[3H]HEMADO--a novel tritiated agonist selective for the human adenosine A3 receptor.

Abstract Adenosine A 3 receptors are promising drug targets for a number of conditions like inflammatory diseases including asthma, ischemic injury or certain types of cancer. Consequently, intense efforts are dedicated to the development of selective A 3 agonists and antagonists. The only tritiated agonist that is available for radioligand binding is the nonselective [ 3 H]5′- N -ethylcarboxamidoadenosine ([ 3 H]NECA). Based on a recently characterized series of 2-substituted adenosine receptor agonists we developed a tritiated selective A 3 radioligand with high affinity. From this series 2-hexyn-1-yl- N 6 -methyladenosine (HEMADO) with a K i -value of 1.1 nM at the human A 3 subtype was chosen. HEMADO is 300-fold selective versus the A 1 subtype, and 1100-fold and more than 25,000-fold selective compared to the adenosine A 2A and A 2B receptors, respectively. The tritiated derivative [ 3 H]HEMADO exhibited the same affinity as the unlabeled precursor. In concentrations up to 10 nM no specific binding to adenosine A 1 , A 2A or A 2B receptors was observed confirming the high selectivity for adenosine A 3 receptors. Characterization of [ 3 H]HEMADO in radioligand binding studies revealed reversible binding to the human adenosine A 3 subtype. In saturation binding studies for the A 3 subtype a K D -value of 1.1 nM was determined. Nonspecific binding at a radioligand concentration of 1 nM amounted to 1–2% of total binding. Competition binding with a panel of adenosine receptor ligands clearly confirmed the correct A 3 pharmacology of the binding site labeled by [ 3 H]HEMADO. With [ 3 H]HEMADO we present a tritiated agonist with high affinity and A 3 -selectivity and very low nonspecific binding. [ 3 H]HEMADO is a useful tool for specific screening for A 3 receptor agonists and antagonists in improved radioligand binding assays with the human subtype.