1812 - Functional Characterization of An Allosteric Enhancer of the Adenosine A2a Receptor That Inhibits Pro-Inflammatory Cytokine Production

Tuesday, November 8, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Ajith A. Welihinda and Edward P. Amento, Molecular Medicine Research Institute, Sunnyvale, CA
Presentation Number: 1812

Background/Purpose: Adenosine, an endogenous nucleoside, is produced at high levels at inflamed sites as a by-product of cellular activation and breakdown. Adenosine, mediates its immune suppressive activity primarily through the adenosine A2a receptor (A2aR), a member of the G-protein coupled receptor family of transmembrane receptors. High affinity A2aR agonists have demonstrated anti-inflammatory efficacy, however, their therapeutic utility is hindered by lack of adenosine receptor subtype selectivity upon systemic exposure. We sought to harness the inherent immune suppressive effects of adenosine by enhancing the responsiveness of A2aR to endogenously produced adenosine. Using a mutually exclusive array of cell-based assays, we examined a series of compounds originally derived from plant sources to identify potential A2aR allosteric enhancers. The present study describes allosteric properties of a representative compound.

Method: Compounds that inhibited inflammatory cytokines by mononuclear cells without affecting known anti-inflammatory pathways were screened for the enhancement of A2aR-mediated cAMP production in cells over-expressing the receptor. A2aR deficient CD4+ T cells were used to identify those compounds whose anti-inflammatory activity depended solely upon the intact receptor. Binding affinity of A2aR was determined using [14C]CGS21680.  A2aR-mediated G-protein activation was evaluated using [35S]GTP-γS binding to A2aR membranes. Human monocytes were isolated from PBMCs by depleting other cell types. CD4+ T cells were isolated from splenocytes by negative selection. Cytokine levels in culture media and intracellular cAMP levels were quantitated by ELISA. 

Result:A subset of compounds potentiated cAMP production by CHO cells stably expressing the human A2aR only in the presence of adenosine. A representative compound, AEA061, was chosen to establish allosteric modulation as the basis of the activity. AEA061 enhanced both the potency and efficacy of adenosine at the A2aR. Binding studies demonstrated that the compound increased the affinity as well as the Bmax of the hA2aR to agonists. In addition, AEA061 elevated agonist-mediated G-protein activation as shown by increased [35S]GTP-γS incorporation into A2aR expressing cell membranes. Consistent with the immunomodulatory role of the A2aR, AEA061-dependent activation of the receptor attenuated TNF-α production stimulated by distinctly different signaling pathways in monocytes/macrophages. Moreover, AEA061 attenuated IFN-γ production by anti-CD3-stimulated wild-type CD4+ T cells but not by A2aR deficient CD4+ T cells. 

Conclusion: Our observations support the hypothesis that AEA061 and its analogs allosterically modulate the A2aR to potentiate cAMP production. The allosteric enhancement of A2aR leads to inhibition of pro-inflammatory cytokines. The potential to enhance natural immune suppression through the adenosine-A2aR pathway may provide a means to focus anti-inflammatory activity at disease sites where adenosine is abundant. Allosteric modulation of A2aR presents a novel and unique approach for the treatment of RA and associated inflammatory conditions.

Keywords: T cells, adenosine receptors, inflammation, interferons and tumor necrosis factor (TNF)

Disclosure: A. A. Welihinda, None; E. P. Amento, None.