Structure-Based Design of Potent and Highly Selective NUAK1 Inhibitors by Exploiting a Unique Glutamate Switch for the Prevention of Tumor Growth, Migration, and Invasion

NUAK1, an AMPK-related kinase overexpressed in cancers, plays a crucial role in tumor metastasis and cell survival, making it an attractive cancer therapeutic target. Herein, we report potent, selective NUAK1 inhibitors via structure-guided repurposing of a covalent JAK3 inhibitor. By capitalizing on the critical structural differenceCys909 in JAK3 versus Glu139 in NUAK1we substituted the electrophilic warhead with glutamate-favoring moieties, a modification that confers selective NUAK1 targeting. Supporting this design rationale, cocrystal structures verify the specific engagement of these moieties with the Glu139 residue of NUAK1. Among the synthesized analogs, candidate compound 10i exhibits subnanomolar NUAK1 inhibition (IC50 = 0.49 nM) and kinome-wide selectivity. Besides, 10i suppresses proliferation, migration, and invasion of triple-negative breast cancer cells, reverses EMT markers, and shows robust antitumor efficacy in mouse xenografts. This study provides a promising lead and validates Glu139 as an anchor for selective NUAK1 targeting.