Vickery, H. R. | Search bioRxiv

bioRxiv2024

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 14-3-3{sigma} client proteins CRAF, TAZ, and estrogen receptor (ER), which represent three specific binding modes. We have measured stabilization of 14-3-3{sigma}/client complexes by molecular glues with EC50 values between 100 nM and 1 M in cells, which align with the EC50 values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3{sigma} allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3{sigma} 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=55 SRC="FIGDIR/small/573792v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1f1531org.highwire.dtl.DTLVardef@a862cdorg.highwire.dtl.DTLVardef@ee8f6dorg.highwire.dtl.DTLVardef@a780e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

A Systematic Approach to the Discovery of Protein-Protein Inter-action Stabilizers

Protein-protein interactions (PPIs) are responsible for the proper function of biological processes and, when dysregulated, commonly lead to disease. PPI stabilization has only recently been systematically explored for drug discovery despite being a powerful approach to selectively target intrinsically disordered proteins and hub proteins, like 14-3-3, with multiple interaction partners. Disulfide tethering is a site-directed fragment-based drug discovery (FBDD) methodology for screening small molecules in a quantitative, high-throughput manner. We explore the scope of the disulfide tethering technology for the discovery of selective fragments as starting points for the development of potent small molecule PPI stabilizers and molecular glues using the hub protein 14-3-3{sigma}. The complexes with 5 biologically and structurally diverse phospho-peptides, derived from the 14-3-3 client proteins ER, FOXO1, C-RAF, USP8, and SOS1, were screened for hit identification. Stabilizing fragments could be found for 4/5 client complexes with a diversified hit-rate and stabilizing efficacy for the different 14-3-3/client phospho-peptides. Extensive structural elucidation revealed the ability and adaptivity of the peptide to make productive interactions with the tethered fragments as key criterion for cooperative complex formation. We validated eight fragment stabilizers, six of which showed selectivity for one phospho-peptide client, and structurally characterized two nonselective hits and four fragments that selectively stabilized C-RAF or FOXO1. The most efficacious of these fragments increased 14-3-3{sigma}/C-RAF phospho-peptide affinity by 430-fold. Disulfide tethering to the wildtype C38 in 14-3-3{sigma} provided diverse structures for future optimization of 14-3-3/client stabilizers and highlighted a systematic method to discover molecular glues.

biochemistry↗