Molecular mechanism of allosteric modulation: insight from a binding kinetics study at the human adenosine A1 receptor

Guo D, Venhorst S, Massink A, Van Velthoven Jpd, Georges Vauquelin, Ijzerman Ap, Heitman Lh

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    19 Citations (Scopus)


    Background and purpose: Many G protein-coupled receptors (GPCRs) 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-) labeling 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 A1 receptor (A1R) for this purpose.
    Experimental approach: We used a competition association assay to examine the binding kinetics of several unlabeled A1R orthosteric agonists 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 in between. The relevance of the competition association assay for the bitopic ligands' binding kinetics was also explored by analyzing simulated data.
    Key results: The binding kinetics of an unlabeled 'cold' orthosteric ligand was influenced upon the addition of an allosteric modulator and such an effect was 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 A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well.
    Original languageEnglish
    Pages (from-to)5295-5312
    Number of pages18
    JournalBritish Journal of Pharmacology
    Publication statusPublished - 2014


    • Binding kinetics
    • residence time
    • allosteric modulation
    • GPCR
    • adenosine A1 receptor
    • bitopic ligand
    • competition association assay
    • simulations


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