title: Dual Screening of BPTF and Brd4 Using Protein-Observed Fluorine NMR Uncovers New Bromodomain Probe Molecules.
abstract: Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a (19)F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of (19)F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 μM for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed (19)F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant St. SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Urick
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant St. SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Laura M L
authors.initials: LM
authors.lastname: Hawk
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant St. SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Melissa K
authors.initials: MK
authors.lastname: Cassel
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant St. SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Neeraj K
authors.initials: NK
authors.lastname: Mishra
authors.affiliation: Department of Chemistry, University of Massachusetts Boston , 100 Morrissey Boulevard, Boston, Massachusetts 02125, United States.
authors.forename: Shuai
authors.initials: S
authors.lastname: Liu
authors.affiliation: Lillehei Heart Institute , Department of Medicine, 2231 6th Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Neeta
authors.initials: N
authors.lastname: Adhikari
authors.affiliation: Department of Chemistry, University of Massachusetts Boston , 100 Morrissey Boulevard, Boston, Massachusetts 02125, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Cold Spring Harbor Laboratory , 1 Bungtown Road, Cold Spring Harbor, New York 11724, United States.
authors.forename: Camila O
authors.initials: CO
authors.lastname: dos Santos
authors.affiliation: Lillehei Heart Institute , Department of Medicine, 2231 6th Street SE, Minneapolis, Minnesota 55455, United States.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Hall
authors.affiliation: Department of Chemistry, University of Minnesota , 207 Pleasant St. SE, Minneapolis, Minnesota 55455, United States.
authors.forename: William C K
authors.initials: WC
authors.lastname: Pomerantz
mesh_terms: D034961:Antigens, Nuclear / Q000737:chemistry*; D001667:Binding, Competitive; D018797:Cell Cycle Proteins; D002460:Cell Line; D016503:Drug Delivery Systems; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005461:Fluorine / Q000737:chemistry; D006801:Humans; D020128:Inhibitory Concentration 50; D008279:Magnetic Resonance Imaging; D015394:Molecular Structure; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013696:Temperature; D014157:Transcription Factors / Q000737:chemistry*; D000092468:Bromodomain Containing Proteins
chemical_list: D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D005461:Fluorine; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D014157:Transcription Factors; C441289:BRD4 protein, human; D000092468:Bromodomain Containing Proteins; C406238:fetal Alzheimer antigen