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

Nadya Smotrov

Automated Lead Optimization, Merck Research Laboratories, Boston, Massachusetts

Anjili Mathur

Pharmacology, Merck Research Laboratories, Boston, Massachusetts

Ilona Kariv

Automated Lead Optimization, Merck Research Laboratories, Boston, Massachusetts

Christopher M. Moxham

Automated Lead Optimization, Merck Research Laboratories, Boston, Massachusetts

Nathan Bays

Automated Lead Optimization, Merck Research Laboratories, Boston, Massachusetts, nathan_bays{at}merck.com

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 (AlphaScreenTM) technology. Using AlphaScreenTM, the authors were able to detect both global and site-specific phosphorylation of c-Met in transformed cell lines. Data obtained from the AlphaScreenTM 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 AlphaScreenTM 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 AlphaScreenTM assay allows its adaptation to measure cellular activity of other kinases. (Journal of Biomolecular Screening 2009:404-411)

Key Words: AlphaScreenTM • kinase assay • c-Met • tyrosine phosphorylation

Journal of Biomolecular Screening, Vol. 14, No. 4, 404-411 (2009)
DOI: 10.1177/1087057109331803
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