NanoBRET They conducted NanoBRET experiments to test the interaction between two proteins, ERα and 14-3-3σ. They generated four constructs for each protein, resulting in a total of eight constructs. They tested these constructs in combination, using a 1:10 ratio of NanoLuc:HaloTag. They then tested selected combinations at ratios of 1:1, 1:10, 1:100, and 1:1000. They transfected the plasmids using jetOPTIMUS following Polyplus recommendations and plated the cells in white, flat-bottom TC-treated 384-well microplates after transfection in Gibco FluoroBrite DMEM with 4% fetal bovine serum. For ERα experiments, they used 4% charcoal dextran stripped FBS. They performed each experiment in triplicate and included no-acceptor controls and samples with the HaloTag NanoBRET 618 Ligand. They read the plates on an EnVision XCite 2105 plate reader at 618 nm (HaloTag) and 460 nm (NanoLuc) using specific filters and a mirror.

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.