Discovery of 14‐3‐3 PPI Stabilizers by Extension of an Amidine‐Substituted Thiophene Fragment

Protein‐protein interaction (PPI) modulation is a promising approach in drug discovery with the potential to expand the ‘druggable’ proteome and develop new therapeutic strategies. While there have been significant advancements in methodologies for developing PPI inhibitors, there is a relative scarcity of literature describing the ‘bottom‐up’ development of PPI stabilizers (Molecular Glues). The hub protein 14‐3‐3 and its interactome provide an excellent platform for exploring conceptual approaches to PPI modulation, including evolution of chemical matter for Molecular Glues. In this study, we employed a fragment extension strategy to discover stabilizers for the complex of 14‐3‐3 protein and an Estrogen Receptor alpha‐derived peptide (ERα). A focused library of analogues derived from an amidine‐substituted thiophene fragment enhanced the affinity of the 14‐3‐3/ERα complex up to 6.2‐fold. Structure‐activity relationship (SAR) analysis underscored the importance of the newly added, aromatic side chain with a certain degree of rigidity. X‐ray structural analysis revealed a unique intermolecular π–π stacking binding mode of the most active analogues, resulting in the simultaneous binding of two molecules to the PPI binding pocket. Notably, analogue 11 displayed selective stabilization of the 14‐3‐3/ERα complex. 14‐3‐3 PPI stabilizers were discovered through a fragment extension approach. The stabilizer exhibited moderate stabilization effect and selectivity towards a 14‐3‐3/ERα peptide complex.