Rational design of a sensitivity-enhanced tracer for discovering efficient APC-Asef inhibitors

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 mu M) and wide signal dynamic range (span = 251 mp). By using tracer 7 in fluorescencepolarization assays for APC-Asef inhibitor screening, we discover a best-in-class inhibitor, MAI-516, with an IC50 of 0.041 +/- 0.004 mu M 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.

Automated summary

This study identifies a best-in-class inhibitor for the protein-protein interaction crucial to colorectal cancer metastasis and develops a sensitivity-enhanced tracer for fluorescence polarization assays to accurately detect high-activity inhibitors.