Development of a NanoBRET assay for evaluation of 14-3-3σ molecular glues

We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC50 values between 100 nM and 1 μM in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors. ![Figure][1] ### Competing Interest Statement The authors declare the following competing financial interest: M.R.A. is a co-founder and director of Ambagon Therapeutics. * PPI : Protein-protein interaction PROTAC : Proteolysis targeting chimera MS : Mass spectrometry FA : Fluorescence anisotropy pS/T : Phosphoserine/phosphothreonine NanoBRET : NanoLuciferase bioluminescence resonance energy transfer FRET : Fluorescence resonance energy transfer NanoBiT : NanoLuc binary technology 4PL : 4 parameter logistic NES : Nuclear export signal mBU : milliBRET units XMU : XMU-MP-1 FC-A : Fusicoccin-A ΔDBD-ERα : ERα without DNA binding domain [1]: pending:yes