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NanoBRET binding assay for histamine H2 receptor ligands using live recombinant HEK293T cells.
Grätz, Lukas; Tropmann, Katharina; Bresinsky, Merlin; Müller, Christoph; Bernhardt, Günther; Pockes, Steffen.
Afiliación
  • Grätz L; Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
  • Tropmann K; Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
  • Bresinsky M; Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
  • Müller C; Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
  • Bernhardt G; Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany.
  • Pockes S; Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053, Regensburg, Germany. steffen.pockes@ur.de.
Sci Rep ; 10(1): 13288, 2020 08 06.
Article en En | MEDLINE | ID: mdl-32764682
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.
Asunto(s)

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: ADN Recombinante / Receptores Histamínicos H2 / Nanotecnología / Transferencia Resonante de Energía de Fluorescencia Límite: Humans Idioma: En Revista: Sci Rep Año: 2020 Tipo del documento: Article País de afiliación: Alemania

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: ADN Recombinante / Receptores Histamínicos H2 / Nanotecnología / Transferencia Resonante de Energía de Fluorescencia Límite: Humans Idioma: En Revista: Sci Rep Año: 2020 Tipo del documento: Article País de afiliación: Alemania