Assessment of biased agonism at the A(3) adenosine receptor using beta-arrestin and miniG alpha(i) recruitment assays

The A(3) adenosine receptor (A(3)AR) is a G protein-coupled receptor that is involved in a wide variety of physiological and pathological processes, such as cancer. However, the use of compounds pharmacologically targeting this receptor remains limited in clinical practice, despite extensive efforts for compound synthesis. Moreover, the possible occurrence of biased agonism further complicates the interpretation of the functional characteristics of compounds. Hence the need for simple assays, which are comparable in terms of the used cell lines and read-out technique. We previously established a stable beta-arrestin 2 (beta arr2) bioassay, employing a simple, luminescent read-out via functional complementation of a split nanoluciferase enzyme. Here, we developed a complementary, new bioassay in which coupling of an engineered miniGa(i) protein to activated A(3)AR is monitored using a similar approach. Application of both bioassays for the concurrent determination of the potencies and efficacies of a set of 19 N-6-substituted adenosine analogues not only allowed for the characterization of structure-activity relationships, but also for the quantification of biased agonism. Although a broad distribution in potency and efficacy values was obtained within the test panel, no significant bias was observed toward either the beta arr2 or miniGa(i) pathway.

Publication year:2020

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