Single-molecule tracking-based drug screening

The single-molecule tracking of transmembrane receptors in living cells has provided significant insights into signaling mechanisms, such as mobility and clustering upon their activation/inactivation, making it a potential screening method for drug discovery. Here we show that single-molecule tracking-based screening can be used to explore compounds both detectable and undetectable by conventional methods for disease-related receptors. Using an automated system for a fast large-scale single-molecule analysis, we screened for epidermal growth factor receptor (EGFR) from 1,134 of FDA approved drugs. The 18 hit compounds included all EGFR-targeted tyrosine kinase inhibitors (TKIs) in the library that suppressed any phosphorylation-dependent mobility shift of EGFR, proving the concept of this approach. The remaining hit compounds are not reported as EGFR-targeted drugs and did not inhibit EGF-induced EGFR phosphorylation. These non-TKI compounds affected the mobility and/or clustering of EGFR without EGF and induced EGFR internalization, to impede EGFR-dependent cell growth. Thus, single-molecule tracking provides a new modality for discovering novel therapeutics on various receptor functions with previously untargeted mechanisms.