Characterization of [H]LUF5834: A novel non-ribose high-affinity agonist radioligand for the adenosine A1 receptor

The adenosine A₁ receptor is a promising therapeutic target for neurological disorders such as cognition deficits and is involved in cardiovascular preconditioning. Classically adenosine receptor agonists were all derivatives of adenosine, and thought to require a D-ribose moiety. More recently, however, the discovery of non-adenosine agonists for the human adenosine A₁ receptor (hA₁R) has challenged this dogma (Beukers et al., 2004). In this study we characterize the tritiated form of one of these compounds, [³H]LUF5834, as the first non-ribose partial agonist radioligand with nanomolar affinity for the hA₁R. Due to its partial agonist efficacy, [³H]LUF5834 labeled both G protein-coupled and uncoupled receptors with a similar high affinity. Using [³H]LUF5834 we performed competition binding experiments to characterize a range of A₁R ligands varying in efficacy from the full agonist CPA to the inverse agonist DPCPX. Surprisingly, in the control condition both agonists and inverse agonists displayed biphasic isotherms. With the addition of 1 mM GTP the high affinity isotherm of agonists or the low affinity isotherm of inverse agonists was lost revealing the mechanism of action of such inverse agonists at the A₁R. Consequently, [³H]LUF5834 represents a novel high affinity radioligand for the A₁R and may prove a useful tool to provide estimates of inverse agonist efficacy at this receptor.