Structure-affinity relationships for ligand binding at the human A 2A adenosine receptor have been probed using site-directed mutagenesis in the transmembrane helical domains (TMs). The mutant receptors were expressed in COS-7 cells and characterized by binding of the radioligands

The binding affinities at rat A 1 , A 2a , and A 3 adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [ 3 H]PIA or [ 3 H]CGS 21680 in rat brain membranes or [ 125 I]AB-MECA in CHO cells stably transfected with rat A 3 receptors. Several new classes of adenosine antagonists (e.g. 5-oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11-hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidinone, folic acid, and cytochalasin H and J bound to A 3 receptors selectively. Moreover, cytochalasin A, which bound to A 1 adenosine receptors with K i value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A 1 receptor binding.