TY - JOUR
T1 - A fluorescence polarization based screening assay for identification of small molecule inhibitors of the PICK1 PDZ domain
AU - Thorsen, Thor S
AU - Madsen, Kenneth L
AU - Dyhring, Tino
AU - Bach, Anders
AU - Peters, Dan
AU - Strømgaard, Kristian
AU - Rønn, Lars Christian B
AU - Gether, Ulrik
PY - 2011/8/1
Y1 - 2011/8/1
N2 - 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.
AB - 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.
M3 - Journal article
C2 - 21534917
SN - 1386-2073
VL - 14
SP - 590
EP - 600
JO - Combinatorial Chemistry and High Throughput Screening
JF - Combinatorial Chemistry and High Throughput Screening
IS - 7
ER -
