title: A fluorescence polarization based screening assay for identification of small molecule inhibitors of the PICK1 PDZ domain.
abstract: PDZ (PSD-95/Discs-large/ZO-1 homology) domains represent putative targets in several diseases including cancer, stroke, addiction and neuropathic pain. Here we describe the application of a simple and fast screening assay based on fluorescence polarization (FP) to identify inhibitors of the PDZ domain in PICK1 (protein interacting with C kinase 1). We screened 43,380 compounds for their ability to inhibit binding of an Oregon Green labeled C-terminal dopamine transporter peptide (OrG-DAT C13) to purified PICK1 in solution. The assay was highly reliable with excellent screening assay parameters (Z'≈0.7 and Z≈0.6). Out of ~200 compounds that reduced FP to less than 80% of the control wells, six compounds were further characterized. The apparent affinities of the compounds were determined in FP competition binding experiments and ranged from ~5.0 µM to ~193 µM. Binding to the PICK1 PDZ domain was confirmed for five of the compounds (CSC-03, CSC-04, CSC-43, FSC-231 and FSC-240) in a non-fluorescence based assay by their ability to inhibit pull-down of PICK1 by a C-terminal DAT GST fusion protein. CSC-03 displayed the highest apparent affinity (5.0 µM) in the FP assay, and was according to fluorescence resonance energy transfer (FRET) experiments capable of inhibiting the interaction between the C-terminus of the GluR2 subunit of the AMPA-type glutamate receptor and PICK1 in live cells. Additional experiments suggested that CSC-03 most likely is an irreversible inhibitor but with specificity for PICK1 since it did not bind three different PDZ domains of PSD-95. Summarized, our data suggest that FP based screening assays might be a widely applicable tool in the search for small molecule inhibitors of PDZ domain interactions.
authors.affiliation: Molecular Neuropharmacology Group, Department of Neuroscience and Pharmacology, University of Copenhagen, Blegdamsvej, Denmark.
authors.forename: Thor S
authors.initials: TS
authors.lastname: Thorsen
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Madsen
authors.forename: Tino
authors.initials: T
authors.lastname: Dyhring
authors.forename: Anders
authors.initials: A
authors.lastname: Bach
authors.forename: Dan
authors.initials: D
authors.lastname: Peters
authors.forename: Kristian
authors.initials: K
authors.lastname: Strømgaard
authors.forename: Lars Christian B
authors.initials: LC
authors.lastname: Rønn
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays; D006801:Humans; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects*; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D009687:Nuclear Proteins; C498297:PICk1 protein, human; D047428:Protein Kinase Inhibitors