title: Screening for inhibitors of low-affinity epigenetic peptide-protein interactions: an AlphaScreen-based assay for antagonists of methyl-lysine binding proteins.
abstract: The histone code comprises many posttranslational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. They focus on the development of an AlphaScreen-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen beads to clear the hurdle to assay development presented by the low micromolar binding constants of the histone binding proteins for their cognate peptides. The assay is applicable to other families of methyl-lysine binding proteins, and it has the potential to be used in screening efforts toward the discovery of novel small molecules with utility as research tools for cellular reprogramming and ultimately drug discovery.
authors.affiliation: Center for Integrative Chemical Biology and Drug Discovery, Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Tim J
authors.initials: TJ
authors.lastname: Wigle
authors.forename: J Martin
authors.initials: JM
authors.lastname: Herold
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Senisterra
authors.forename: Masoud
authors.initials: M
authors.lastname: Vedadi
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Kireev
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowsmith
authors.forename: Stephen V
authors.initials: SV
authors.lastname: Frye
authors.forename: William P
authors.initials: WP
authors.lastname: Janzen
mesh_terms: D001331:Automation; D001667:Binding, Competitive / Q000187:drug effects; D001681:Biological Assay / Q000379:methods*; D002021:Buffers; D044127:Epigenesis, Genetic / Q000187:drug effects; D006657:Histones / Q000378:metabolism; D008024:Ligands; D008239:Lysine / Q000378:metabolism*; D008745:Methylation / Q000187:drug effects; D008904:Miniaturization; D009363:Neoplasm Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D010865:Pilot Projects; D011136:Polysorbates; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000032:analysis* / Q000494:pharmacology*; D012965:Sodium Chloride; D013997:Time Factors; D017186:Titrimetry
chemical_list: D002021:Buffers; D006657:Histones; D008024:Ligands; D009363:Neoplasm Proteins; D010455:Peptides; D011136:Polysorbates; D011506:Proteins; D054852:Small Molecule Libraries; D012965:Sodium Chloride; D008239:Lysine