Positive allosteric modulation of the adenosine A_2a receptor attenuates inflammation

Adenosine is produced at high levels at inflamed sites as a by-product of cellular activation and breakdown. Adenosine mediates its anti-inflammatory activity primarily through the adenosine A_2a receptor (A_2aR), a member of the G-protein coupled receptors. A_2aR agonists have demonstrated anti-inflammatory efficacy, however, their therapeutic utility is hindered by a lack of adenosine receptor subtype selectivity upon systemic exposure. We sought to harness the anti-inflammatory effects of adenosine by enhancing the responsiveness of A_2aR to endogenously produced adenosine through allosteric modulation. We have identified a family of positive allosteric modulators (PAMs) of the A_2aR. Using one member of this PAM family, AEA061, we demonstrate that A_2aRs are amenable to allosteric enhancement and such enhancement produces increased A_2aR signaling and diminished inflammation in vivo.

A_2aR activity was evaluated using a cell-based cAMP assay. Binding affinity of A_2aR was determined using [³H]CGS 21680. A_2aR-mediated G-protein activation was quantified using [³⁵S]GTP-γS. The effect of AEA061 on cytokine production was evaluated using primary monocytes and splenocytes. The anti-inflammatory effect of AEA061 was evaluated in the LPS-induced mouse model of inflammation.

AEA061 had no detectable intrinsic agonist activity towards either rat or human A_2aRs. AEA061 enhanced the efficacy of adenosine to rat and human A_2aRs by 11.5 and 2.8 fold respectively. AEA061 also enhanced the maximal response by 4.2 and 2.1 fold for the rat and the human A_2aR respectively. AEA061 potentiated agonist-mediated Gα activation by 3.7 fold. Additionally, AEA061 enhanced both the affinity as well as the B_max at the human A_2aR by 1.8 and 3 fold respectively. Consistent with the anti-inflammatory role of the A_2aR, allosteric enhancement with AEA061 inhibited the production of TNF-α, MIP-1α, MIP-1β, MIP-2, IL-1α, KC and RANTES by LPS-stimulated macrophages and/or splenocytes. Moreover, AEA061 reduced circulating plasma TNF-α and MCP-1 levels and increased plasma IL-10 in endotoxemic A_2aR intact, but not in A_2aR deficient, mice.

AEA061 increases affinity and B_max of A_2aR to adenosine, thereby increasing adenosine potency and efficacy, which translates to enhanced A_2aR responsiveness. Since the A_2aR negatively regulates inflammation, PAMs of the receptor offer a novel means of modulating inflammatory processes.