A2AR expressing cells labeled with Terbium

Tag-lite cells transiently expressing the A2A receptor labeled with Terbium for use in receptor binding applications. The cells are provided ready to use.

Fully functional cell line
Transient expression
Ready for binding

Tag-lite cells transiently expressing the A2A receptor labeled with Terbium for use in receptor binding applications. The cells are provided ready to use.

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Overview

One of four adenosine receptors found in the brain, A2A is a GPCR. A2A receptor is thought to play a role in acute and chronic neurodegenerative disorders such as cerebral ischemia or Parkinson's disease. A2A may also be involved in seizures and sleep disorders.

Cells expressing the A2A receptor are provided pre-labeled with Terbium, and can be used to conduct receptor binding studies on the aforementioned receptor.

Benefits

PRE-LABELED WITH TERBIUM
FROZEN CELLS, READY TO BE USED
CELLS/LIGAND VALIDATED FOR BINDING

Assay principle

Running your A2A receptor binding assay using Tag-lite is as easy as it can gets. Simply dispense 10 µL of labeled cells into each well, followed by 5 µL of labeled ligand and 5 µL of the compound you wish to test. Like all HTRF assays, Tag-lite assays do not require any washing steps. A diagram of the procedure to be followed is given on the right.

Diagram of A2A receptor binding assay using the Tag-lite protocol

Saturation binding (KD)

A saturation binding assay measures total and non-specific binding for increasing concentrations of ligand under equilibrium conditions. To perform the assay, the fluorescent ligand is titrated into a solution containing a fixed amount of labeled cells and then incubated to equilibrium. The HTRF ratio obtained from this titration is the total binding.

Diagram of a saturation binding assay using Tag-lite

Representative data obtained when running a saturation binding assay

Watch this video explaining how to run a saturation binding assay using Tag-lite.

Competitive binding (KI)

A competitive binding assay is performed to measure the dissociation constant, Ki. To perform the assay, the compound is titrated into a solution containing a fixed concentration of fluorescent ligand and a fixed amount of cells.

Diagram of a competitive binding assay using Tag-lite

Representative data obtained when running a competitive binding assay

Watch this video explaining how to run a competitive binding assay using Tag-lite.

Kd and Ki validation

Examples of data obtained using A2A labeled cells and their matching fluorescent ligand (L0058RED). ZM-241385 was used as reference ligand. Results may vary from one HTRF® compatible reader to another.

Representative saturation binding and competitive binding graph

Representative competitive binding graph

Compound profiling on Adenosine A1, A2A, A2B and A3 receptors

Adenosine receptors designated by four subtypes A1, A2A, A2B and A3 were shown to be promising pharmacological targets for the development of therapeutic compounds for various therapeutic areas such as cancer, neurodegenerative disease and COPD/Asthma. Thus, a wide range of compounds, and particularly antagonists have been largely studied.

The Tag-Lite® binding assay platform offers the complete adenosine receptors subset A1, A2A, A2B and A3 to characterize compounds in term of binding affinity (Ki) and selectivity profile.

The set of compounds have been profiled on A1, A2A, A2B and A3 receptors using their respective fluorescent ligands L0067RED, L0058RED, L0068RED and L0069GRE.The selectivity profiles of several compounds involved in different therapeutic areas were assessed with this Tag-Lite platform.

The table below summarizes the resulting Ki obtained for each receptor subtype for the full panel of compounds. The results are confirming the expected selectivity and the range of affinities for these reference compounds.

Compound	Pharmacological class	Compounds affinities (Ki - nM) for the different adenosine receptor subtypes
		A1	A2A	A2B	A3
XAC	Non selective antagonist	12	15	20	50
Theophylline	Non selective antagonist	4000	5000	18000	140000
DPCPX	A1 antagonist	7.6	170	145	800
Vipadenant	A2A antagonist	70	0.9	55	20000
Ciforadenant (CPI-444)	A2A antagonist	150	3.4	660	1600
Preladenant (SCH 420814)	A2A antagonist	> 20 000	5	> 20 000	>20 000
Istradefylline (KW-6002)	A2A antagonist	>10 000	40	>10 000	>10 000
ZM241385	A2A antagonist	260	0.7	40	750
AZD4635	A2A antagonist	900	15	660	> 10 000
MRS1706	A2B inverse agonist	150	325	30	> 300
MRS1220	A3 antagonist	325	37	> 500	1.6