Xiaowei Jin

Molecular characterization of recombinant human adenosine receptors

The four human adenosine receptors (ARs), A1, A2A, A2B, and A3, have been stably expressed in mammalian cells. Radioligand binding properties were examined for all four subtypes, including, for the first time the A2BAR. The A1AR‐selective radioligand, [3H]8‐cyclopentyl‐1,3‐dipropylxanthine ([3H]CPX; A1; KD = 2 nM) also can be used to characterize recombinant A2BAR (KD = 40 nM). Theophylline and enprofylline both bind to A2BAR with KI of 7 μM, well within the therapeutic ranges of these compounds used to treat asthma. We have identified C8‐(N‐methylisopropyl)‐amino‐N6‐(5′‐endohydroxy)‐endonorbornan‐2‐yl‐9‐methyladenine (WRC‐0571) as the most selective antagonist of A1AR. Recombinant ARs have been extended with hexahistidine (H) and the FLAG (F) epitope to make H/F‐A1 and H/F‐A2A receptors that have been purified to near homogeneity under conditions that preserve radioligand binding. [3H]Adenosine binds to purified H/F‐A1AR‐G protein complexes with a KD of 0.95 ± 0.3 nM; binding is not affected by the adenosine deaminase inhibitor, erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine (EHNA). Phosphoaminoacid analysis of H/F‐A1AR and H/F‐A2AAR purified from mammalian cells incubated in [32P]phosphate indicates that both receptors are phosphorylated on serine residues. Monoclonal antibodies were raised by using the purified H/F‐A2AAR as an antigen. Antibodies with higher affinity than antipeptide antisera were generated and used for Western blotting and rat brain immunocytochemistry. Drug Dev. Res. 39:243–252, 1996.

The structure and function of A1 and A2B adenosine receptors.

Of the four G protein coupled adenosine receptor (AR) subtypes, the A1 is best suited for studies of reconstitution with G proteins. Recombinant A1 receptors extended with hexahistidine and FLAG have been purified to near homogeneity. In reconstitution assays using pure recombinant G protein subunits, the composition of the gamma subunit influences coupling to purified A1ARs. The least well characterized AR is the A2B. New data indicate that A(2B)ARs can trigger the degranulation of canine and human mast cell lines. Recombinant human A(2B)ARs are blocked by the anti-asthma drugs theophylline and enprofylline at concentrations that are used therapeutically to treat asthma. Although A(2B)ARs have long been known to stimulate adenylyl cyclase, they also can activate phospholipase C and mobilize Ca2+ by signaling through Gq/11. There is great potential for new therapies based on compounds that selectively target individual AR subtypes.

Piperazine derivatives of [1,2,4]triazolo[1,5-a][1,3,5]triazine as potent and selective adenosine A2a receptor antagonists.

The [1,2,4]triazolo[1,5-a]triazine derivative 3, more commonly known in the field of adenosine research as ZM-241385, has previously been demonstrated to be a potent and selective adenosine A2a receptor antagonist, although with limited oral bioavailability. This [1,2,4]triazolo[1,5-a]triazine core structure has now been improved by incorporating various piperazine derivatives. With some preliminary optimization, the A2a binding affinity of some of the best piperazine derivatives is almost as good as that of compound 3. The selectivity level over the adenosine A1 receptor subtype for some of the more active analogues is also fairly high, > 400-fold in some cases. Many compounds within this piperazine series of [1,2,4]triazolo[1,5-a]triazine have now been shown to have good oral bioavailability in the rat, with some as high as 89% (compound 35). More significantly, some piperazines derivatives of [1,2,4]triazolo[1,5-a]triazine also possessed good oral efficacy in rodent models of Parkinsons disease. For instance, compound 34 was orally active in the rat catalepsy model at 3 mg/kg. In the 6-hydroxydopamine-lesioned rat model, this compound was also quite effective, with a minimum effective dose of 3 mg/kg po.