An Exploration of Chemical Properties Required for Cooperative Stabilization of the 14-3-3 Interaction with NF-κB—Utilizing a Reversible Covalent Tethering Approach

[Image: see text] Protein–protein modulation has emerged as a proven approach to drug discovery. While significant progress has been gained in developing protein–protein interaction (PPI) inhibitors, the orthogonal approach of PPI stabilization lacks established methodologies for drug design. Here, we report the systematic ″bottom-up″ development of a reversible covalent PPI stabilizer. An imine bond was employed to anchor the stabilizer at the interface of the 14-3-3/p65 complex, leading to a molecular glue that elicited an 81-fold increase in complex stabilization. Utilizing protein crystallography and biophysical assays, we deconvoluted how chemical properties of a stabilizer translate to structural changes in the ternary 14-3-3/p65/molecular glue complex. Furthermore, we explore how this leads to high cooperativity and increased stability of the complex.