The systematic stabilization of protein-protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.

Protein-protein interactions (PPIs) are responsible for the proper function of biological processes and, when dysregulated, commonly lead to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for screening small molecules in a quantitative, high-throughput manner. We explore the scope of the disulfide tethering technology for the discovery of selective fragments as starting points for the development of potent small molecule PPI stabilizers and molecular glues using the hub protein 14-3-3s;. The complexes with 5 biologically and structurally diverse phospho-peptides, derived from the 14-3-3 client proteins ERa;, FOXO1, C-RAF, USP8, and SOS1, were screened for hit identification. Stabilizing fragments could be found for 4/5 client complexes with a diversified hit-rate and stabilizing efficacy for the different 14-3-3/client phospho-peptides. Extensive structural elucidation revealed the ability and adaptivity of the peptide to make productive interactions with the tethered fragments as key criterion for cooperative complex formation. We validated eight fragment stabilizers, six of which showed selectivity for one phospho-peptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious of these fragments increased 14-3-3s;/C-RAF phospho-peptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3s; provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover 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...

Dysregulation of protein–protein interactions (PPIs) commonly leads to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for identifying reversibly covalent small molecules. We explored the scope of disulfide tethering for the discovery of selective PPI stabilizers (molecular glues) using the hub protein 14-3-3σ. We screened complexes of 14-3-3 with 5 biologically and structurally diverse phosphopeptides derived from the 14-3-3 client proteins ERα, FOXO1, C-RAF, USP8, and SOS1. Stabilizing fragments were found for 4/5 client complexes. Structural elucidation of these complexes revealed the ability of some peptides to conformationally adapt to make productive interactions with the tethered fragments. We validated eight fragment stabilizers, six of which showed selectivity for one phosphopeptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious fragment increased 14-3-3σ/C-RAF phosphopeptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3σ provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.

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