Discovery of Dimer-Dependent Aminoacrylamide Molecular Glues for 14–3–3 Protein–Protein Interactions

Molecular glues (MGs) offer a promising strategy for stabilizing protein-protein interactions (PPIs), particularly within the 14-3-3 protein family, which regulates diverse cellular processes and is implicated in many disease pathways. This study reports on the discovery of an aminoacrylamide MG (<b>7</b>) for 14-3-3 PPIs. Structure-activity relationship analysis using a fluorescence polarization (FP) assay revealed that both a basic amine and acrylamide moiety are essential for activity. However, further investigation using FP, mass spectrometry, and a thermal shift assay revealed that <b>7</b> has a cysteine-independent mode of action, distinguishing it from other covalent 14-3-3 MGs. Furthermore, its activity is reliant on 14-3-3 dimerization suggesting that it targets the 14-3-3 dimer interface. Aminoacrylamide <b>7</b> differentially affected interactions with ERα, LRRK2, and AHA2, suggesting that 14-3-3 dimerization plays an important role in 14-3-3 client recognition. These findings further validate the 14-3-3 dimer interface as a novel MG target and underscore the complexities of 14-3-3 molecular recognition and small-molecule modulation.