Functional mapping of the 14-3-3 hub protein as a guide to design 14-3-3 molecular glues

Molecular glues represent an evolution in drug discovery, however, targeted stabilization of protein complexes remains challenging, owing to a paucity of drug design rules. The functional mapping of hotspots has been critical to protein-protein interaction (PPI) inhibitor research, however, the orthogonal approach to stabilize PPIs has not exploited this information. Utilizing the hub protein 14-3-3 as a case study we demonstrate that functional mapping of hotspots provides a triage map for 14-3-3 molecular glue development. Truncation and mutation studies allowed deconvoluting the energetic contributions of sidechain and backbone interactions of a 14-3-3-binding non-natural peptide. Three central 14-3-3 hotspots were identified and their thermodynamic characteristics profiled. In addition to the phospho-binding pocket; (i) Asn226, (ii) Lys122 and (iii) the hydrophobic patch formed by Leu218, Ile219 and Leu222 were critical for protein complex formation. Exploiting this hotspot information allowed a peptide-based molecular glue that elicits high cooperativity (α = 36) and selectively stabilizes the 14-3-3/ChREBP PPI to be uniquely developed.