title: A dual-mode fluorescence strategy for screening HAT modulators.
abstract: Histone acetyltransferases (HATs) are an important class of epigenetic enzymes involved in chromatin restructuring and transcriptional regulation. We describe in this paper a novel approach for the identification and characterization of HAT inhibitors using both fluorescence resonance energy transfer (FRET) and fluorescence polarization. Expressed protein ligation (EPL) was used to label HATs PCAF and p300 with Dabcyl (Dab) as FRET acceptors. Methoxycoumarin (Mca) is conjugated to HAT substrate analogues to function as fluorescent donors, namely, H3CoA20Mca for interacting with PCAF and LysCoAMca for p300. When a ligand-protein interaction occurs, the fluorescent intensity of the donor fluorophore decreases due to FRET quenching by the Dab acceptor. Meanwhile, the formation of ligand-protein complexes causes reduction of the molecular mobility of the donor fluorophore, resulting in increased fluorescence anisotropy. Thus, dual modes of fluorescence measurement, FRET and anisotropy, are integrated in the same assay system. In particular, we demonstrated that both FRET and anisotropy measurements can be used to effectively detect and characterize HAT inhibitors. The developed strategy should be useful in the search of new anticancer drugs that target the substrate interfaces of the HAT targets, as well as find values in mechanistic study of HATs.
authors.affiliation: Department of Chemistry, Georgia State University, PO Box 4098, Atlanta, Georgia 30302, USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Xie
authors.forename: Emilia N
authors.initials: EN
authors.lastname: Elangwe
authors.forename: Sabrina
authors.initials: S
authors.lastname: Asher
authors.forename: Yujun George
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D003374:Coumarins / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005453:Fluorescence; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D005786:Gene Expression Regulation; D051548:Histone Acetyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D050880:p300-CBP Transcription Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D000099224:p300-CBP-Associated Factor
chemical_list: D003374:Coumarins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D051548:Histone Acetyltransferases; D009842:Oligopeptides; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor