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Published on: November 29, 2018
Development of a cell-based assay for measurement of c-Met phosphorylation using AlphaScreen technology and
Nadya Smotrov1, Anjili Mathur, Ilona Kariv
1Automated Lead Optimization, Merck Research Laboratories, Boston, Massachusetts 02115, USA.
Abstract:
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.
Insights
A new Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) effectively monitors c-Met phosphorylation, a key cancer target. This assay aids in developing new anticancer therapies by measuring kinase activity in cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- c-Met is a receptor tyrosine kinase (RTK) crucial for cell functions.
- Aberrant c-Met signaling drives cancer initiation and progression, making it a therapeutic target.
- Assessing c-Met kinase activity via phosphorylation is vital for drug development.
Purpose of the Study:
- To develop and validate a novel assay for monitoring c-Met phosphorylation.
- To compare the novel assay with existing high-content imaging (HCI) methods.
- To evaluate the assay's utility in drug discovery for cancer therapy.
Main Methods:
- Utilized Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen) technology.
- Detected global and site-specific c-Met phosphorylation in cancer cell lines.
- Miniaturized the assay to a 384-well format and assessed its performance (S/B, Z').
Main Results:
- Successfully detected c-Met phosphorylation using the AlphaScreen assay.
- Demonstrated comparable results to high-content imaging (HCI) methods.
- Measured c-Met kinase activity in situ following treatment with c-Met inhibitors.
Conclusions:
- The AlphaScreen assay is a robust method for quantifying endogenous c-Met phosphorylation.
- This assay facilitates lead optimization in anticancer drug discovery.
- The modular design allows adaptation for studying other kinases.
