A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes
| Autor: | Höring, Carina, Seibel, Ulla, Tropmann, Katharina, Grätz, Lukas, Mönnich, Denise, Pitzl, Sebastian, Bernhardt, Günther, Pockes, Steffen, Strasser, Andrea |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Protein Conformation
Histamine Antagonists split-luciferase complementation (SLC) Ligands Article Receptors G-Protein-Coupled Histamine Agonists lcsh:Chemistry histamine receptor ligands Radioligand Assay 615 Pharmazie GTP-Binding Proteins Drug Discovery Animals Humans Luciferases HIGH CONSTITUTIVE ACTIVITY H-4 RECEPTOR INVERSE AGONISM GUINEA-PIG 1ST POTENT H-2-RECEPTOR ACTIVATION H-1-RECEPTOR SELECTIVITY ANTAGONISTS histamine receptors mini-G protein recruitment G protein-coupled receptors (GPCRs) bioluminescence lcsh:QH301-705.5 Molecular Mimicry Recombinant Proteins ddc:615 Kinetics HEK293 Cells lcsh:Biology (General) lcsh:QD1-999 Guanosine 5'-O-(3-Thiotriphosphate) Receptors Histamine |
| Zdroj: | International Journal of Molecular Sciences Volume 21 Issue 22 International Journal of Molecular Sciences, Vol 21, Iss 8440, p 8440 (2020) |
| Popis: | In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTP&gamma S, [&gamma 32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z&rsquo factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTP&gamma S binding assay. |
| Databáze: | OpenAIRE |
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