Validation of a Na+-shift binding assay for estimation of the intrinsic efficacy of ligands at the A2Aadenosine receptor.
BACKGROUND
Determination of the intrinsic efficacy of ligands at the A2Areceptor is important for selecting drug candidates, e.g. in the case of inflammatory diseases where agonists are searched for or in Parkinson disease (antagonists).


METHODS
Three functional binding assays were compared with up to seven ligands with different efficacies: the GTP-shift method based on the decrease of affinity observed with agonists when GTP is added to the competition binding assay; the Kiratio method based on the different affinity states of the receptor when using an agonist or antagonist radioligand and the Na+-shift assay based on the difference of affinity of agonists when tested in a medium containing a divalent cation (50mM MgCl2) favoring the G protein coupled agonist-receptor complex or sodium (100mM NaCl) as negative allosteric modulator.


RESULTS
The Na+-shift assay proposed herein successfully discriminated the full agonists CGS21680, NECA and adenosine (IC50ratio=13-14) from the weak inverse agonists ZM241385 and IBMX (IC50ratio=0.85) and the partial agonists LUF5834 and regadenoson (IC50ratios equal to 3 and 10, respectively).


CONCLUSIONS
We conclude that the Na+-shift assay proposed herein for the A2Areceptors has been validated and represents a rapid, economic and efficient functional binding assay to be used in a drug development program for early estimation of the intrinsic efficacy of hits.
Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil. Electronic address: fnoel@pharma.ufrj.br.
François
F
Noël
Laboratório de Farmacologia Bioquímica e Molecular, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil.
Fernando M
FM
do Monte
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D058908:Adenosine A2 Receptor Agonists / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D008024:Ligands; D008297:Male; D010627:Phenethylamines / Q000378:metabolism; D011687:Purines / Q000378:metabolism; D011720:Pyrazoles / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D043705:Receptor, Adenosine A2A / Q000378:metabolism*; D015203:Reproducibility of Results; D012964:Sodium / Q000378:metabolism*; D014227:Triazines / Q000378:metabolism; D014230:Triazoles / Q000378:metabolism
D058908:Adenosine A2 Receptor Agonists; D008024:Ligands; D010627:Phenethylamines; D011687:Purines; D011720:Pyrazoles; D043705:Receptor, Adenosine A2A; D014227:Triazines; D014230:Triazoles; C097270:ZM 241385; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; C430916:regadenoson; D012964:Sodium; D000241:Adenosine
1,4-bis-[2-(5-phenyloxazolyl)]-benzene (POPOP); 2,5-diphenyloxazole (POP); 2-Amino-4-(4-hydroxyphenyl)-6-[(1H-imidazol-2-ylmethyl)thio]-3,5-pyridinecarbonitrile (LUF5834). 5′-(N-ethylcarboxamido)adenosine (NECA); 3-Isobutyl-1-methylxanthine (IBMX); 4-(−2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM241385); 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS21680); A(2A); Adenosine; Binding; Efficacy; GTP-shift; Na(+); Screening; guanosine 5′-triphosphate (GTP)