The recent determination of X-ray structures of pharmacologically relevant GPCRs has made these targets accessible to structure-based ligand discovery. Here we explore whether novel chemotypes may be discovered for the A2A adenosine receptor, based on complementarity to its recently determined structure. The A2A adenosine receptor signals in the periphery and the CNS, with agonists explored as anti-inflammatory drugs and antagonists explored for neurodegenerative diseases. We used molecular docking to screen a 1.4 million compound database against the X-ray structure computationally and tested 20 high-ranking, previously unknown molecules experimentally. Of these 35% showed substantial activity with affinities between 200 nM and 9 μM. For the most potent of these new inhibitors, over 50-fold specificity was observed for the A2A versus the related A1 and A3 subtypes. These high hit rates and affinities at least partly reflect the bias of commercial libraries toward GPCR-like chemotypes, an issue that we attempt to investigate quantitatively. Despite this bias, many of the most potent new ligands were novel, dissimilar from known ligands, providing new lead structures for modulation of this medically important target.

We previously synthesized a series of potent and selective A 3 adenosine receptor (AR) agonists (North-methanocarba nucleoside 5′-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed "click" chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A 3 AR activation was preserved in these multivalent conjugates, which bound with apparent K i 0.1-0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A 3 AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A 3 and P2Y 14 receptors (via amide-linked uridine-5′-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.