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

2011 ◽  
Vol 14 (7) ◽  
pp. 590-600 ◽  
Author(s):  
Thor S. Thorsen ◽  
Kenneth L. Madsen ◽  
Tino Dyhring ◽  
Anders Bach ◽  
Dan Peters ◽  
...  
Keyword(s):  
Small Molecule ◽  
Fluorescence Polarization ◽  
Small Molecule Inhibitors ◽  
Pdz Domain ◽  
Screening Assay

scholarly journals Optimization of Fluorescently Labeled Nrf2 Peptide Probes and the Development of a Fluorescence Polarization Assay for the Discovery of Inhibitors of Keap1-Nrf2 Interaction

2011 ◽  
Vol 17 (4) ◽  
pp. 435-447 ◽  
Author(s):  
Daigo Inoyama ◽  
Yu Chen ◽  
Xinyi Huang ◽  
Lesa J. Beamer ◽  
Ah-Ng Tony Kong ◽  
...  
Keyword(s):  
Small Molecule ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Dynamic Range ◽  
Antioxidant Response ◽  
Binding Affinities ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Peptide Amide

Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z′ factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.

scholarly journals High-Throughput Screening Assay for Identification of Small Molecule Inhibitors of Aurora2/STK15 Kinase

2004 ◽  
Vol 9 (5) ◽  
pp. 391-397 ◽  
Author(s):  
Chongbo Sun ◽  
Yvette Newbatt ◽  
Leon Douglas ◽  
Paul Workman ◽  
Wynne Aherne ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Tumor Development ◽  
Screening Assay ◽  
Amino Terminal ◽  
Terminal Domain ◽  
High Throughput Screening Assay ◽  
Amino Terminal Domain

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, γ-33P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate® to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z′ factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.

scholarly journals A High-Throughput Cellular Screening Assay for Small-Molecule Inhibitors and Activators of Cytoplasmic Dynein-1-Based Cargo Transport

2020 ◽  
Vol 25 (9) ◽  
pp. 985-999
Author(s):  
John Vincent ◽  
Marian Preston ◽  
Elizabeth Mouchet ◽  
Nicolas Laugier ◽  
Adam Corrigan ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Cytoplasmic Dynein ◽  
Lead Discovery ◽  
Screening Assay ◽  
Age Related ◽  
The Uk

Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein’s cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.

Fluorescence Polarization for the Evaluation of Small-Molecule Inhibitors of PCAF BRD/Tat-AcK50 Association

ChemMedChem ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. 928-931 ◽  
Author(s):  
Ping Hu ◽  
Xinghui Wang ◽  
Baiqun Zhang ◽  
Shuai Zhang ◽  
Qiang Wang ◽  
...  
Keyword(s):  
Small Molecule ◽  
Fluorescence Polarization ◽  
Small Molecule Inhibitors

scholarly journals Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling

2021 ◽  
Author(s):  
Nestor Kamdem ◽  
Yvette Roske ◽  
Dmytro Kovalskyy ◽  
Maxim O. Platonov ◽  
Oleksii Balinskyi ◽  
...  
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Pdz Domain ◽  
Wnt Signalling ◽  
Dependent Manner ◽  
Pdz Domains ◽  
X Ray Crystallography ◽  
Structure Activity ◽  
Dose Dependent ◽  
Anthranilic Acid Derivatives

Abstract. Dishevelled (Dvl) proteins are important regulators of the Wnt signalling pathway, interacting through their PDZ domains with the Wnt receptor Frizzled. Blocking the Dvl PDZ/Frizzled interaction represents a potential approach for cancer treatment, which stimulated the identification of small molecule inhibitors, among them the anti-inflammatory drug Sulindac and Ky-02327. Aiming to develop tighter binding compounds without side effects, we investigated structure-activity relationships of sulfonamides. X-ray crystallography showed high complementarity of anthranilic acid derivatives in the GLGF loop cavity and space for ligand growth towards the PDZ surface. Our best binding compound inhibits Wnt signalling in a dose-dependent manner as demonstrated by TOP-GFP assays (IC50 ~50 µM), and Western blotting of β-catenin levels. Real-time PCR showed reduction in the expression of Wnt-specific genes. Our compound interacted with Dvl-1 PDZ (Kd = 2.4 µM) stronger than Ky-02327 and may be developed into a lead compound interfering with the Wnt pathway.

scholarly journals An RNAi-based high-throughput screening assay to identify small molecule inhibitors of hepatitis B virus replication

2017 ◽  
Vol 292 (30) ◽  
pp. 12577-12588 ◽  
Author(s):  
Subhanita Ghosh ◽  
Abhinav Kaushik ◽  
Sachin Khurana ◽  
Aditi Varshney ◽  
Avishek Kumar Singh ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Virus Replication ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
B Virus
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