A Cell-Free, Nonisotopic, High-Throughput Assay for Inhibitors of Type-I Interleukin-1 Receptor

1996 ◽  
Vol 237 (1) ◽  
pp. 70-75 ◽  
Author(s):  
Edoardo Sarubbi ◽  
Stephen D. Yanofsky ◽  
Ronald W. Barrett ◽  
Maurizio Denaro
Keyword(s):  
High Throughput ◽  
Interleukin 1 ◽  
Type I ◽  
A Cell ◽  
High Throughput Assay

scholarly journals A Cell-Based High-Throughput Assay for Gap Junction Communication Suitable for Assessing Connexin 43–Ezrin Interaction Disruptors Using IncuCyte ZOOM

2016 ◽  
Vol 22 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Aleksandra R. Dukic ◽  
David W. McClymont ◽  
Kjetil Taskén
Keyword(s):  
Gap Junction ◽  
High Throughput ◽  
Connexin 43 ◽  
General Mechanism ◽  
Protein Protein Interaction ◽  
Gap Junction Communication ◽  
Anchoring Protein ◽  
A Cell ◽  
Rat Liver Epithelial Cells ◽  
High Throughput Assay

Connexin 43 (Cx43), the predominant gap junction (GJ) protein, directly interacts with the A-kinase-anchoring protein (AKAP) Ezrin in human cytotrophoblasts and a rat liver epithelial cells (IAR20). The Cx43-Ezrin–protein kinase (PKA) complex facilitates Cx43 phosphorylation by PKA, which triggers GJ opening in cytotrophoblasts and IAR20 cells and may be a general mechanism regulating GJ intercellular communication (GJIC). Considering the importance of Cx43 GJs in health and disease, they are considered potential pharmaceutical targets. The Cx43-Ezrin interaction is a protein-protein interaction that opens possibilities for targeting with peptides and small molecules. For this reason, we developed a high-throughput cell-based assay in which GJIC can be assessed and new compounds characterized. We used two pools of IAR20 cells, calcein loaded and unloaded, that were mixed and allowed to attach. Next, GJIC was monitored over time using automated imaging via the IncuCyte imager. The assay was validated using known GJ inhibitors and anchoring peptide disruptors, and we further tested new peptides that interfered with the Cx43-Ezrin binding region and reduced GJIC. Although an AlphaScreen assay can be used to screen for Cx43-Ezrin interaction inhibitors, the cell-based assay described is an ideal secondary screen for promising small-molecule hits to help identify the most potent compounds.

scholarly journals A Cell-Based High-Throughput Assay for the Screening of Small-Molecule Inhibitors of p53–MDM2 Interaction

2011 ◽  
Vol 16 (4) ◽  
pp. 450-456 ◽  
Author(s):  
Jing Li ◽  
Shuyong Zhang ◽  
Linghuan Gao ◽  
Ying Chen ◽  
Xin Xie
Keyword(s):  
High Throughput ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Direct Inhibition ◽  
A Cell ◽  
Luciferase Reporter System ◽  
Novel Strategy ◽  
High Throughput Assay ◽  
Two Hybrid ◽  
Rule Out

The p53 tumor suppressor is a potent transcription factor that regulates cell growth inhibition and apoptosis. The oncoprotein MDM2 suppresses p53 activity by direct inhibition of its transcriptional activity and enhances the degradation of p53 via the ubiquitin–proteosome pathway. Overexpression of MDM2, found in many human tumors, impairs p53-mediated cell death effectively. Inhibition of the p53–MDM2 interaction can stabilize p53 and may offer a novel strategy for cancer therapy. To search for new inhibitors of the p53–MDM2 interaction, the authors developed a cell-based high-throughput assay system based on mammalian two-hybrid technology. They also used a dual-luciferase reporter system to rule out false- positive hits due to the cytotoxic effect of compounds. Using this assay, they screened a library consisting of 3840 compounds and identified one compound that activates p53 pathway and induces growth arrest in tumor cells.

scholarly journals Transcriptional-Based Screens for Pathway-Specific, High-Throughput Target Discovery in Endothelial Cells

2004 ◽  
Vol 9 (8) ◽  
pp. 704-711 ◽  
Author(s):  
Robert L. Yauch ◽  
Edward E. Kadel ◽  
Cory Nicholas ◽  
Selwyna Tetangco ◽  
Douglas O. Clary
Keyword(s):  
Endothelial Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Target Identification ◽  
Gene Products ◽  
Target Discovery ◽  
Drug Target Identification ◽  
A Cell ◽  
Cytokine Stimulation ◽  
High Throughput Assay

With the sequence of the human genome at hand, target discovery strategies are needed that can rapidly identify novel gene products involved in human disease pathways. In this article, the authors describe a cell-based, high-throughput assay that can identify gene products capable of modulating the vascular endothelial growth factor (VEGF) and tumor necrosis factor • (TNFa) signaling pathways in human endothelial cells. The assay uses real-time PCRtechnology tomeasure downstreamreporter mRNA transcripts induced upon cytokine stimulation in a 96-well plate format and has been adapted for use with recombinant adenoviruses. The authors specifically demonstratemodulation of cytokine-driven reporter transcripts using drug inhibitors and through adenoviral-mediated expression of known signaling intermediates of the respective pathways. In addition, they have used an arrayed library of 350 recombinant adenoviruses to screen for novel modulators of the VEGF and TNF• pathways. The high-throughput screening capacity and sensitivity of this system make it a useful tool for new drug target identification.

A cell‐based high‐throughput assay for screening inhibitors of human papillomavirus‐16 long control region activity

2005 ◽  
Vol 20 (1) ◽  
pp. 148-150 ◽  
Author(s):  
David Lembo ◽  
Manuela Donalisio ◽  
Marco De Andrea ◽  
Maura Cornaglia ◽  
Sara Scutera ◽  
...  
Keyword(s):  
Human Papillomavirus ◽  
Control Region ◽  
High Throughput ◽  
Long Control Region ◽  
Human Papillomavirus 16 ◽  
A Cell ◽  
High Throughput Assay

Development of a High-Throughput Assay to Identify Inhibitors of ENPP1

2021 ◽  
pp. 247255522098232
Author(s):  
Meera Kumar ◽  
Robert G. Lowery
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Type I Interferon ◽  
Direct Detection ◽  
Innate Immune ◽  
Assay Method ◽  
Type I ◽  
Immune Response To Cancer ◽  
Z Value ◽  
High Throughput Assay

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP2/GMP2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP2/GMP2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z′ value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2′3′cGAMP compared with 2′3′c-diGMP and showed no activity with 3′3′c-diAMP. The Transcreener AMP2/GMP2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

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