Fluorescence Anisotropy-Based Tethering for Discovery of Protein–Protein Interaction Stabilizers

• DOI: 10.1021/acschembio.0c00646
• Publication Date: 2020-11-16T15:30:53Z
• Authors: Eline Sijbesma; Bente A. Somsen; Galen P. Miley; Iris A. Leijten-v...; Luc Brunsveld; Michelle R. Arkin; Christian Ottmann
• Keywords: Tethering; Bimolecular fluorescence complementation

Protein-protein interaction (PPI) networks are fundamental for cellular processes. Small-molecule PPI enhancers have been shown to be powerful tools fundamentally study PPIs and as starting points potential new therapeutics. Yet, systematic approaches their discovery not widely available, the design prerequisites of "molecular glues" poorly understood. Covalent fragment-based screening can identify chemical these at specific sites in interfaces. We recently reported a mass spectrometry-based disulfide-trapping (tethering) approach cysteine residue hub protein 14-3-3, an important regulator phosphorylated client proteins. Here, we invert strategy report development functional read-out identification based on fluorescence anisotropy (FA-tethering) with reactive handle now client-derived peptide. Using DNA-binding domain nuclear receptor Estrogen Related Receptor gamma (ERRγ), target native positioned 14-3-3 interface several fragments that form disulfide bond ERRγ stabilize complex up 5-fold. Crystallography indicates bind pocket comprised phosphopeptide. FA-tethering presents streamlined methodology discover molecular glues complexes.