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 μ M to ∼ 193 μ 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 μ 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.
Keywords: PDZ domains, protein-protein interactions, fluorescence polarization, small molecule inhibitors
Combinatorial Chemistry & High Throughput Screening
Title: A Fluorescence Polarization Based Screening Assay for Identification of Small Molecule Inhibitors of the PICK1 PDZ Domain
Volume: 14 Issue: 7
Author(s): Thor S. Thorsen, Kenneth L. Madsen, Tino Dyhring, Anders Bach, Dan Peters, Kristian Stromgaard and Ulrik Gether
Affiliation:
Keywords: PDZ domains, protein-protein interactions, fluorescence polarization, small molecule inhibitors
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 μ M to ∼ 193 μ 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 μ 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.
Export Options
About this article
Cite this article as:
S. Thorsen Thor, L. Madsen Kenneth, Dyhring Tino, Bach Anders, Peters Dan, Stromgaard Kristian and Gether Ulrik, A Fluorescence Polarization Based Screening Assay for Identification of Small Molecule Inhibitors of the PICK1 PDZ Domain, Combinatorial Chemistry & High Throughput Screening 2011; 14 (7) . https://dx.doi.org/10.2174/138620711796367201
DOI https://dx.doi.org/10.2174/138620711796367201 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
Call for Papers in Thematic Issues
Artificial Intelligence Methods for Biomedical, Biochemical and Bioinformatics Problems
Recently, a large number of technologies based on artificial intelligence have been developed and applied to solve a diverse range of problems in the areas of biomedical, biochemical and bioinformatics problems. By utilizing powerful computing resources and massive amounts of data, methods based on artificial intelligence can significantly improve the ...read more
Emerging trends in diseases mechanisms, noble drug targets and therapeutic strategies: focus on immunological and inflammatory disorders
Recently infectious and inflammatory diseases have been a key concern worldwide due to tremendous morbidity and mortality world Wide. Recent, nCOVID-9 pandemic is a good example for the emerging infectious disease outbreak. The world is facing many emerging and re-emerging diseases out breaks at present however, there is huge lack ...read more
Exploring Spectral Graph Theory in Combinatorial Chemistry
Scope of the Thematic Issue: Combinatorial chemistry involves the synthesis and analysis of a large number of diverse compounds simultaneously. Traditional methods rely on brute force experimentation, which can be time-consuming and resource-intensive. Spectral Graph Theory, a branch of mathematics dealing with the properties of graphs in relation to the ...read more
Integrating Network Pharmacology and Traditional Medicine: A New Perspective in Drug Mechanism Research
Network pharmacology is a network construction and network topology analysis strategy that combines pharmacology and pharmacodynamics. In recent years, network pharmacology has emerged as a powerful tool that can be integrated with pharmacology. Natural products commonly function in multicomponent, multitarget, and multipathway systems. Some examples encompass Ayurveda, traditional Chinese medicines ...read more
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
Microbes, Immunity and Multiple Sclerosis: An Update
Current Immunology Reviews (Discontinued) The Large Conductance Calcium- and Voltage-activated Potassium Channel (BK) and Epilepsy
CNS & Neurological Disorders - Drug Targets The Interactions Between Alzheimer’s Disease and Major Depression: Role of Ca<sup>2+</sup> Channel Blockers and Ca<sup>2+</sup>/cAMP Signalling
Current Drug Research Reviews Neuroprotective Role of Natural Polyphenols
Current Topics in Medicinal Chemistry A Perspective in Renewable Energy Production from Biomass Pyrolysis - Challenges and Prospects
Current Organic Chemistry Vitamin D Combined with Resveratrol Prevents Cognitive Decline in SAMP8 Mice
Current Alzheimer Research In-Silico Identification of Drug Lead Molecule Against Pesticide Exposed-neurodevelopmental Disorders Through Network-Based Computational Model Approach
Current Bioinformatics Contextualizing Genetics for Regional Heart Failure Care
Current Cardiology Reviews Obstructive Sleep Apnea and Autoimmune Rheumatic Disease: Is there Any Link?
Inflammation & Allergy - Drug Targets (Discontinued) Evidences for the Anti-panic Actions of Cannabidiol
Current Neuropharmacology Glucocorticoids, the Etiology of Obesity and the Metabolic Syndrome
Current Alzheimer Research Synthesis and Biological Activities of Naturally Functionalized Polyamines: An Overview
Current Medicinal Chemistry Manipulation of Microglial Activation as a Therapeutic Strategy in Alzheimers Disease
Current Medicinal Chemistry Subject Index Volume 2
Current Pediatric Reviews Auditory Verbal Learning Test is Superior to Rey-Osterrieth Complex Figure Memory for Predicting Mild Cognitive Impairment to Alzheimer’s Disease
Current Alzheimer Research The Effect of Pharmacotherapy for Attention Deficit Hyperactivity Disorder on Risk of Seizures in Pediatric Patients as Assessed in an Insurance Claims Database
Current Drug Safety Hydrolytic Activity of Amyloid-beta and its Inhibition with Short Peptides
Current Medicinal Chemistry - Central Nervous System Agents The Impact of Metabolic Disease Associated with Metabolic Syndrome on Human Pregnancy
Current Pharmaceutical Biotechnology Does the MK2-dependent Production of TNFα Regulate mGluR-dependent Synaptic Plasticity?
Current Neuropharmacology Non-transfusion Dependent Thalassemias: A Developing Country Perspective
Current Pediatric Reviews