title: Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair.
abstract: A new class of biosensors, fluorogen activating proteins (FAPs), has been successfully used to track receptor trafficking in live cells. Unlike the traditional fluorescent proteins (FPs), FAPs do not fluoresce unless bound to their specific small-molecule fluorogens, and thus FAP-based assays are highly sensitive. Application of the FAP-based assay for protein trafficking in high-throughput flow cytometry resulted in the discovery of a new class of compounds that interferes with the binding between fluorogens and FAP, thus blocking the fluorescence signal. These compounds are high-affinity, nonfluorescent analogs of fluorogens with little or no toxicity to the tested cells and no apparent interference with the normal function of FAP-tagged receptors. The most potent compound among these, N,4-dimethyl-N-(2-oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)benzenesulfonamide (ML342), has been investigated in detail. X-ray crystallographic analysis revealed that ML342 competes with the fluorogen, sulfonated thiazole orange coupled to diethylene glycol diamine (TO1-2p), for the same binding site on a FAP, AM2.2. Kinetic analysis shows that the FAP-fluorogen interaction is more complex than a homogeneous one-site binding process, with multiple conformational states of the fluorogen and/or the FAP, and possible dimerization of the FAP moiety involved in the process.
authors.affiliation: Department of Pathology, University of New Mexico, Albuquerque, NM, USA Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Vanderbilt Specialized Chemistry Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Shaun R
authors.initials: SR
authors.lastname: Stauffer
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Robyn L
authors.initials: RL
authors.lastname: Stanfield
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: Phillip H
authors.initials: PH
authors.lastname: Tapia
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: Oleg
authors.initials: O
authors.lastname: Ursu
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Gregory W
authors.initials: GW
authors.lastname: Fisher
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Szent-Gyorgyi
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: Annette
authors.initials: A
authors.lastname: Evangelisti
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Waller
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: J Jacob
authors.initials: JJ
authors.lastname: Strouse
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: Mark B
authors.initials: MB
authors.lastname: Carter
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: Cristian
authors.initials: C
authors.lastname: Bologa
authors.affiliation: Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
authors.forename: Kristine
authors.initials: K
authors.lastname: Gouveia
authors.affiliation: Vanderbilt Specialized Chemistry Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Mike
authors.initials: M
authors.lastname: Poslusney
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Alan S
authors.initials: AS
authors.lastname: Waggoner
authors.affiliation: Vanderbilt Specialized Chemistry Center, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: Craig W
authors.initials: CW
authors.lastname: Lindsley
authors.affiliation: Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA, USA.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Jarvik
authors.affiliation: Department of Pathology, University of New Mexico, Albuquerque, NM, USA Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA Lsklar@salud.unm.edu.
authors.forename: Larry A
authors.initials: LA
authors.lastname: Sklar
mesh_terms: D001681:Biological Assay / Q000379:methods; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011485:Protein Binding / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000494:pharmacology*; D020298:U937 Cells
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D054852:Small Molecule Libraries
keywords: biosensor; crystal structure; drug discovery; fluorogen activating protein; protein trafficking