Abstract
We report an innovative design concept for small-molecule fluorescent sensors that are highly selective for protein targets. The sensor's enhanced selectivity stems from a "C-clamping" paradigm where three different binding domains cooperatively interact with a protein target. We established the sensor's performance both in vitro and in live cells using epidermal growth factor receptor (EGFR) tyrosine kinase as a proof-of-concept target. Detailed combined quantum mechanics/molecular mechanics computational studies strongly support both the experimentally established specificity and the "C-clamp" binding mechanism. To demonstrate the sensor's practical utility, we developed a single-compound assay for the quantitative profiling of EGFR kinase inhibitors upon fluorescent imaging in live cells. When bound to the protein target, the sensor is emissive in the near-infrared region and yields a turn-on quantitative fluorescent response toward small-molecule tyrosine kinase inhibitors. Furthermore, this sensing system produces differentiated responses to a series of clinically relevant EGFR kinase inhibitors in native environments. Overall, we envision that this work will empower the development of small-molecule systems for highly specific protein recognition and sensing, become an invaluable tool for assessing small-molecule/protein engagement, and be extended toward live-cell screening and fluorescent imaging of other important biomolecular targets.