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. 2011 Sep 15;82(6):658-68.
doi: 10.1016/j.bcp.2011.06.017. Epub 2011 Jun 21.

Functionally biased modulation of A(3) adenosine receptor agonist efficacy and potency by imidazoquinolinamine allosteric enhancers

Zhan-Guo Gao  1 , Dennis Verzijl, Annelien Zweemer, Kai Ye, Anikó Göblyös, Adriaan P Ijzerman, Kenneth A Jacobson
Affiliations

Functionally biased modulation of A(3) adenosine receptor agonist efficacy and potency by imidazoquinolinamine allosteric enhancers

Zhan-Guo Gao et al. Biochem Pharmacol. .

Abstract

Allosteric modulators for the G(i)-coupled A(3) adenosine receptor (AR) are of considerable interest as therapeutic agents and as pharmacological tools to probe various signaling pathways. In this study, we initially characterized the effects of several imidazoquinolinamine allosteric modulators (LUF5999, LUF6000 and LUF6001) on the human A(3) AR stably expressed in CHO cells using a cyclic AMP functional assay. These modulators were found to affect efficacy and potency of the agonist Cl-IB-MECA differently. LUF5999 (2-cyclobutyl derivative) enhanced efficacy but decreased potency. LUF6000 (2-cyclohexyl derivative) enhanced efficacy without affecting potency. LUF6001 (2-H derivative) decreased both efficacy and potency. We further compared the agonist enhancing effects of LUF6000 in several other A(3) AR-mediated events. It was shown that although LUF6000 behaved somewhat differently in various signaling pathways, it was more effective in enhancing the effects of low-efficacy than of high-efficacy agonists. In an assay of cyclic AMP accumulation, LUF6000 enhanced the efficacy of all agonists examined, but in the membrane hyperpolarization assay, it only enhanced the efficacy of partial agonists. In calcium mobilization, LUF6000 did not affect the efficacy of the full agonist NECA but was able to switch the nucleoside antagonist MRS542 into a partial agonist. In translocation of β-arrestin2, the agonist-enhancing effect LUF6000 was not pronounced. In an assay of ERK1/2 phosphorylation LUF6000 did not show any effect on the efficacy of Cl-IB-MECA. The differential effects of LUF6000 on the efficacy and potency of the agonist Cl-IB-MECA in various signaling pathway were interpreted quantitatively using a mathematical model.

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Figures

Fig. 1
Fig. 1
A3 AR-mediated signaling explored in the present study. AC, adenylyl cyclase; PLC, phospholipase C.
Fig. 2
Fig. 2
Chemical structures of the agonists, antagonists, and allosteric modulators of the A3 AR used in the present study.
Fig. 3
Fig. 3
Effect of LUF6000 and its analogues on the potency and efficacy of the A3 AR agonist, Cl-IB-MECA, measured in a cyclic AMP accumulation assay using intact CHO cells stably expressing the human A3 AR. Agonist effect on the y-axis represents percent inhibition of cyclic AMP production. Cells were pretreated with LUF6000 (A) or its analogues (B, C), 20 min before the addition of an agonist. Data were representative of 2-4 separate experiments providing similar results performed in duplicate.
Fig. 4
Fig. 4
Functional enhancing effect of LUF6000 in a cyclic AMP functional assay on a full agonist NECA (A), partial agonists, inosine (B) and MRS541 (C) (nucleoside) and LUF5833 (D, non-nucleoside), and nucleoside antagonists MRS542 (E) and CCPA (F) in a cyclic AMP functional assay of the A3AR. Agonist effect on the y-axis represents percent inhibition of cyclic AMP production. Cells were pretreated with LUF6000 20 min before the addition of an agonist. Results were expressed as mean ± SEM from three separate experiments providing similar results performed in duplicate.
Fig. 4
Fig. 4
Functional enhancing effect of LUF6000 in a cyclic AMP functional assay on a full agonist NECA (A), partial agonists, inosine (B) and MRS541 (C) (nucleoside) and LUF5833 (D, non-nucleoside), and nucleoside antagonists MRS542 (E) and CCPA (F) in a cyclic AMP functional assay of the A3AR. Agonist effect on the y-axis represents percent inhibition of cyclic AMP production. Cells were pretreated with LUF6000 20 min before the addition of an agonist. Results were expressed as mean ± SEM from three separate experiments providing similar results performed in duplicate.
Fig. 5
Fig. 5
Effects of LUF6000 on agonist-induced membrane potential changes in CHO cells stably expressing the human A3 AR. Cells were pretreated with LUF6000 20 min before the addition of agonists. Data were from three separate experiments providing similar results performed in duplicate. Cells were incubated with 50 μl membrane potential dye/probenecid for 60 min at room temperature. The compound plate was prepared using dilutions of various compounds in Hank's Buffer (pH 7.4). Cell fluorescence (Excitation = 535 and Emission = 565 nm) was monitored following exposure to the compound. Cell fluorescence changes are reported as the maximum fluorescence value after exposure minus the basal fluorescence value before exposure.
Fig. 6
Fig. 6
Effect of LUF6000 (10 μM) on agonist-induced calcium mobilization in CHO cells stably expressing the human A3 AR. Cells were pretreated with LUF6000 20 min before the addition of agonists. Data were from 3-4 separate experiments providing similar results performed in duplicate.
Fig. 7
Fig. 7
Effect of LUF6000 (10 μM) on agonist-induced translocation of β-arrestin2 in CHO cells stably expressing the human A3 AR. Cells were pretreated with LUF6000 20 min before the addition of agonists. Data were from 3-4 separate experiments providing similar results performed in duplicate.
Fig. 8
Fig. 8
Effect of LUF6000 on Cl-IB-MECA-induced ERK1/2 phosphorylation in CHO cells expressing the human A3 ARs. Results are from 2 separate experiments performed in duplicate. Cells were pre-incubated with various concentrations of LUF6000 for 20 min before addition of agonists.
Fig. 9
Fig. 9
Simulation of pharmacological curves with MatLab. The equations from Hall (2000) were used to derive conditions that vary efficacy and potency. Parameters in these equations are listed in the text. A. cyclic AMP; B, membrane hyper polarization; C, calcium mobilization; D, arrestin translocation; E, ERK1/2 activation.
Fig. 10
Fig. 10
Analysis of the maximum agonist efficacy in the presence and absence of LUF6000. By using the parameters selected for simulation of Figure 2A (β=1; δ=15; γ=0.156) and leaving α as a variable, the equation can be restated as: Max[B]→∞ — Max[B]=0 = 2.34α/(1+ 2.34α) — 0.015α/(1+0.015α).
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