Dynamic Imaging of Small Molecule Induced Protein–Protein Interactions in Living Cells with a Fluorophore Phase Transition Based Approach

Protein–protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule induced protein–protein association and dissociation in living cells. Here we have applied a fluorophore phase transition based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drug (IMiD) induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiD analogue (e.g., CC-885) induced PPI between cereblon and GSPT1. Furthermore, SPPIER can visualize bifunctional molecules (e.g. PROTAC)-induced PPI between an E3 ubiquitin ligase and a target protein. Lastly, SPPIER can be modified to image small molecule induced protein–protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intense brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.