Development of NanoBRET-Binding Assays for FKBP-Ligand Profiling in Living Cells
| Autor: | Jürgen M. Kolos, Michael Walz, Thomas M. Geiger, Angela Kuehn, Niklas Gutfreund, Felix Hausch, Monika T. Gnatzy |
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| Rok vydání: | 2021 |
| Předmět: |
Endogeny
010402 general chemistry Ligands 01 natural sciences Biochemistry Tacrolimus Binding Proteins Very Important Paper Fluorescence Resonance Energy Transfer luminescence Humans Nanotechnology receptor occupancy Molecular Biology 010405 organic chemistry Ligand Chemistry target engagement Communication NanoBRET Organic Chemistry Target engagement FKBP52 Communications 0104 chemical sciences Cytosol FKBP HEK293 Cells Immunophilin Molecular Medicine Intracellular Protein Binding |
| Zdroj: | Chembiochem |
| ISSN: | 1439-7633 |
| Popis: | FK506‐binding proteins (FKBPs) are promising targets for a variety of disorders and infectious diseases. High FKBP occupancy is thought to be necessary for ligands to effectively compete with the endogenous intracellular functions of FKBPs. Here, we report the development of NanoBRET assays for the most prominent cytosolic FKBPs, FKBP12, 12.6, 51 and 52. These assays allowed rapid profiling of FKBP ligands for target engagement and selectivity in living cells. These assays confirmed the selectivity of SAFit‐type ligands for FKBP51 over FKBP52 but revealed a substantial offset for the intracellular activity of these ligands compared to bicyclic ligands or natural products. Our results stress the importance to control for intracellular FKBP occupancy and provide the assays to guide further FKBP ligand optimization. Intracellular inhibition insights: NanoBRET assays were developed to measure target engagement of cytosolic FK506‐binding proteins, confirming selectivities and revealing permeation issues of FKBP ligands. |
| Databáze: | OpenAIRE |
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