title: Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor.
abstract: OBJECTIVE
  Many 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)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 A1 receptor for this purpose.
  
  
  METHODS
  We used a competition association assay to examine the binding kinetics of several unlabelled orthosteric agonists of the A1 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands.
authors.forename: Dong
authors.initials: D
authors.lastname: Guo
authors.forename: Suzanne N
authors.initials: SN
authors.lastname: Venhorst
authors.forename: Arnault
authors.initials: A
authors.lastname: Massink
authors.forename: Jacobus P D
authors.initials: JP
authors.lastname: van Veldhoven
authors.forename: Georges
authors.initials: G
authors.lastname: Vauquelin
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
authors.forename: Laura H
authors.initials: LH
authors.lastname: Heitman
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D003198:Computer Simulation; D003412:Cricetulus; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding; D043682:Receptor, Adenosine A1 / Q000378:metabolism*
chemical_list: D008024:Ligands; D043682:Receptor, Adenosine A1