Abstract
Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H2 receptor with different fluorescently labeled squaramide-type compounds synthesized in the course of this study. Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pKd = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pKi values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.
Highlights
The histamine H 2 receptor ( H2R), which is activated endogenously by the biogenic amine histamine (1, Fig. 1), is a long known member of rhodopsin-like receptors, the largest and best studied group of G proteincoupled receptors (GPCRs)[1,2,3,4]
In this study we report the development of a NanoBRET binding assay for the histamine H 2 receptor including the synthesis and characterization of suitable fluorescent H 2R ligands
As a homogeneous live cell-based assay, this assay allows for a convenient determination of affinity constants of putative H2 receptor ligands, independent of their mode of action without any washing or separation steps
Summary
The histamine H 2 receptor ( H2R), which is activated endogenously by the biogenic amine histamine (1, Fig. 1), is a long known member of rhodopsin-like receptors (class A), the largest and best studied group of G proteincoupled receptors (GPCRs)[1,2,3,4]. We synthesized three differently labeled fluorescent ligands (8–10, Fig. 1), structurally derived from BMY-25368 (5, Fig. 1), a potent and long-acting histamine H 2 receptor antagonist developed by Brystol-Myers in the 1 980s26, and radioligand [3H]UR-DE257 (7, Fig. 1) from our laboratory[27,28]. These fluorescent tracers were tested for their suitability in the BRET binding assay
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