AlphaScreen™

c-Met is a receptor tyrosine kinase (RtK) with a critical role in many fundamental cellular processes, including cell proliferation and differentiation. Deregulated c-Met signaling has been implicated in both the initiation and progression of human cancers and therefore represents an attractive target for anticancer therapy. Monitoring the phosphorylation status of relevant tyrosine residues provides an important method of assessing c-Met kinase activity. this report describes a novel assay to monitor c-Met phosphorylation in cells using Amplified Luminescent proximity homogeneous Assay (AlphaScreen) technology. Using AlphaScreen, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreen assay were compared to data obtained from a high-content imaging (hcI) method developed in parallel to monitor c-Met phosphorylation at the single cell level. the AlphaScreen assay was miniaturized to a 384-well format with acceptable signal-to-background ratio (S/B) and Z′ statistics and was employed to measure c-Met kinase activity in situ after treatment with potent c-Met-specific kinase inhibitors. the authors discuss the utility of quantifying endogenous cellular c-Met phosphorylation in lead optimization and how the modular design of the AlphaScreen assay allows its adaptation to measure cellular activity of other kinases. (Journal of Biomolecular Screening 2009:404-411)

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Development of a Cell-Based Assay for Measurement of c-Met Phosphorylation Using AlphaScreenTM Technology and High-Content Imaging Analysis

Smotrov

Mathur

Kariv

et al. 2009

SLAS Discovery

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Volume‐Sensitive Organic Osmolyte/Anion Channels in Cancer: Novel Approaches to Studying Channel Modulation Employing Proteomics Technologies

Davies

Belsey

Kozlowski

2004

Annals of the New York Academy of Sciences

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Key elements of tumor development include proliferation, migration, invasiveness, and angiogenesis. Activation of the volume-sensitive organic osmolyte/anion channel (VSOAC) has been suggested to play a role in all of these processes. VSOACs may therefore represent an important therapeutic target in the etiology of cancer. However, pharmacological inhibitors of VSOAC are nonselective and of low potency, highlighting the importance of identifying novel regulators of the channel. The use of electrophysiological methods coupled with techniques such as pull-down assays, yeast 2-hybrid, and functional protein arrays have already proved valuable in studying protein-protein interactions in a variety of systems. Some of these methods have been used to identify small molecules that modulate the function of other types of ion channels. Given that several proteins have already been identified as putative modulators of VSOACs, proteomics technologies may prove useful in elucidating the molecular identity of VSOACs and helpful in identifying novel modulators of channel function. In this paper, we review the involvement of VSOACs in tumor development processes and its regulation by pharmacological agents and cellular proteins. Proteomic approaches to study protein-protein interactions and how such approaches may be used to study VSOACs are also discussed. We speculate on how modulation of protein-protein interactions may result in the identification of a novel class of compounds for modulating VSOACs.

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AlphaScreen™

Cited by 2 publications

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Development of a Cell-Based Assay for Measurement of c-Met Phosphorylation Using AlphaScreenTM Technology and High-Content Imaging Analysis

Development of a Cell-Based Assay for Measurement of c-Met Phosphorylation Using AlphaScreenTM Technology and High-Content Imaging Analysis

Volume‐Sensitive Organic Osmolyte/Anion Channels in Cancer: Novel Approaches to Studying Channel Modulation Employing Proteomics Technologies

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