Identification of Selective Class II Histone Deacetylase Inhibitors Using a Novel Dual-Parameter Binding Assay Based on Fluorescence Anisotropy and Lifetime

Abstract

IntroductIon The family of histone deacetylases (hdacs) has emerged as an important novel class of targets for pharmaceutical intervention on different types of cancer.1 there are 11 zinc-dependent hdacs subdivided in classes i, iia, iib, and iV and 7 hdacs that depend on nad + , the so-called sirtuins. hdacs and their antagonistic opponents, histone acetyl transferases (hats), regulate a variety of genes and cellular functions through balancing the acetylation status of histones and many nonhistone proteins.2 recently, two inhibitors of hdacs (hdacis), vorinostat (ZolinZa; merck, Whitehouse station, nJ) and romidepsin (istodax; celgene, summit, nJ), have been approved for the treatment of cutaneous t-cell lymphoma by the Us food and drug administration. the therapeutic interest in hdacis is also reflected by more than 87 open clinical trials.3 although cancer will remain the most important therapeutic area of hdacis for years, there is an exceeding body of evidence that hdacs play a pivotal role in immunological networks, inflammation, 4 neurological disorders, 5 memory, 6 and muscle/ heart diseases. 7 in addition, hdac orthologues in parasitic protozoa may serve as malaria and leishmania targets.8 the bacterial histone deacetylase homologue hdah from Bordetella has been discovered by hildmann et al.9,10 and extensively investigated by them and our group.11,12 hdah is structurally and functionally closely related to hdac6. the enzyme shares 35% identity with the second deacetylase domain of hdac6, and the crystal structure of hdah contains a deacetylase domain very similar to other hdac isoforms with resolved structures. 13 moreover, hdah and hdac6 display similar substrate and inhibitor selectivities.10 consequently, hdah has been used as a proven model system for hdac6 and hdacs in general. 12,14 the outstanding potential of hdacis with defined isoform selectivity profiles as drugs against a plurality of diseases vindicates an increased effort in the development of high-throughput capable assays and screening campaigns. histone deacetylases (hdacs) are important epigenetic factors regulating a variety of vital cellular functions such as cell cycle progression, differentiation, cell migration, and apoptosis. consequently, hdacs have emerged as promising targets for cancer therapy. the drugability of hdacs has been shown by the discovery of several structural classes of inhibitors (hdacis), particularly by the recent approval of two hdacis, vorinostat (ZolinZa) and romidepsin (istodax), for the treatment of cutaneous t-cell lymphoma by the Us food and drug administration. the outstanding potential of hdacis, with a defined isoform selectivity profile as drugs against a plurality of diseases, vindicates increased effort in developing highthroughput capable assays for screening campaigns. in this study, a dual-competition assay exploiting changes in fluorescence anisotropy and lifetime was used to screen the loPac (sigma-aldrich, st louis, mo) library against the bacterial histone deacetylase homologue hdah from...

Class IIa HDACs repressive activities on MEF2‐depedent transcription are associated with poor prognosis of ER + breast tumors

“…For this study, we used N ‐lauroyl‐( l )‐phenylalanine, a recently identified class IIa specific histone deacetylase inhibitor (HDI; ref. 30). The deacetylase activity associated with immunoprecipitated HDAC4, but not with immunoprecipitated HDAC3, was inhibited by the HDI (Supplemental Fig.…”

“…Interestingly, the prosurvival role of class IIa HDACs could have a therapeutic perspective. A class IIa HDAC inhibitor (30) elicited an antiproliferative response and apoptosis only in MCF7 and ZR‐75‐1 ER + cells. This response was coupled to the up‐regulation of several MEF2 target genes, with Nur77 the more reactive.…”

Fluorescence anisotropy (polarization): from drug screening to precision medicine

“…19 Introduced in 1970s and with the first dedicated instruments in 1980s, FA has become a standard way of quantitatively measuring biomarkers (first clinical utility of FA), elucidating the mechanism of drug action, and screening potential drug candidates. 20, 21 In the last decade, a variety of FA designs and formats have been utilized to study enzymes, 22–24 as well as protein-protein 25 and protein-DNA interactions, with the goal of developing drugs. 2627 A number of recent reviews cover different aspects of these applications in great detail.…”

Selective class IIa HDAC inhibitors: myth or reality

“…-N-lauroyl-(l)-phenylalanine is a class IIa HDACi active in the lM range (Fig. 3) [81]. It was identified during a screening of a commercial available library of compounds.…”

“…It was identified during a screening of a commercial available library of compounds. The specificity was scored not merely by classical measurements of HDAC activity but also through a fluorescence assay, which exploits the competition between a fluorescent substrate and the putative inhibitor for each purified HDAC [81]. This molecule shows anti-tumoral properties against ER?…”