Positive allosteric modulation of the adenosine A(2a) receptor attenuates inflammation

BACKGROUND: 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(2a)R), a member of the G-protein coupled receptors. A(2a)R 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(2a)R to endogenously produced adenosine through allosteric modulation. We have identified a family of positive allosteric modulators (PAMs) of the A(2a)R. Using one member of this PAM family, AEA061, we demonstrate that A(2a)Rs are amenable to allosteric enhancement and such enhancement produces increased A(2a)R signaling and diminished inflammation in vivo. METHODS: A(2a)R activity was evaluated using a cell-based cAMP assay. Binding affinity of A(2a)R was determined using [(3)H]CGS 21680. A(2a)R-mediated G-protein activation was quantified using [(35)S]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. RESULTS: AEA061 had no detectable intrinsic agonist activity towards either rat or human A(2a)Rs. AEA061 enhanced the efficacy of adenosine to rat and human A(2a)Rs 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(2a)R 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(2a)R by 1.8 and 3 fold respectively. Consistent with the anti-inflammatory role of the A(2a)R, 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(2a)R intact, but not in A(2a)R deficient, mice. CONCLUSIONS: AEA061 increases affinity and B(max) of A(2a)R to adenosine, thereby increasing adenosine potency and efficacy, which translates to enhanced A(2a)R responsiveness. Since the A(2a)R negatively regulates inflammation, PAMs of the receptor offer a novel means of modulating inflammatory processes.