Assay-Dependent Inverse Agonism at the A(3) Adenosine Receptor: When Neutral Is Not Neutral

TheA(3) adenosine receptor (A(3)AR) is implicatedin a variety of (patho)physiological conditions. While most researchhas focused on agonists and antagonists, inverse agonism at A(3)AR has been scarcely studied. Therefore, this study aimedat exploring inverse agonism, using two previously engineered celllines (hA(3)ARLgBiT-SmBiT & beta;arr2 and hA(3)ARLgBiT-SmBiTminiG & alpha;(i)), both employing the NanoBiT technology. The previously establishedinverse agonist PSB-10 showed a decrease in basal signal in the & beta;-arrestin2 (& beta;arr2) but not the miniG & alpha;(i) recruitment assay,indicative of inverse agonism in the former assay. Control experimentsconfirmed the specificity and reversibility of this observation. Evaluationof a set of presumed neutral antagonists (MRS7907, MRS7799, XAC, andMRS1220) revealed that all displayed concentration-dependent signaldecreases when tested in the A(3)AR-& beta;arr2 recruitmentassay, yielding EC50 and E (max) values for inverse agonism. Conversely, in the miniG & alpha;(i) recruitment assay, no signal decreases were observed. Toassess whether this observation was caused by the inability of theligands to induce inverse agonism in the G protein pathway, or ratherby a limitation inherent to the employed A(3)AR-miniG & alpha;(i) recruitment assay, a GloSensor cAMP assay was performed.The outcome of the latter also suggests inverse agonism by the presumedneutral antagonists in this latter assay. These findings emphasizethe importance of prior characterization of ligands in the relevanttest system. Moreover, it showed the suitability of the NanoBiT & beta;arr2recruitment and the GloSensor cAMP assays to capture inverse agonismat the A(3)AR, as opposed to the NanoBiT miniG & alpha;(i) recruitment assay.