A Fluorogenic Histone Deacetylase Assay Well Suited for High-Throughput Activity Screening

Wegener, Dennis; Wirsching, Frank; Riester, Daniel; Schwienhorst, Andreas

doi:10.1016/s1074-5521(02)00305-8

Cited by 266 publications

(249 citation statements)

References 43 publications

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“…The Fluor de Lys assay is a relatively new biochemical method for measuring deacetylation of a chemically modified acetylated peptide substrate coupled to aminomethylcoumarin. Upon deacetylation, the aminomethylcoumarin group is proteolytically cleaved resulting in fluorescence (45).…”

Section: Resultsmentioning

confidence: 99%

Substrate-specific Activation of Sirtuins by Resveratrol

Kaeberlein

McDonagh

Heltweg

et al. 2005

Journal of Biological Chemistry

708

555

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Resveratrol, a small molecule found in red wine, is reported to slow aging in simple eukaryotes and has been suggested as a potential calorie restriction mimetic. Resveratrol has also been reported to act as a sirtuin activator, and this property has been proposed to account for its anti-aging effects. We show here that resveratrol is a substrate-specific activator of yeast Sir2 and human SirT1. In particular, we observed that, in vitro, resveratrol enhances binding and deacetylation of peptide substrates that contain Fluor de Lys, a non-physiological fluorescent moiety, but has no effect on binding and deacetylation of acetylated peptides lacking the fluorophore. Consistent with these biochemical data we found that in three different yeast strain backgrounds, resveratrol has no detectable effect on Sir2 activity in vivo, as measured by rDNA recombination, transcriptional silencing near telomeres, and life span. In light of these findings, the mechanism accounting for putative longevity effects of resveratrol should be reexamined.

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Section: Resultsmentioning

confidence: 99%

Substrate-specific Activation of Sirtuins by Resveratrol

Kaeberlein

McDonagh

Heltweg

et al. 2005

Journal of Biological Chemistry

708

555

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“…Typical fluorogenic substrates for class I HDACs consist of an eacetylated lysine residue (alone or in a short peptide) coupled through a carboxy-terminal amide to a fluorescent moiety such as 7-amino-4-methylcoumarin (AMC), for example, cell-permeable Boc-Lys(Ac)-AMC 29,30 . These synthetic substrates undergo a two-step conversion.…”

Section: Increasedmentioning

confidence: 99%

“…Second, proteases cleave the Lys-AMC amide bond to release the AMC, which fluoresces. In tissue culture-based assays, the second step is performed after cell lysis by addition of excessive amount of trypsin, which only recognizes deacetylated lysine 29,30 . As the deacetylated Boc-Lys-AMC was the identical substrate used in our CTSL assay, we postulated that the substrate Boc-Lys(Ac)-AMC could also be processed in live cells through the two-step conversion by endogenous HDAC and CTSL activities.…”

Section: Increasedmentioning

confidence: 99%

Selective cancer targeting with prodrugs activated by histone deacetylases and a tumour-associated protease

et al. 2013

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Eradication of cancer cells while minimizing damage to healthy cells is a primary goal of cancer therapy. Highly selective drugs are urgently needed. Here we demonstrate a new prodrug strategy for selective cancer therapy that utilizes increased histone deacetylase (HDAC) and tumour-associated protease activities produced in malignant cancer cells. By coupling an acetylated lysine group to puromycin, a masked cytotoxic agent is created, which is serially activated by HDAC and an endogenous protease cathepsin L (CTSL) that remove the acetyl group first and then the unacetylated lysine group liberating puromycin. The agent selectively kills human cancer cell lines with high HDAC and CTSL activities. In vivo studies confirm tumour growth inhibition in prodrug-treated mice bearing human cancer xenografts. This cancer-selective cleavage of the masking group is a promising strategy for the next generation of anticancer drug development that could be applied to many other cytotoxic agents.

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“…The fluorescence analysis method was used for HDAC enzyme activity assay in vivo which was well suited for high-throughput screening of HDAC inhibitors. 31 HDAC releases the acetate moiety from 3-acetylated lysine residues of the substrate peptides, then the deacetylated peptides containing now unprotected lysine residues are recognized by trypsin and subsequently cleaved to release 7-amino-4-methylcoumarin (AMC). The fluorescence measurement is done at excitation of 390 nm, emission of 460 nm by fluorometry (Varioskan, Thermo).…”

Section: Methodsmentioning

confidence: 99%