A Fluorescence‐Based High‐Throughput Screening Method for EGFR Inhibitors Using an EGFR‐Targeted Environment‐Sensitive Fluorescent Probe

Fluorescence technology offers a highly sensitive, rapid, and efficient approach for drug screening at the cellular level, enabling the direct quantification of drug‐target binding interactions through precise measurement of fluorescence intensity changes. In our previous research, a small‐molecule fluorescent probe targeting epidermal growth factor receptor (EGFR) was successfully developed, featuring strong targeting specificity, low background signal, and an “off–on” response feature. Based on the EGFR‐overexpressing A431 cell line, this study established a cell‐based screening system for EGFR inhibitors and successfully identified flavokawain A (FKA), a novel potential EGFR inhibitor. FKA notably reduces the expression levels of p ‐EGFR and its downstream molecule p ‐Erk in the EGFR signaling pathway. It demonstrates a potent inhibitory effect on the proliferation of A431 cells, effectively suppressing clone formation and cellular migration, and exhibits a strong apoptosis‐inducing effect against A431 cells. This fluorescence‐based high‐throughput screening method has demonstrated its effectiveness in identifying novel EGFR inhibitors from a wide range of natural materials. It holds significant application potential and plays a crucial role in expediting the discovery of EGFR inhibitors from medicinal sources.