Protein-protein interactions (PPIs) are key regulators of various cellular processes. Modulating PPIs with small molecules has gained increasing attention in drug discovery, particularly targeting the 14-3-3 protein family, which interacts with several hundred client proteins and plays a central role in cellular networks. However, targeting a specific PPI of the hub protein 14-3-3, with its plethora of potential client proteins, poses a significant selectivity challenge. This not only involves the selectivity of 14-3-3 PPIs with other client proteins, but also the selective stabilization of a specific 14-3-3 binding site within a protein partner featuring several binding sites. The interaction of 14-3-3 with Tau, characterized by different phospho-site driven binding modes, forms a valuable, disease-relevant, 14-3-3 multivalent model PPI to explore this selectivity issue. This work presents the identification and early-stage optimization of small molecule fragment-like stabilizers for a specific binding site of the 14-3-3/Tau PPI. Using different biophysical assays, the stabilizing potency of the imine-bond forming molecules was mapped and X-ray crystallography studies provided structural data on the binding mode of the ternary complexes. Exploiting the unique topologies and functionalities of the different binding sites enabled the engineering of selectivity for this initial molecular glue matter for the pS214 binding site, over a second 14-3-3 binding site in Tau (pS324). These reversible covalent tool compounds will allow for the further exploration of the role of 14-3-3 in Tau aggregation.
