UCSF

Protein-protein interactions (PPIs) are vital for biology as they govern numerous essential biological processes, including signal transduction, enzymatic activity regulation, protein localization, and complex formation, collectively orchestrating cellular functions and maintaining homeostasis. The hub protein 14-3-3 interacts with hundreds of client proteins, including key signaling proteins CRAF and SOS1 and transcription factors Estrogen Receptor α (ERα), FOXO1, and TAZ. These 14-3-3/client interactions can be altered in disease, leading to undesirable activity and signaling. The Arkin Lab has pioneered a mass spectrometry-based site-directed disulfide tethering screen to identify PPI stabilizers and inhibitors. We have used this screen to discover stabilizers of various 14-3-3σ/client interactions including, CRAF, FOXO1, ERα, SOS1, and USP8. Disulfide fragment stabilizers lead to a 4 – 250-fold stabilization of 14-3-3σ/client complexes. To further characterize the effects of these stabilizers, we have developed Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) assays to measure 14-3-3σ/CRAF, TAZ, and ERα interactions, where we have measured stabilizer EC50 values between 100 nM – 1 μM. We have optimized 14-3-3σ/CRAF stabilizers for selectivity and potency and measured increased PPI formation in cells, decreased CRAF kinase activity, and decreased signaling through the mitogen-activated protein kinase (MAPK) pathway. Finally, we have expanded the disulfide tethering technology to a new class of hub proteins to discover stabilizers of additional PPIs to modulate their interaction in disease.