Enhancement of inosine-mediated A 2A R signaling through positive allosteric modulation.

Inosine is an endogenous nucleoside that is produced by metabolic deamination of adenosine. Inosine is metabolically more stable (half-life 15h) than adenosine (half-life <10s). Inosine exerts anti-inflammatory and immunomodulatory effects similar to those observed with adenosine. These effects are mediated in part through the adenosine A2A receptor (A2A R). Relative to adenosine inosine exhibits a lower affinity towards the A2A R. Therefore, it is generally believed that inosine is incapable of activating the A2A R through direct engagement, but indirectly activates the A2A R upon metabolic conversion to higher affinity adenosine. A handful of studies, however, have provided evidence for direct inosine engagement at the A2A R leading to activation of downstream signaling events and inhibition of cytokine production. Here, we demonstrate that under conditions devoid of adenosine, inosine as well as an analog of inosine 6-S-[(4-Nitrophenyl)methyl]-6-thioinosine selectively and dose-dependently activated A2A R-mediated cAMP production and ERK1/2 phosphorylation in CHO cells stably expressing the human A2A R. Inosine also inhibited LPS-stimulated TNF-α, CCL3 and CCL4 production by splenic monocytes in an A2A R-dependent manner. In addition, we demonstrate that a positive allosteric modulator (PAM) of the A2A R enhanced inosine-mediated cAMP production, ERK1/2 phosphorylation and inhibition of pro-inflammatory cytokine and chemokine production. The cumulative effects of allosteric enhancement of adenosine-mediated and inosine-mediated A2A R activation may be the basis for the sustained anti-inflammatory and immunomodulatory effects observed in vivo and thereby provide insights into potential therapeutic interventions for inflammation- and immune-mediated diseases.