Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Traditional drug discovery for receptor tyrosine kinase (RTK) inhibitors often uses recombinant kinases, leading to limited cellular activity.
• Screening RTKs within their native cellular environment is a desirable alternative for more effective drug discovery.

Purpose of the Study:

• To develop and validate a novel biosensor for measuring endogenous epidermal growth factor receptor (EGFR) activity in live cells.
• To adapt this biosensor for high-throughput screening of chemical and RNA interference (RNAi) libraries for novel EGFR modulators.

Main Methods:

• Utilized a green fluorescent protein-labeled SRC homology 2 domain-based biosensor to report on EGFR activity in A549 cells.
• Measured EGFR function by quantifying the formation of green fluorescent granules upon receptor activation.
• Validated assay specificity through chemical inhibition of EGFR and RNAi-dependent knockdown.

Main Results:

• Demonstrated that EGFR activation in live cells correlates with the formation of green fluorescent granules.
• Confirmed assay specificity by observing the complete abrogation of granule formation upon EGFR inhibition or knockdown.
• Identified Gefitinib and PD 153035 as hits in a pilot screen, validating the biosensor's efficacy.

Conclusions:

• The developed biosensor provides a reliable method for assessing endogenous EGFR activity in a cellular context.
• This approach facilitates high-throughput screening for novel EGFR modulators, potentially improving drug discovery outcomes.
• The strategy offers a valuable tool for identifying new therapeutic agents targeting EGFR signaling pathways.