Horing, Carina and Seibel, Ulla and Tropmann, Katharina and Gratz, Lukas and Monnich, Denise and Pitzl, Sebastian and Bernhardt, Gunther and Pockes, Steffen and Strasser, Andrea (2020) A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 21 (22): 8440. ISSN , 1422-0067
Full text not available from this repository. (Request a copy)Abstract
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 ([S-35]GTP gamma S, [gamma-P-32]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 pEC(50) values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z' factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [S-35]GTP gamma S binding assay.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | HIGH CONSTITUTIVE ACTIVITY; H-4 RECEPTOR; INVERSE AGONISM; GUINEA-PIG; 1ST POTENT; H-2-RECEPTOR; ACTIVATION; H-1-RECEPTOR; SELECTIVITY; ANTAGONISTS; histamine receptors; split-luciferase complementation (SLC); mini-G protein recruitment; G protein-coupled receptors (GPCRs); histamine receptor ligands; bioluminescence |
| Subjects: | 600 Technology > 615 Pharmacy |
| Divisions: | Chemistry and Pharmacy > Institute of Pharmacy Chemistry and Pharmacy > Institute of Pharmacy > Alumni or Retired Professors > Pharmaceutical/Medicinal Chemistry II (Prof. Buschauer) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 08 Mar 2021 09:47 |
| Last Modified: | 08 Mar 2021 09:47 |
| URI: | https://pred.uni-regensburg.de/id/eprint/43445 |
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