2. Academic Validation
  3. Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair

Discovery of Small-Molecule Nonfluorescent Inhibitors of Fluorogen-Fluorogen Activating Protein Binding Pair

  • J Biomol Screen. 2016 Jan;21(1):74-87. doi: 10.1177/1087057115609145.
Yang Wu 1,  Shaun R Stauffer 2,  Robyn L Stanfield 3,  Phillip H Tapia 4,  Oleg Ursu 4,  Gregory W Fisher 5,  Christopher Szent-Gyorgyi 5,  Annette Evangelisti 4,  Anna Waller 4,  J Jacob Strouse 4,  Mark B Carter 4,  Cristian Bologa 4,  Kristine Gouveia 4,  Mike Poslusney 2,  Alan S Waggoner 6,  Craig W Lindsley 2,  Jonathan W Jarvik 6,  Larry A Sklar 7
Affiliations

Affiliations

  • 1 Department of Pathology, University of New Mexico, Albuquerque, NM, USA Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
  • 2 Vanderbilt Specialized Chemistry Center, Vanderbilt University Medical Center, Nashville, TN, USA.
  • 3 Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.
  • 4 Center for Molecular Discovery, University of New Mexico, Albuquerque, NM, USA.
  • 5 Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA.
  • 6 Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, USA Department of Biological Science, Carnegie Mellon University, Pittsburgh, PA, USA.
  • 7 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.
PMID: 26442911 DOI: 10.1177/1087057115609145
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.

Keywords

biosensor; crystal structure; drug discovery; fluorogen activating protein; protein trafficking.

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