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. 2014 Dec;171(23):5295-312.
doi: 10.1111/bph.12836. Epub 2014 Sep 5.

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

Dong Guo  1 , Suzanne N Venhorst, Arnault Massink, Jacobus P D van Veldhoven, Georges Vauquelin, Adriaan P IJzerman, Laura H Heitman
Affiliations

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

Dong Guo et al. Br J Pharmacol. 2014 Dec.

Abstract

Background and purpose: 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.

Experimental approach: 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.

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 A1 receptor. This assay may have general applicability to study allosteric modulation at other GPCRs as well.

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Figures

Figure 1
Figure 1
Chemical structures of compounds used in the present study. CCPA, NECA and LUF5519 are ribose-containing adenosine A1 receptor agonists, whereas LUF5834 is a non-ribose agonist (Lohse et al., ; Müller, ; Beukers et al., 2004); PD81,723 and BC-1 are allosteric enhancers for adenosine A1 receptor agonists (Romagnoli et al., 2008). LUF6232, LUF6234 and LUF6258 are in-house synthesized bitopic ligands for the adenosine A1 receptor with four-, five- and nine-carbon linker, respectively (Narlawar et al., 2010). LUF7161 and LUF7160 are newly synthesized monovalent ligands with five-atom linker and nine-atom linker respectively.
Figure 2
Figure 2
Schematic representation of the monovalent, ‘c’, and bitopic, ‘ab’, ligand–target site interactions. The bitopic ligand bears two distinct pharmacophores: ‘a’ and ‘b’. ‘AB’ is the target with distinct binding sites ‘A’ and ‘B’. ‘a’ and ‘c’ only bind to ‘A’ in a competitive manner and ‘b’ only binds to ‘B’. The abbreviated notation for the free target and target complexes is in parentheses. k+a, k+b and k+c (in M−1·min−1) are the microscopic association rate constants and k−a, k−b and k−c (in min−1) are the microscopic dissociation rate constants for a-A, b-B and c-A binding respectively. αab and αbc are cooperativity factors affecting the association process of different pharmacophores. α'ab and α'bc are cooperativity factors affecting the dissociation process of different pharmacophores.
Figure 3
Figure 3
(A) Displacement of specific [3H]-DPCPX binding from the adenosine A1 receptor at 25°C by CCPA in the absence or presence of 10 μM PD81,723, 10 μM BC-1, 1 mM GTP or a combination thereof. (B) Displacement of specific [3H]-DPCPX binding from the adenosine A1 receptor at 25°C by NECA in the absence or presence of 10 μM PD81,723, 10 μM BC-1, 1 mM GTP or a combination thereof. (C) Displacement of specific [3H]-DPCPX binding from the adenosine A1 receptor at 25°C by LUF5834 in the absence or presence of 10 μM PD81,723 or BC-1. (D) Displacement of specific [3H]-DPCPX binding from the adenosine A1 receptor at 25°C by 100 nM CCPA (normalized as 100%) in the presence of increased concentrations of PD81,723 or BC-1. Representative graphs from one experiment performed in duplicate.
Figure 4
Figure 4
[3H]-DPCPX competition association assay in the absence or presence of three different concentrations of unlabelled CCPA. (A) Control experiment. (B) Experiment in the presence of 1 mM GTP. (C) Experiment in the presence of 10 μM PD81,723. Representative graphs from one experiment performed in duplicate (see Table 4 for kinetic values).
Figure 5
Figure 5
(A) [3H]-DPCPX competition association assay in the absence or presence of unlabeled CCPA with or without different concentrations of PD81,723. (B) [3H]-DPCPX competition association assay in the absence or presence of unlabelled NECA with or without 10 μM PD81,723. (C) [3H]-DPCPX competition association assay in the absence or presence of unlabelled LUF5834 with or without 10 μM PD81,723. (D) [3H]-DPCPX competition association assay in the absence or presence of unlabelled CCPA with or without 1 μM BC-1. (E) [3H]-DPCPX competition association assay in the absence or presence of unlabeled CCPA, NECA or LUF5834 with or without 10 μM BC-1. (F) [3H]-DPCPX competition association assay in the absence or presence of unlabelled CCPA with or without 33 μM BC-1. Data were fitted to Equation 2 described in the Methods section to calculate the kon (k3) and koff (k4) values of unlabelled ligands by using the respective k1 and k2 values of [3H]-DPCPX under different conditions. Representative graphs from one experiment performed in duplicate (see Table 5 for kinetic values).
Figure 6
Figure 6
(A) [3H]-DPCPX competition association assay in the absence or presence of unlabeled LUF5519 with or without 10 or 33 μM PD81,723. (B) [3H]-DPCPX competition association assay in the absence or presence of unlabelled LUF6232 with or without 10 or 33 μM PD81,723. (C) [3H]-DPCPX competition association assay in the absence or presence of unlabelled LUF6234 with or without 10 or 33 μM PD81,723. (D) [3H]-DPCPX competition association assay in the absence or presence of unlabelled LUF6258 with or without 10 or 33 μM PD81,723. (E) [3H]-DPCPX competition association assay in the absence or presence of unlabelled LUF7161 with or without 10 or 33 μM PD81,723. Data were fitted to the equations described in the methods to calculate the kon (k3) and koff (k4) values of unlabelled ligands by using the k1 and k2 values of [3H]-DPCPX. Representative graphs from one experiment performed in duplicate (see Table 6 for kinetic values).
Figure 7
Figure 7
Proposed diagram of three distinct binding modes for the bitopic ligands in the absence or presence of further added allosteric modulator. Mode 1: the linker length of a bitopic ligand is sufficient to occupy both sites on the receptor, yet in a non-optimal manner. Mode 2: the linker length of a bitopic ligand is not sufficient to occupy both sites on the receptor. Two ligands could possibly bind to one receptor with either the orthosteric part to the ‘O-site’ or with the allosteric part to the ‘A-site’ on the receptor. Mode 3: the linker length of a bitopic is optimal to occupy both orthosteric and allosteric sites of the receptor. Upon the addition of excess allosteric modulator, the allosteric modulator can prevent the rebinding of the freshly dissociated allosteric pharmacophore in Mode 1 due to the bitopic ligand's less optimal binding pose or occupy the ‘A-site’ on the receptor and thus ‘displace’ the bitopic ligand binding to the receptor via its allosteric pharmacophore in Mode 2. In contrast, the binding of the bitopic ligand with optimal linker length (Mode 3) is less affected in the presence of the allosteric modulator.
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