Functionally biased modulation of A3 adenosine receptor agonist efficacy and potency by imidazoquinolinamine allosteric enhancers

Abstract

Allosteric modulators for the Gi-coupled A3 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 A3 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 A3 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.

Allosteric Modulation of Endogenous Metabolites as an Avenue for Drug Discovery

“…Likewise, resting inosine levels in the brain and the heart can reach concentrations as high as 10 M and at least 30-fold higher in ischemic conditions (Bä ckström et al, 2003). Evidence for modulation of inosine at the A 1 -AR (in addition to previous evidence for modulation at the A 3 -AR (Gao et al, 2011) provides additional proof that targeting metabolites is viable. Furthermore, circulating GLP-1(9 -36)NH 2 concentrations are Ͼ10-fold higher than that of GLP-1(7-36)NH 2 (Göke et al, 1993).…”

“…Therefore, it is plausible that the metabolites investigated in this study could also have effects at these other subtypes. Certainly this is true for inosine, for which allosteric potentiation of cAMP signaling at the A 3 -AR has been reported (Gao et al, 2011). One advantage of allosteric ligands is their ability to provide selectivity, and, therefore, use of a selective modulator should, in theory, only modulate the metabolite at the subtype where the allosteric ligand binds.…”

“…However, a hitherto-unappreciated extension of this phenomenon is the possibility that endogenous metabolites of GPCR agonists, which may normally be minimally active in their own right, can also be influenced by allosteric modulators. Indeed, a recent study reported that the allosteric compound N- (3,4-dichlorophenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine (LUF6000) can enhance signaling by inosine (the metabolite of adenosine) at the adenosine A 3 receptor (A 3 -AR) (Gao et al, 2011). Although drug discovery programs focusing on developing small molecule allosteric drugs invariably screen for compounds that modulate responses mediated by the predominant orthosteric receptor agonist, it is currently not routine to incorporate similar studies on endogenous metabolites.…”

Distinct Signaling Patterns of Allosteric Antagonism at the P2Y1 Receptor

“…The method used was essentially as previously described (Gao et al, 2011(Gao et al, , 2014. 1321N1 astrocytoma cells or U2OS cells expressing the human P2Y 1 R (30,000 cells/100 ml) were seeded in a 96-well plate in complete growth medium.…”

“…The preparation of membranes from U2OS cells expressing human P2Y 1 R was as previously described (Gao et al, 2011 35 S]GTPgS, 0.5% bovine serum albumin, test agonists, and membrane suspension (10 mg protein/tube). Antagonists were added 20 minutes before the addition of agonists.…”