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

High Resolution Image Download MS PowerPoint Slide 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 ( 7 ) 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 7 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 7 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.