Bioluminescence-based assays for quantifying endogenous protein interactions in live cells

Area of Science:

• Cellular Biology
• Biochemistry
• Drug Discovery

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions and are implicated in various diseases.
• Current methods often use artificial overexpression, not reflecting native cellular conditions.
• Targeting PPIs is a key strategy for developing new therapeutics.

Purpose of the Study:

• To evaluate NanoLuc Binary Technology (NanoBiT) and NanoLuc Bioluminescence Resonance Energy Transfer (NanoBRET) for quantifying endogenous PPIs in live cells.
• To establish a robust workflow for generating reporter cell lines for studying PPI dynamics.
• To improve the relevance and predictive power of live-cell assays for drug discovery.

Main Methods:

• CRISPR-mediated genome engineering to tag endogenous proteins (EGFR/GRB2, KRas/CRAF) with NanoBiT/NanoBRET tags.
• Utilizing DLD-1 and HCT 116 cell lines for assays in monolayer cultures and 3D spheroid models.
• Employing endpoint, kinetic measurements, and luminescence imaging to quantify PPIs.

Main Results:

• Successfully generated cell lines with endogenously tagged proteins for NanoBiT/NanoBRET assays.
• Demonstrated the quantification of PPIs in both standard cell cultures and more complex 3D spheroid models.
• Validated the workflow for studying PPIs in isogenic cell lines relevant to cancer.

Conclusions:

• Developed a versatile and modular workflow for creating endogenously regulated PPI reporter cell lines.
• This method offers a more biologically relevant platform for live-cell PPI analysis.
• The approach provides a valuable tool for understanding signaling pathways and characterizing PPI-targeting drugs.