Abstract
Protein-protein interactions (PPIs) are integral to cellular signaling networks and are frequently disrupted in cancer, neurodegeneration, inflammation, and metabolic disorders. Targeting dysregulated PPIs presents a promising strategy for the development of therapeutic compounds. However, traditional drug discovery platforms often rely on plasmid-driven overexpression models that fail to replicate the complexity and dynamics of PPI in native cellular contexts. This study aims to evaluate the use of NanoLuc Binary Technology (NanoBiT) and NanoLuc Bioluminescence Resonance Energy Transfer (NanoBRET) for quantifying interactions of endogenously regulated proteins in live cells. To achieve this, CRISPR-mediated genome engineering was used to integrate NanoBiT and NanoBRET fusion tags at the loci for EGFR/GRB2 and KRas/CRAF in DLD-1 and HCT 116 cell lines. Assays using the engineered cell lines were then conducted in monolayer cultures using endpoint and kinetic measurements, as well as luminescence imaging. The approach was further expanded to investigate PPI in cancer-associated isogenic cell lines and 3D spheroid models that better preserve additional aspects of cellular organization. Collectively, these findings establish a robust and modular workflow for generating endogenously regulated PPI reporter cell lines to improve the relevance and predictive power of live-cell assays. By capturing interaction dynamics in a more representative background, this approach offers a potentially valuable tool for elucidating signaling mechanisms and characterizing therapeutic compounds targeting PPIs.