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Validation of a Na +-shift binding assay for estimation of the intrinsic efficacy of ligands at the A 2A adenosine receptor

Validation of a Na +-shift binding assay for estimation of the intrinsic efficacy of ligands at the A 2A adenosine receptor

François Noël, Fernando M do Monte

J Pharmacol Toxicol Methods. Mar-Apr 2017;84:51-56.

PMID: 27810394

Abstract:

Introduction:
Determination of the intrinsic efficacy of ligands at the A2A receptor 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 Ki ratio 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 (IC50 ratio=13-14) from the weak inverse agonists ZM241385 and IBMX (IC50 ratio=0.85) and the partial agonists LUF5834 and regadenoson (IC50 ratios equal to 3 and 10, respectively).
Discussion:
We conclude that the Na+-shift assay proposed herein for the A2A receptors 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP333962917	LUF5834		333962-91-7	Price

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