Functional efficacy of adenosine A₂A receptor agonists is positively correlated to their receptor residence time

Abstract

BACKGROUND AND PURPOSE The adenosine A(2A) receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A(2A) receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A(2A) receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACH We set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A(2A) receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A(2A) receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTS A simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A(2A) receptor ligands at their receptor. A correlation was observed between the receptor residence time of A(2A) receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A(2A) receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONS This study indicates that the molecular basis of different agonist efficacies at the A(2A) receptor lies within their different residence times at this receptor.

Figure 1

Chemical structures of the 10 A_2A receptor agonists used in this study.

Figure 2

Saturation experiment for [³H]-ZM241385 (∼0.2–10 nM, final concentration) binding to HEK293hA_2AR membranes at 5°C; total binding, non-specific binding and specific binding are shown. Representative graph from one experiment performed in duplicate.

Figure 3

(A) Displacement of specific [³H]-ZM241385 binding from the hA_2A receptor by ZM241385 and NECA in the absence (closed symbols) or presence of 100 µM GTP (open symbols). (B) Displacement of specific [³H]-ZM241385 binding from the hA_2A receptors by the other nine agonists. Representative graphs from one experiment performed in duplicate.

Figure 4

The association and dissociation of [³H]-ZM241385 at the hA_2A receptor at (A) 25°C and (B) 5°C. Representative graphs from one experiment performed in duplicate.

Figure 5

Competition association experiment with [³H]-ZM241385 in the absence of ligand and in the presence of onefold K_i value, threefold K_i value or tenfold K_i value of unlabelled ZM241385. Representative graphs from one experiment performed in duplicate (see Table 1 for kinetic values).

Figure 6

[³H]-ZM241385 competition association experiments in the absence (A) or presence (B) of 100 µM GTP with NECA concentrations 1-, 3- or 10-fold its K_i value. Data were fitted to the equations described in the methods to calculate NECA's k_on and k_off values in the absence or presence of GTP. Representative graphs from one experiment performed in duplicate (see Table 2 for kinetic values).

Figure 7

(A) [³H]-ZM241385 competition association binding in the absence of ligand and in the presence of unlabelled CGS21680, LUF5834, LUF5550 or UK432,097. Data were fitted to the equations described in the methods to calculate the k_on and k_off values of unlabelled ligands. Representative graphs from one experiment performed in duplicate (see Table 3 for kinetic values). (B) Correlation between affinities (K_i) and ‘kinetic K_D’ values of all the tested compounds. K_i values were taken from the displacement experiments at equilibrium and K_D values were derived from the competition association experiments.

Figure 8

(A) Representative graph of normalized cell-electrode impedance (expressed as CI) after the addition of different concentrations of CGS21680 in a label-free impedance-based assay: agonist stimulation of HEK293hA_2AR cells results in an increased CI. The change in CI was normalized over time against the vehicle control. (B) Concentration–response curves for difference A_2A receptor agonists at HEK293hA_2AR derived from peak-analysis of CI changes in the xCELLigence RTCA system. The cellular response was normalized and shown as % of the maximal CI by 1 µM CGS21680. (C) Concentration–response curves of cAMP stimulation by ten A_2A receptor agonists in a cAMP assay. Data were normalized and shown as % of the maximal cAMP production by 10 µM CGS21680 in HEK293hA_2AR cells (=100%). Representative graphs from one experiment performed in duplicate.

Figure 9

Correlation of the functional efficacy (E_max) derived from label-free whole-cell assay against (A) Log K_i (r²= 0.13, P= 0.32) and (B) Log RT (residence time) (r²= 0.90, P < 0.0001). (C) Correlation of the functional efficacy derived from cAMP assay against Log RT (r²= 0.74, P < 0.001). (D) Correlation of the functional efficacy derived from label-free whole-cell assay against the functional efficacy derived from cAMP assay (r²= 0.79, P < 0.001). Data used in these plots are detailed in Tables 3 and 4. Data are expressed as mean ± SEM from at least three independent experiments.

Figure 10

Proposed schematic representation of (A) obstacles at the bottom of the binding pocket and (B) the stabilization effects of the ribose moiety after agonist binding to the receptor. Molecule in blue is ZM241385; molecule in orange is UK432,097. The length of arrows in blue (ZM241385) and orange (UK432,097) indicates the speed of ligand association and dissociation. The relative position of these two molecules is co-ordinated according to the surrounding amino acids based on the crystal structures reported by Jaakola et al. (2008; PDB code: 3EML) and Xu et al. (2011; PDB code: 3QAK). In comparison to ZM241385 (non-ribose compound), UK432,097 (ribose-containing compound), binds deeper in the main binding pocket, hence before receptor activation obstacles such as water molecules or structural H-bond networks slow down the association of UK432,097 (A). After ligand binding to the receptor, the ribose part of UK432,097 is stabilized by H-bonds and non-polar interactions and stays in an entropy favourable state after the displacement of explicit water molecules (as present in the ZM241385 crystal structure). This would represent a molecular mechanism accounting for the slower dissociation of ribose-containing agonists compared to antagonists (B).