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.