Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3s/ERa Protein-Protein Interaction from Nonselective Fragments

The stabilization of protein-protein interactions (PPIs) has emerged as a promising strategy in chemical biology and drug discovery. The identification of suitable starting points for stabilizing native PPIs and their subsequent elaboration into selective and potent molecular glues lacks structure-guided optimization strategies. We have previously identified a disulfide fragment that stabilized the hub protein 14-3-3s bound to several of its clients, including ERa and C-RAF. Here, we show the structure-based optimization of the nonselective fragment toward selective and highly potent small-molecule stabilizers of the 14-3-3s/ERa complex. The more elaborated molecular glues, for example, show no stabilization of 14-3-3s/C-RAF up to 150 mu M compound. Orthogonal biophysical assays, including mass spectrometry and fluorescence anisotropy, were used to establish structure-activity relationships. The binding modes of 37 compounds were elucidated with X-ray crystallography, which further assisted the concomitant structure-guided optimization. By targeting specific amino acids in the 14-3-3s/ERa interface and locking the conformation with a spirocycle, the optimized covalent stabilizer 181 achieved potency, cooperativity, and selectivity similar to the natural product Fusicoccin-A. This case study showcases the value of addressing the structure, kinetics, and cooperativity for molecular glue development.

Automated summary

The stabilization of protein-protein interactions (PPIs) is a promising strategy in chemical biology and drug discovery. In this study, selective and potent small-molecule stabilizers for the 14-3-3s/ERa complex were optimized through structure-based optimization. By targeting specific amino acids and locking the conformation, the optimized covalent stabilizer achieved potency, cooperativity, and selectivity similar to a natural product. The study highlights the value of addressing structure, kinetics, and cooperativity in molecular glue development.