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

    Onderzoeksoutput: Articlepeer review

    19 Citaten (Scopus)

    Samenvatting

    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.
    Originele taal-2English
    Pagina's (van-tot)5295-5312
    Aantal pagina's18
    TijdschriftBritish Journal of Pharmacology
    Volume171
    StatusPublished - 2014

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