Bioluminescence Resonance Energy Transfer Based G Protein-Activation Assay to Probe Duration of Antagonism at the Histamine H3 Receptor.
| Title: | Bioluminescence Resonance Energy Transfer Based G Protein-Activation Assay to Probe Duration of Antagonism at the Histamine H3 Receptor. |
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| Authors: | Mocking TAM; Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.; Buzink MCML; Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.; Leurs R; Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.; Vischer HF; Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), Division of Medicinal Chemistry, Faculty of Science, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands. h.f.vischer@vu.nl. |
| Source: | International journal of molecular sciences [Int J Mol Sci] 2019 Jul 30; Vol. 20 (15). Date of Electronic Publication: 2019 Jul 30. |
| Publication Type: | Journal Article |
| Language: | English |
| Journal Info: | Publisher: MDPI Country of Publication: Switzerland NLM ID: 101092791 Publication Model: Electronic Cited Medium: Internet ISSN: 1422-0067 (Electronic) Linking ISSN: 14220067 NLM ISO Abbreviation: Int J Mol Sci Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: Basel, Switzerland : MDPI, [2000- |
| MeSH Terms: | Bioluminescence Resonance Energy Transfer Techniques/*methods ; Histamine H3 Antagonists/*pharmacology ; Receptors, Histamine H3/*metabolism; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; G-Protein-Coupled Receptor Kinase 3/genetics ; G-Protein-Coupled Receptor Kinase 3/metabolism ; Luminescent Proteins/genetics ; Luminescent Proteins/metabolism ; HEK293 Cells ; Humans ; Protein Binding |
| Abstract: | Duration of receptor antagonism, measured as the recovery of agonist responsiveness, is gaining attention as a method to evaluate the 'effective' target-residence for antagonists. These functional assays might be a good alternative for kinetic binding assays in competition with radiolabeled or fluorescent ligands, as they are performed on intact cells and better reflect consequences of dynamic cellular processes on duration of receptor antagonism. Here, we used a bioluminescence resonance energy transfer (BRET)-based assay that monitors heterotrimeric G protein activation via scavenging of released Venus-Gβ1γ2 by NanoLuc (Nluc)-tagged membrane-associated-C-terminal fragment of G protein-coupled receptor kinase 3 (masGRK3ct-Nluc) as a tool to probe duration of G protein-coupled receptor (GPCR) antagonism. The Gαi-coupled histamine H3 receptor (H3R) was used in this study as prolonged antagonism is associated with adverse events (e.g., insomnia) and consequently, short-residence time ligands might be preferred. Due to its fast and prolonged response, this assay can be used to determine the duration of functional antagonism by measuring the recovery of agonist responsiveness upon washout of pre-bound antagonist, and to assess antagonist re-equilibration time via Schild-plot analysis. Re-equilibration of pre-incubated antagonist with agonist and receptor could be followed in time to monitor the transition from insurmountable to surmountable antagonism. The BRET-based G protein activation assay can detect differences in the recovery of H3R responsiveness and re-equilibration of pre-bound antagonists between the tested H3R antagonists. Fast dissociation kinetics were observed for marketed drug pitolisant (Wakix®) in this assay, which suggests that short residence time might be beneficial for therapeutic targeting of the H3R. |
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| Grant Information: | 718.014.002 Nederlandse Organisatie voor Wetenschappelijk Onderzoek |
| Contributed Indexing: | Keywords: G protein-coupled receptor (GPCR); histamine H3 receptor (H3R); ligand binding kinetics; re-equilibration; residence time |
| Substance Nomenclature: | 0 (Bacterial Proteins); 0 (Histamine H3 Antagonists); 0 (Luminescent Proteins); 0 (Receptors, Histamine H3); 0 (yellow fluorescent protein, Bacteria); EC 2.7.11.15 (G-Protein-Coupled Receptor Kinase 3) |
| Entry Date(s): | Date Created: 20190802 Date Completed: 20191223 Latest Revision: 20200225 |
| Update Code: | 20260130 |
| PubMed Central ID: | PMC6695674 |
| DOI: | 10.3390/ijms20153724 |
| PMID: | 31366084 |
| Database: | MEDLINE |
Journal Article