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Updated: May 2, 2026
Using High Content Imaging to Quantify Target Engagement in Adherent Cells
Published on: November 29, 2018
High-throughput high-content imaging assays for identification and characterization of selective AXL pathway
Huaping Tang1, Jing Yang, Ding Ren Shen
11 Department of Leads Discovery and Optimization, Bristol-Myers Squibb , Princeton, New Jersey.
Abstract:
Receptor tyrosine kinases (RTKs) regulate a wide range of important biological activities, including cell proliferation, differentiation, migration, and apoptosis. Abnormalities in RTKs are involved in numerous diseases, including cancer and other proliferative disorders. AXL belongs to the TAM (Tyso3, AXL, and Mer) family of RTKs. The AXL signaling pathway represents an attractive target for the treatment of diseases, such as cancer. Using phospho-AKT as readout, a high-throughput 384-well cell-based assay was established in the NCI-H1299 human non-small cell lung carcinoma cell line to evaluate compound potency in inhibiting AXL pathway activation. In addition, a counter screen assay was established in the same cellular background to differentiate AXL kinase inhibitors from AXL receptor antagonists, which block the interaction of AXL and its natural ligand GAS6. These cell-based functional assays are useful tools in the identification and optimization of small molecules and biological reagents for potential therapeutics for the treatment of GAS6/AXL-related diseases.
Insights
This study developed cell-based assays to find drugs targeting the AXL signaling pathway, crucial in cancer. These assays help identify inhibitors of AXL kinase and receptor activity for treating related diseases.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Receptor tyrosine kinases (RTKs) control vital cellular functions and are implicated in diseases like cancer.
- AXL, a member of the TAM family of RTKs, plays a role in various cancers, making its signaling pathway a therapeutic target.
Purpose of the Study:
- To establish high-throughput cell-based assays for evaluating inhibitors of the AXL signaling pathway.
- To develop methods for distinguishing between AXL kinase inhibitors and AXL receptor antagonists.
Main Methods:
- A high-throughput 384-well cell-based assay using NCI-H1299 cells and phospho-AKT as a readout was established to measure AXL pathway inhibition.
- A counter-screen assay was developed to differentiate kinase inhibitors from receptor antagonists that block GAS6/AXL interaction.
Main Results:
- The developed assays successfully evaluated compound potency against AXL pathway activation.
- The assays can differentiate between AXL kinase inhibitors and receptor antagonists.
Conclusions:
- The cell-based functional assays are effective tools for identifying and optimizing small molecules and biological reagents.
- These assays support the development of therapeutics for GAS6/AXL-related diseases, particularly cancers.
