Abstract

The adenomatous polyposis coli (APC)–Rho guanine nucleotide exchange factor 4 (Asef) protein–protein interaction (PPI) is essential for colorectal cancer metastasis, making it a promising drug target. Herein, we obtain a sensitivity-enhanced tracer (tracer 7) with a high binding affinity ( K d  = 0.078 μM) and wide signal dynamic range (span = 251 mp). By using tracer 7 in fluorescence-polarization assays for APC–Asef inhibitor screening, we discover a best-in-class inhibitor, MAI-516, with an IC 50 of 0.041 ± 0.004 μM and a conjugated transcriptional transactivating sequence for generating cell-permeable MAIT-516. MAIT-516 inhibits CRC cell migration by specifically hindering the APC–Asef PPI. Furthermore, MAIT-516 exhibits no cytotoxic effects on normal intestinal epithelial cell and colorectal cancer cell growth. Overall, we develop a sensitivity-enhanced tracer for fluorescence polarization assays, which is used for the precise quantification of high-activity APC–Asef inhibitors, thereby providing insight into PPI drug development.