Thermodynamics of A2B adenosine receptor binding discriminates agonistic from antagonistic behaviour.

Thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees of the binding equilibrium of 12 ligands (six agonists and six antagonists) to the A(2B) adenosine receptor subtype have been determined by affinity measurements carried out on HEK 293 cells stably transfected with human A(2B) adenosine receptors at six different temperatures (4, 10, 15, 20, 25, 30 degrees C) and van't Hoff plot analysis have been performed. Affinity constants were obtained from saturation experiments of [(3)H]MRE 2029-F20 or by its displacement in inhibition assays for the other compounds. van't Hoff plots were essentially linear in the temperature range investigated, showing that the DeltaC(p) degrees of the binding equilibrium is nearly zero. Thermodynamic parameters are in the range 7< or =DeltaH degrees < or =23 kJ mol(-1)and 123< or =DeltaS degrees < or =219 JK(-1)mol(-1) for agonists and -40 < or =DeltaH degrees < or =-20 kJ mol(-1) and 10< or =DeltaS degrees < or =91 JK(-1)mol(-1) for antagonists indicating that agonistic binding is always totally entropy-driven while antagonistic binding is enthalpy and entropy-driven. In the -TDeltaS degrees versus DeltaH degrees plot the thermodynamic data are clearly arranged in separate clusters for agonists and antagonists, which, therefore, turn out to be thermodynamically discriminated.

Pharmacological characterization of novel adenosine ligands in recombinant and native human A2B receptors.

The present study was designed to evaluate the effects of novel and recognised compounds at human recombinant A(2B) adenosine receptors expressed in Chinese hamster ovary (hA(2B)CHO), in human embryonic kidney 293 (hA(2B)HEK-293) and at endogenous A(2B) receptors in human mast cells (HMC-1). Saturation binding experiments performed using the new high affinity A(2B) adenosine radioligand [(3)H]-N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetra hydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide ([(3)H]-MRE 2029F20) revealed a single class of binding sites in hA(2B)CHO, hA(2B)HEK-293 and HMC-1 cells with K(D) (nM) of 1.65+/-0.18, 2.83+/-0.34, 2.62+/-0.27 and B(max) (fmol/mg protein) of 36+/-4, 475+/-50 and 128+/-15, respectively. The pharmacological profile of new compounds, determined in inhibition binding experiments in hA(2B)HEK-293 cells using [(3)H]-MRE 2029F20, showed a rank order of potency typical of the A(2B) receptors with K(i) values in the range 3.2-28nM. In functional assays, recognised agonists and antagonists were studied by evaluating their capability to modulate the cAMP production in hA(2B)CHO and in HMC-1 cells. Novel compounds were able to decrease NECA-stimulated cAMP production in hA(2B)CHO and in HMC-1 cells showing a high potency. New compounds were also able to inhibit cAMP levels in the absence of NECA and in the presence of forskolin stimulation in hA(2B)CHO and in HMC-1 cells. In HEK-293 cells MRE 2029F20 reduced cAMP basal levels with an IC(50) value of 2.9+/-0.3nM. These results suggest that novel compounds are antagonists with an inverse agonist activity in recombinant and native human A(2B) receptors.