Homogenous non-isotopic assays for histone deacetylase activity

Abstract

This article is intended to provide a short overview of recent developments in the area of in vitro assays for histone deacetylase (HDAC) activity as an extension of a recent patent evaluation [1]. HDAC inhibitors are a promising class of new anticancer agents [2]. Traditional assays for the in vitro measurement of zinc-dependent HDAC (class I and II) activity involve radioactively-labelled histones from chicken reticulocytes or cell culture, or chemically [³H]-acetylated oligopeptides as the substrates. Measurement requires the extraction of [³H]-acetic acid, which is not suitable for high sample throughput. A high-throughput version with a biotinylated and tritiated acetyl-histone peptide fragment is available that makes use of a scintillation proximity assay, but this still bears the disadvantage of radioactivity and laborious/expensive substrate preparation (reviewed in [2]). Fluorescent oligopeptides of the general structure Boc-X-X-(Ac)Lys-AMC have recently been introduced as HDAC substrates. A lysine endopeptidase is added in a second step and coumarin released only if the lysine has been deacetylated. This allows for a homogeneous assay [3] and a similar kit is marketed by CycLex. A small molecule substrate, Boc-(Ac)Lys-AMC, called MAL, has been introduced, which is deacetylated by HDACs. Originally detection relied on extraction, which is useful only for a small number of samples [4,5]. A homogeneous version using this substrate is now also available. The amine derivatisation reagent, naphthalenedicarbaldehyde (NDA), is added after incubation leading to intramolecular fluorescence quenching only in the metabolite [6]. The recent class of NAD⁺-dependent HDACs (sirtuins, class III) have a catalytic mechanism that is different from HDACs [7] but they are also able to convert certain oligopeptides (announced by CycLex) and the MAL analogue, Z(Ac)Lys-AMC (ZMAL) [8]. Another homogeneous kit is available from Biomol but the nature of the substrate is not revealed. It can be converted by HDACs from all three classes. These convenient new assays should drive further inhibitor development in this interesting class of transcriptional regulators.