A3 Human Adenosine GPCR GTPgammaS Agonist & Antagonist LeadHunter Assay - TW

Procedure Summary:

Human recombinant adenosine A3 receptors expressed in CHO-K1 cells are used. Test compound and/or vehicle is pre-incubated with 0.02 mg/ml membranes‡ and 1 µM GDP in modified HEPES pH 7.4 buffer for 20 minutes. SPA beads are then added for another 60 minutes at 30°C. The reaction is initiated by addition of 0.3 nM [35S]GTPgS for another 30 minute incubation period. Test compound-induced increase of [35S]GTPgS binding by 50 percent or more (≥50%) relative to the 3 µM 2-CI-IB-MECA response indicates possible A3 receptor agonist activity. Test compound-induced inhibition of 0.1 µM 2-CI-IB-MECA-induced increase of [35S]GTPgS binding response by 50 percent or more (≥50%) indicates receptor antagonist activity. Compounds are screened at 10, 1, 0.1, 0.01 and 0.001 µM. Note: ‡Membrane protein may change from lot to lot, the concentration used will be adjusted if necessary.

Incubation:

30 min at 30°C

Control Inhibitor:

MRS 1220

Control Activator:

Cl-IB-MECA

Criteria For Significance:

≥ 50% Increase in bound [35S]GTPgammaS relative to 2-Cl-IB-MECA response, ≥ 50% Inhibition of 2-Cl-IB-MECA-induced bound [35S]GTPgammaS

Test Concentration / Dose:

Compounds are screened at 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0.003 μM

Test Sample Requirements:

75 μl of 10 mM stock or 1 mg (pre-weighed) powder (assuming FW ≦500 and purity 100%)

Minimum Order Quantity:

16