title: A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E-receptor interactions.
abstract: The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE-receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring the IgE-receptor interaction. The TR-FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR-FRET assay can also be used to follow the kinetics of IgE-Fc-FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR-FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
authors.forename: Beomkyu
authors.initials: B
authors.lastname: Kim
authors.forename: Svetlana S
authors.initials: SS
authors.lastname: Tarchevskaya
authors.forename: Alexander
authors.initials: A
authors.lastname: Eggel
authors.forename: Monique
authors.initials: M
authors.lastname: Vogel
authors.forename: Theodore S
authors.initials: TS
authors.lastname: Jardetzky
mesh_terms: D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D017455:Receptors, IgE / Q000032:analysis / Q000737:chemistry
chemical_list: D000888:Antibodies, Anti-Idiotypic; D017455:Receptors, IgE; C026408:anti-IgE antibodies; D007073:Immunoglobulin E