Protein-protein interactions (PPI) drive biology. Small-molecule modulators of protein complexes can therefore provide important biological insight and lead to new therapies. There have been big strides in development of inhibitors of PPI; the next frontier is the development of selective and drug-like PPI stabilizers. Molecular glue degraders, natural products, and seredipitous discoveries of PPI stabilizers have demonstrated the value of stabilization for chemical biology and drug discovery. However, systematic and prospective approaches have been lacking. We have used a site-directed fragment discovery approach called disulfide trapping to discover and optimize selective stabilizers for the adaptor protein 14-3-3 binding to diverse client protein complexes. Binding of 14-3-3 regulates the function of hundreds of phosphorylated clients, including difficult-to-drug targets such as transcription factors and intrinsically disordered proteins. Disulfide-trapping based screens took advantage of cysteine residues on 14-3-3 near the client-binding sites and cysteine residues on the clients themselves. Tethering to 14-3-3 identified selective stabilizers for single 14-3-3/client complexes and 'priveleged pharmacophores' that bind to multiple complexes. Starting from these priveleged scaffolds, we used structure-guided design to develop selective inhibitors for multiple complexes. Optimized disulfides were then converted to irreversible electrophilic probes with nanomolar potency and 40-fold stabilization of 14-3-3/client interactions. Furthermore, cysteine mutants allowed us to probe the features of molecular recognition and dissect binding affinity and cooperativity. This systematic and generalizable technology opens the door to discovery of diverse PPI stabilizers. Taken together, protein-protein interaction inhibitors, stabilizers, and allosteric modulators provide a toolbox of probes to study protein-interaction networks.

Systematic and selective stabilization of protein complexes

ACS Fall 2021

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