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Molecular mechanism of allosteric modulation at GPCRs : insight from a binding kinetics study at the human A 1 adenosine receptor
Author(s) -
Guo Dong,
Venhorst Suzanne N,
Massink Arnault,
Veldhoven Jacobus P D,
Vauquelin Georges,
IJzerman Adriaan P,
Heitman Laura H
Publication year - 2014
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.12836
Subject(s) - allosteric regulation , pharmacophore , chemistry , g protein coupled receptor , receptor–ligand kinetics , ligand (biochemistry) , small molecule , adenosine receptor , biophysics , allosteric enzyme , adenosine , allosteric modulator , receptor , stereochemistry , biochemistry , biology , agonist
Background and Purpose Many GPCR s can be allosterically modulated by small‐molecule ligands. This modulation is best understood in terms of the kinetics of the ligand–receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands. Here, we describe the application of a so‐called competition association assay at the adenosine A 1 receptor for this purpose. Experimental Approach We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A 1 receptor in the absence or presence of two allosteric modulators. We also tested three bitopic ligands, in which an orthosteric and an allosteric pharmacophore were covalently linked with different spacer lengths. The relevance of the competition association assay for the binding kinetics of the bitopic ligands was also explored by analysing simulated data. Key Results The binding kinetics of an unlabelled orthosteric ligand were affected by the addition of an allosteric modulator and such effects were probe‐ and concentration‐dependent. Covalently linking the orthosteric and allosteric pharmacophores into one bitopic molecule had a substantial effect on the overall on‐ or off‐rate. Conclusion and Implications The competition association assay is a useful tool for exploring the allosteric modulation of the human adenosine A 1 receptor. This assay may have general applicability to study allosteric modulation at other GPCR s as well.