A Fluorescence Polarization Based Screening Assay for Identification of Small Molecule Inhibitors of the PICK1 PDZ Domain

作者：Thorsen Thor S; Madsen Kenneth L; Dyhring Tino; Bach Anders; Peters Dan; Stromgaard Kristian; Ronn Lars Christian B; Gether Ulrik^*

来源：Combinatorial Chemistry & High Throughput Screening, 2011, 14(7): 590-600.

DOI：10.2174/138620711796367201

• PDZ domains
• protein-protein interactions
• fluorescence polarization
• small molecule inhibitors

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'approximate to 0.7 and Z approximate to 0.6). Out of similar to 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 similar to 5.0 mu M to similar to 193 mu M. 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 mu M) 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.

• 出版日期2011-8