SNAP tag A1 AdenosineR cloned plasmid
Tag-lite plasmid cloned with a SNAP tag A1 Adenosine receptor
Tag-lite cells transiently expressing the A1 receptor labeled with Terbium for use in receptor binding applications. The cells are provided ready to use.
A1 receptor is one of the four adenosine receptor family (including A2A, A2B and A3 receptors). It has vital role in cardiac and renal processes. It regulates neurotransmitters in the brain, such as dopamine and glutamate. Thus, it is considered as an interesting target in treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.
Cells expressing the A1 receptor are provided pre-labeled with Terbium, and can be used to conduct receptor binding studies on the aforementioned receptor.
Running your A1 receptor binding assay using Tag-lite is as easy as it can get. Simply dispense 10 µL of labeled cells (C1TT1A1) into each well, followed by 5 µL of labeled ligand (L0067RED) 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.
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.
Examples of data obtained using Adenosine A1 receptor labeled cells and their matching fluorescent ligand (L0067RED). DPCPX (8-Cyclopentyl-1,3-dipropylxanthine) was used for Ki determination in the competitive binding assay. Results may vary from one HTRF® compatible reader to another.
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|
|XAC||Non selective antagonist||12||15||20||50|
|Theophylline||Non selective antagonist||4000||5000||18000||140000|
|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|
|AZD4635||A2A antagonist||900||15||660||> 10 000|
|MRS1706||A2B inverse agonist||150||325||30||> 300|
|MRS1220||A3 antagonist||325||37||> 500||1.6|
The graphs show the pattern of the dose-response curves obtained for five compounds displaying different selectivity profile.
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