title: Assays for the identification and evaluation of histone acetyltransferase inhibitors.
abstract: There is presently enormous interest in the function and regulatory roles of histone acetyltransferase enzymes. Along with deacetylases it is now evident that these enzymes play a key role in many cellular processes including chromatin remodeling and gene transcription. As such, effective small molecule enzyme inhibitors would be useful tools for molecular pharmacology and may also be suitable for further development into agents for the treatment of diseases such as cancer. A high-throughput assay based on the use of scintillating microplates (FlashPlates) suitable for screening libraries of compounds for inhibitors of acetylase activity is described here. Confirmation of activity of selected compounds is achieved with a conventional filter assay, the details of which are also described. In addition, an assay suitable for confirming that cellular protein acetylation has been altered by inhibition of acetylases or deacetylases is also presented. On the same plate, cells are grown, exposed to compound, fixed, and permeabilized, and protein acetylation is determined using standard ELISA methodology and a europium-labeled second antibody. This latter method provides a medium-throughput alternative to the use of immunoblotting for mechanistic studies.
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Sutton, SM2 5NG, UK. wynne@icr.ac.uk
authors.forename: G
authors.initials: G
authors.lastname: Wynne Aherne
authors.forename: Martin G
authors.initials: MG
authors.lastname: Rowlands
authors.forename: Lindsay
authors.initials: L
authors.lastname: Stimson
authors.forename: Paul
authors.initials: P
authors.lastname: Workman
mesh_terms: D000123:Acetyltransferases / Q000037:antagonists & inhibitors* / Q000145:classification / Q000378:metabolism; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D051548:Histone Acetyltransferases; D056572:Histone Deacetylase Inhibitors; D006657:Histones / Q000378:metabolism; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000145:classification / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D000123:Acetyltransferases; D051548:Histone Acetyltransferases