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

Abstract

Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Recent findings suggest that HDACs could be key targets for chemotherapeutic intervention in malignant diseases. A convenient and sensitive fluorogenic assay for HDAC activity would therefore expedite studies of HDAC in transcriptional regulation and in vitro screening for drug discovery. In this study, novel fluorogenic substrates of HDACs were synthesized with an epsilon-acetylated lysyl moiety and an adjacent MCA moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules became substrates for trypsin, which released highly fluorescent AMC molecules in a subsequent step of the assay. The fluorescence increased in direct proportion to the amount of deacetylated substrate molecules, i.e., HDAC activity. The nonisotopic, homogeneous assay is well suited for high-throughput HDAC inhibitor screening.

Substrate-specific Activation of Sirtuins by Resveratrol

“…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).…”

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

“…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.…”

“…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.…”

Discovery of N-hydroxy-4-(3-phenylpropanamido)benzamide derivative 5j, a novel histone deacetylase inhibitor, as a potential therapeutic agent for human breast cancer

“…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).…”