Epigenetic Targets in Drug Discovery: Cell‐Based Assays for HDAC Inhibitor Hit Validation

Abstract

IntroductionInhibitors of histone deacetylases (HDACs) are emerging as novel potential therapeutic agents for patients with cancers and/or neurological diseases. A number of HDAC inhibitors (HDACIs) have already entered clinical trials; and recently the hydroxamic acid derivative Vorinostat, suberoylanilide hydroxamic acid (SAHA), received FDA approval for the treatment of patients with cutaneous T-cell lymphoma. Furthermore, improved understanding of the roles of various HDAC isoforms in cell processes and in diseases contribute to the strategies for developing second generation inhibitors offering enhanced target specificity and improved toxicity profiles. Reports presenting new classes of HDAC inhibitors with isoform specificity have been developed utilizing in vitro enzymatic screening assays. However, in vitro enzyme activities have limitations and full assessment of isoform specificities of HDACIs requires the application of assays based on functions of multiprotein complexes that may include HDACs and/or other cofactors. In this setting, cell-based assays are needed to permit selective profiling and screening of HDACIs for relative HDAC isoform inhibitory activities for rational drug discovery. The approaches that have been used to assess molecular and biological actions of HDACIs in cells and tissues are reviewed in this chapter.Histone deacetylases are metalloenzymes that catalyze the deacetylation of the e-lysine of acetylated substrates, including histones and nonhistone proteins. Eighteen human HDACs have been identified to date and these are divided into four classes, based on structure, sequence homology, and domain organization. Class I consists of HDACs 1, 2, 3 and 8. These HDACs are homologous to yeast RPD3 [1,2]. Class I HDACs are expressed primarily in the nucleus and play a role in cell proliferation [3,4]. Class II HDACs include isoforms 4, 5, 6, 7, 9 and 10 and are further subdivided into IIa (HDACs 4,5,7,9) and IIb (HDACs 6, 10) [5]. These enzymes are characterized by a large NH 2 -terminal domain or a second catalytic site and play roles in cellular differentiation and development [6]. Class III HDACs were originally

Histone Deacetylase Inhibitor: Antineoplastic Agent and Radiation Modulator

“…Pan-HDACIs, such as SAHA and TSA, appear to target both histones H3/H4 and α-tubulin evidenced by their enhanced acetylation levels, in a dose-dependent manner. In contrast, other HDACIs, such as trapoxin B, sodium butyrate, and FK-228, do not exhibit these characteristics, suggesting their selectivity at the cellular level [17]. Others have reported that depsipeptide exerts modulation of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells [18].…”

Cellular analysis of the action of epigenetic drugs and probes

“…112 These methods are applied to cultured cells, possibly also to animal samples as well as patient derived material. Some directly measure target engagement while others monitor downstream events.…”