienhorst, Andreas

doi:10.1016/s0003-2697(03)00426-3

Cited by 107 publications

(92 citation statements)

References 18 publications

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“…Assay of HDAC Activities and Inhibition Kinetics-The deacetylase activities of HDACs 1, 2, and 3 were measured by assaying enzyme activity using peptidase (Lys-C peptidase and trypsin) and the synthetic substrate acetyl-Lys(Ac)-AMC, as previously described (26,27). Deacetylated lysine-AMC was released by the peptidase and free fluorogenic 4-methylcoumarin-7-amide (MCA) was generated.…”

Section: Methodsmentioning

confidence: 99%

Pimelic Diphenylamide 106 Is a Slow, Tight-binding Inhibitor of Class I Histone Deacetylases

Chou

Herman

Gottesfeld

2008

Journal of Biological Chemistry

192

261

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Histone deacetylase (HDAC) inhibitors, including various benzamides and hydroxamates, are currently in clinical development for a broad range of human diseases, including cancer and neurodegenerative diseases. We recently reported the identification of a family of benzamide-type HDAC inhibitors that are relatively non-toxic compared with the hydroxamates. Members of this class of compounds have shown efficacy in cellbased and mouse models for the neurodegenerative diseases Friedreich ataxia and Huntington disease. Considerable differences in IC 50 values for the various HDAC enzymes have been reported for many of the HDAC inhibitors, leading to confusion as to the HDAC isotype specificities of these compounds. Here we show that a benzamide HDAC inhibitor, a pimelic diphenylamide (106), is a class I HDAC inhibitor, demonstrating no activity against class II HDACs. 106 is a slow, tight-binding inhibitor of HDACs 1, 2, and 3, although inhibition for these enzymes occurs through different mechanisms. Inhibitor 106 also has preference toward HDAC3 with K i of ϳ14 nM, 15 times lower than the K i for HDAC1. In comparison, the hydroxamate suberoylanilide hydroxamic acid does not discriminate between these enzymes and exhibits a fast-on/fast-off inhibitory mechanism. These observations may explain a paradox involving the relative activities of pimelic diphenylamides versus hydroxamates as gene activators.The link between post-translational modifications by reversible histone acetylation and deacetylation and mRNA transcription has been shown to be one of the key mechanisms of epigenetic gene regulation (1). Acetylation of histone lysine residues, controlled by the histone acetyltransferases and histone deacetylases (HDACs), 2 has been a subject of intense recent research (2-5). Generally, histone hypoacetylation causes transcriptional silencing, whereas histone hyperacetylation results in transcriptional activation of various genes (6 -8). Eighteen HDACs have been identified in the human genome, including the zinc-dependent HDACs (class I, class II, and class IV), and the NAD ϩ -dependent enzymes (class III or sirtuins) (9, 10). HDACs 1, 2, 3, and 8 belong to class I, showing homology to the yeast enzyme RPD3. Class II is further divided into class IIa (HDACs 4, 5, 7, and 9) and IIb (HDAC 6 and 10), according to their sequence homology and domain organization. HDAC11 is the lone member of class IV (9, 11). The sirtuins (class III) are related to the yeast Sir2 protein and are involved in regulation of metabolism and aging (10).To date, a number of small molecule inhibitors of the zincdependent HDACs have been identified (12). These compounds can be broadly grouped in four chemical classes: the hydroxamates, the benzamides, butyrate analogs, and cyclic peptides, such as depsipeptide and related compounds (12, 13). Hydroxamate-based inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroamic acid (SAHA; Fig. 1A) are believed to be pan-HDAC inhibitors (14 -16); however, recent studies have shown that TSA and SAHA...

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Section: Methodsmentioning

confidence: 99%

Pimelic Diphenylamide 106 Is a Slow, Tight-binding Inhibitor of Class I Histone Deacetylases

Chou

Herman

Gottesfeld

2008

Journal of Biological Chemistry

192

261

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“…6) as described previously (29,30). In the first reaction catalyzed by the FB188 enzyme, acetate is released from ε-acetylated lysine moieties.…”

Section: Vol 186 2004 Histone Deacetylase-like Amidohydrolase 2329mentioning

confidence: 99%

“…To analyze substrate specificities of the enzyme, the assay was also carried out with Z-His-Arg-Lys(ε-acetyl)-MCA, Ac-Arg-GlyLys(ε-acetyl)-MCA, Ac-Gly-Ala-Lys(ε-acetyl)-MCA, Tos-Gly-Pro-Lys(ε-acetyl)-MCA, and Ac-Gly-Gly-Lys(ε-Ala)-MCA as substrates. With these tripeptide substrates, a novel protocol was used (29).…”

Section: Vol 186 2004 Histone Deacetylase-like Amidohydrolase 2329mentioning

confidence: 99%

A New Amidohydrolase from Bordetella or Alcaligenes Strain FB188 with Similarities to Histone Deacetylases

et al. 2004

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The full-length gene encoding the histone deacetylase (HDAC)-like amidohydrolase (HDAH) from Bordetella or Alcaligenes (Bordetella/Alcaligenes) strain FB188 (DSM 11172) was cloned using degenerate primer PCR combined with inverse-PCR techniques and ultimately expressed in Escherichia coli. The expressed enzyme was biochemically characterized and found to be similar to the native enzyme for all properties examined. Nucleotide sequence analysis revealed an open reading frame of 1,110 bp which encodes a polypeptide with a theoretical molecular mass of 39 kDa. Interestingly, peptide sequencing disclosed that the N-terminal methionine is lacking in the mature wild-type enzyme, presumably due to the action of methionyl aminopeptidase. Sequence database searches suggest that the new amidohydrolase belongs to the HDAC superfamily, with the closest homologs being found in the subfamily assigned acetylpolyamine amidohydrolases (APAH). The APAH subfamily comprises enzymes or putative enzymes from such diverse microorganisms as Pseudomonas aeruginosa, Archaeoglobus fulgidus, and the actinomycete Mycoplana ramosa (formerly M. bullata). The FB188 HDAH, however, is only moderately active in catalyzing the deacetylation of acetylpolyamines. In fact, FB188 HDAH exhibits significant activity in standard HDAC assays and is inhibited by known HDAC inhibitors such as trichostatin A and suberoylanilide hydroxamic acid (SAHA). Several lines of evidence indicate that the FB188 HDAH is very similar to class 1 and 2 HDACs and contains a Zn 2؉ ion in the active site which contributes significantly to catalytic activity. Initial biotechnological applications demonstrated the extensive substrate spectrum and broad optimum pH range to be excellent criteria for using the new HDAH from Bordetella/ Alcaligenes strain FB188 as a biocatalyst in technical biotransformations, e.g., within the scope of human immunodeficiency virus reverse transcriptase inhibitor synthesis.Class 1 and 2 histone deacetylases (HDACs) (19), acetoin utilization proteins, and acetylpolyamine amidohydrolases (APAH) are members of the same superfamily (17) and may very well descend from a common ancestor (14). Sequence comparison reveals that all three classes of proteins share a number of common motifs. Additionally, they show significant functional similarities such as recognition of acetylated aminoalkyl groups and the removal of the acetyl moiety by cleaving an amide bond. Interestingly, the biological role of APAH is still a matter of speculation, although these enzymes are thought to be involved in degradative pathways of polyamines (20), particularly in the deacetylation of acetylpolyamines (23). However, experimental evidence to support this idea is still rather scarce. To date, only the APAH from Mycoplana ramosa has been described in detail. While some APAH were reported to cleave only acetylputrescine in vitro, M. ramosa APAH was described as less specific in the same experiments (23). Besides having a role in the degradative pathways of polyamines, APAH could, i...

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“…To speed-up the isolation of respective candidates in a bioactivity-guided approach, a suitable test method is indispensable. In this study a fluorimetric in vitro enzymatic assay developed earlier (Wegener et al, 2003) was adapted and validated for the investigation of the influence of plant compounds on the activity of class I and class II HDACs. The principle of the assay is based on the following reactions ( Figure 1): an ε-acetylated lysine-based substrate C-terminally coupled with 4-methyl-coumarin-7-amide (MCA) is deacetylated by HDAC.…”

Section: Introductionmentioning

confidence: 99%

“…The deacetylated substance is recognized as substrate by an endopeptidase which releases 7-amino 4-methylcoumarin (AMC). Free AMC, in contrast to acetylated MCA, is highly fluorescent and can be monitored (Wegener et al, 2003;Mazitschek et al, 2008). To the best of our knowledge, this method has so far been employed to test pure compounds only and not yet been tested with complex mixtures nor been optimized and validated for the investigation of plant extracts.…”

Section: Introductionmentioning

confidence: 99%

Optimization and application of a fluorimetric assay for the identification of histone deacetylase inhibitors from plant origin

Krasteva¹,

Heiss²,

Krenn³

2011

Pharmaceutical Biology

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Improved fluorogenic histone deacetylase assay for high-throughput-screening applications

Cited by 107 publications

References 18 publications

Pimelic Diphenylamide 106 Is a Slow, Tight-binding Inhibitor of Class I Histone Deacetylases

Pimelic Diphenylamide 106 Is a Slow, Tight-binding Inhibitor of Class I Histone Deacetylases

A New Amidohydrolase from Bordetella or Alcaligenes Strain FB188 with Similarities to Histone Deacetylases

Optimization and application of a fluorimetric assay for the identification of histone deacetylase inhibitors from plant origin

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