WGA-coated yttrium oxide beads enable an imaging-based adenosine 2a receptor binding scintillation proximity assay suitable for high throughput screening.
Adenosine receptors belong to the superfamily of G protein-coupled receptors and are involved in a variety of physiologic functions. Traditionally, binding assays to detect adenosine 2a (A2a) antagonists and agonists have used filtration methods that are cumbersome to run and not amenable to HTS. We developed scintillation proximity assays (SPA trade mark ) utilizing HEK293 RBHA2AM cell membranes, either wheat germ agglutinin (WGA)-coated yttrium silicate (YSi) or red-shifted yttrium oxide (YO) beads and the A2a-selective radioligand [(3)H]SCH 58261. Both beads gave windows (total binding/nonspecific binding) of >5 and K(d) values of 2-3 nM for the radioligand, in agreement with results obtained by filtration. In contrast, WGA-polyvinyltoluene as well as other bead types had windows of <3 and significant radioligand binding to the uncoated beads. A 384-well WGA-YO bead SPA was optimized utilizing a LEADseeker imaging system and an automated trituration process for dispensing the dense yttrium-based beads. Signals were stable after 4 h, and Z' values were 0.7-0.8. The LEADseeker imaging assay tolerated 2% dimethyl sulfoxide and generated IC(50) values of 3-5 nM for the A2a antagonist CGS 15943, comparable to that obtained by the filtration method. A number of adenosine and xanthine analogues were identified as hits in the Library of Pharmacologically Active Compounds (LOPAC). This imaging-based A2a SPA enables HTS and is a major improvement over the filtration method.
Bryant
Robert
R
Schering-Plough Research Institute, 20156 Galloping Hill Road, Kenilworth, NJ 07033, USA.
McGuinness
Debra
D
Turek-Etienne
Tammy
T
Guyer
Deborah
D
Yu
Liming
L
Howells
Leighton
L
Caravano
Joseph
J
Zhai
Ying
Y
Lachowicz
Jean
J
D058908:Adenosine A2 Receptor Agonists; D058917:Adenosine A2 Receptor Antagonists; D002460:Cell Line; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D011869:Radioligand Assay / Q000379:methods*; D043705:Receptor, Adenosine A2A / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000378:metabolism*; D015019:Yttrium / Q000378:metabolism*
D058908:Adenosine A2 Receptor Agonists; D058917:Adenosine A2 Receptor Antagonists; D043705:Receptor, Adenosine A2A; D014909:Wheat Germ Agglutinins; D015019:Yttrium