2-[2-[4-[2-[2-[1,3-Dihydro-1,1-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl] ethylamino]-5′-N-ethylcarboxamidoadenosine (FITC-APEC): A fluorescent ligand for A_2a-adenosine receptors

Abstract

The fluorescein conjugate, FITC-APEC (2-[2-[4-[2-[2-[1,3-dihydro-1,1-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl] phenyl]ethylamino]-5′-N-ethylcarboxamidoadenosine), is a novel ligand derived from a series of functionalized congeners that act as selective A_2a-adenosine receptor agonists. The binding of FITC-APEC to bovine striatal A_2a-adenosine receptors measured by fluorescence techniques was saturable and of a high affinity, with aB _max of 2.3±0.3 pmol/mg protein andK _D of 57±2 nM. TheK _D value estimated by fluorescence was consistent with theK _i (11±0.3 nM) obtained by competition studies with [³H]CGS 21680. Additionally, theB _max value found by FITC-APEC measurement was in agreement withB _max values obtained using radioligand binding. FITC-APEC exhibited rapid and reversible binding to bovine striatum. The potencies of chemically diverse A_2a-adenosine receptor ligands estimated by inhibition of FITC-APEC binding were in good agreement with their potencies determined using radioligand binding techniques (r=0.97,P=0.0003). FITC-APEC binding was not altered by purine derivatives that do not recognize A_2a-adenosine receptors. These findings demonstrate that the novel fluorescent ligand FITC-APEC can be used in the quantitative characterization of ligand binding to A_2a-adenosine receptors.