scholarly journals CDK10 in Gastrointestinal Cancers: Dual Roles as a Tumor Suppressor and Oncogene

2021 ◽  
Vol 11 ◽  
Author(s):  
Zainab A. Bazzi ◽  
Isabella T. Tai
Keyword(s):  
Cell Proliferation ◽  
Tumor Suppressor ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Screening Assay ◽  
Cellular Processes ◽  
Molecule Inhibitor ◽  
High Throughput Screening Assay ◽  
Kinase Activity Assay

Cyclin-dependent kinase 10 (CDK10) is a CDC2-related serine/threonine kinase involved in cellular processes including cell proliferation, transcription regulation and cell cycle regulation. CDK10 has been identified as both a candidate tumor suppressor in hepatocellular carcinoma, biliary tract cancers and gastric cancer, and a candidate oncogene in colorectal cancer (CRC). CDK10 has been shown to be specifically involved in modulating cancer cell proliferation, motility and chemosensitivity. Specifically, in CRC, it may represent a viable biomarker and target for chemoresistance. The development of therapeutics targeting CDK10 has been hindered by lack a specific small molecule inhibitor for CDK10 kinase activity, due to a lack of a high throughput screening assay. Recently, a novel CDK10 kinase activity assay has been developed, which will aid in the development of small molecule inhibitors targeting CDK10 activity. Discovery of a small molecular inhibitor for CDK10 would facilitate further exploration of its biological functions and affirm its candidacy as a therapeutic target, specifically for CRC.

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

2011 ◽  
Vol 413 (2) ◽  
pp. 90-96 ◽  
Author(s):  
Kenneth Segers ◽  
Hugo Klaassen ◽  
Anastasios Economou ◽  
Patrick Chaltin ◽  
Jozef Anné
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

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 SCAN ™—A High-Throughput Assay for Detecting Small Molecule Binding to RNA Targets

2009 ◽  
Vol 14 (3) ◽  
pp. 219-229 ◽  
Author(s):  
Chris Baugh ◽  
Shaohui Wang ◽  
Bin Li ◽  
James R. Appleman ◽  
Peggy A. Thompson
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
Laboratory Equipment ◽  
Rna Targets ◽  
High Throughput Screening Assay ◽  
Small Molecule Ligands ◽  
Hcv Ires ◽  
High Throughput Assay

A novel optical-based high-throughput screening technology has been developed for increasing the rate of discovering chemical leads against RNA targets. SCAN™ ( Screen for Compounds with Affinity for Nucleic Acids) is an affinity-based assay that identifies small molecules that bind and recognize structured RNA elements. This technology provides the opportunity to conduct high-throughput screening of a new class of targets—RNA. SCAN™ offers many attractive features including a simple homogeneous format, low screening costs, and the ability to use common laboratory equipment. A SCAN™ assay was developed for the HCV IRES Loop IIId RNA domain. A high-throughput screen of our entire compound library resulted in the identification of small molecule ligands that bind to Loop IIId. The Z′ values were greater than 0.8, showing this to be a robust high-throughput screening assay. A correlation between SCAN™ EC50 and KD values is reported suggesting the ability to use the assay for compound optimization. ( Journal of Biomolecular Screening 2009:219-229)

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

scholarly journals A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of gsp

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e90766 ◽  
Author(s):  
Nisan Bhattacharyya ◽  
Xin Hu ◽  
Catherine Z. Chen ◽  
Lesley A. Mathews Griner ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Assay System ◽  
Screening Assay ◽  
High Throughput Screening Assay

A serendipitously identified novel small molecule procoagulant compound giving rise to a high-throughput screening assay based on human plasma

2013 ◽  
Vol 132 (2) ◽  
pp. 248-255 ◽  
Author(s):  
David Gustafsson ◽  
Sofi Nielsen ◽  
Jane McPheat ◽  
Fredrik Wågberg ◽  
Karin Kaspersson ◽  
...  
Keyword(s):  
Human Plasma ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput 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 Monitoring Protein Kinase Activity in Cell Lysates Using a High-Density Peptide Microarray

2009 ◽  
Vol 14 (3) ◽  
pp. 256-262 ◽  
Author(s):  
Xiaoming Han ◽  
Go Yamanouchi ◽  
Takeshi Mori ◽  
Jeong-Hun Kang ◽  
Takuro Niidome ◽  
...  
Keyword(s):  
Protein Kinase ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Disease Diagnosis ◽  
High Density ◽  
Protein Kinase Activity ◽  
Peptide Array ◽  
Screening Assay ◽  
Chip Technology ◽  
High Throughput Screening Assay

Monitoring and targeting protein kinases is widely accepted as a promising approach for disease diagnosis and drug discovery. For this purpose, the authors have developed an original type of peptide array as a high-throughput screening assay for quantitatively evaluating kinase activity. A volume of 2 nL of peptide solution was spotted onto a formyl group-modified glass slide by using an arrayer, which was designed for use with protein chip technology. The phosphorylation was recognized by fluorescence-label antibody and detected with an automatic microarray scanner widely used in DNA chip technology. The system needs low sample volume, provides a high-density peptide array, and supplies high reproducibility. It provided enough sensitivity for inhibitor screening, even though a relatively low concentration of purified kinase was employed. The assay also proved useful for the detection of intracellular kinase activity as well as for the measurement of the fluctuations of intracellular protein kinase activity with drug stimulation. Thus, this peptide array would be applicable for kinase-targeted diagnosis, cell-based drug screening, and signal pathway investigation. ( Journal of Biomolecular Screening 2009:256-262)

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C

2014 ◽  
Vol 20 (5) ◽  
pp. 655-662 ◽  
Author(s):  
Brittany D. Wright ◽  
Catherine Simpson ◽  
Michael Stashko ◽  
Dmitri Kireev ◽  
Emily A. Hull-Ryde ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mobility Shift ◽  
Screening Assay ◽  
Lipid Kinase ◽  
Cellular Processes ◽  
High Throughput Screening Assay ◽  
Lipid Kinases ◽  
Mobility Shift Assay ◽  
Phosphatidylinositol 4

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z’ values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.

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