Biosensor-based affinities and binding kinetics of small molecule antagonists to the adenosine A(2A) receptor reconstituted in HDL like particles.
The options for investigating solubilised G protein-coupled receptors (GPCRs) by biophysical techniques have long been hampered by their instability. A thermostabilised adenosine A2A receptor expressed in insect cells, purified in detergent and reconstituted into high-density lipoprotein (HDL) particles was immobilised onto a Surface Plasmon Resonance sensor chip. This allowed measurement of affinities and kinetics for A2A antagonists with affinities ranging from 50 pM to almost 2 μM. Compared with other formats, reproduction of affinities, and dissociation and association rate constants are good, reasonable and poor respectively, indicating stabilised receptors in HDL particles are useful for investigating specific aspects of GPCR-ligand interactions.
Heptares Therapeutics, Biopark, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
Elena
E
Segala
Heptares Therapeutics, Biopark, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
James C
JC
Errey
Heptares Therapeutics, Biopark, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
Cédric
C
Fiez-Vandal
Heptares Therapeutics, Biopark, Welwyn Garden City, Hertfordshire AL7 3AX, UK.
Andrei
A
Zhukov
Heptares Therapeutics, Biopark, Welwyn Garden City, Hertfordshire AL7 3AX, UK. Electronic address: rob.cooke@heptares.com.
Robert M
RM
Cooke
D058917:Adenosine A2 Receptor Antagonists / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods; D043705:Receptor, Adenosine A2A / Q000235:genetics / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D061987:Sf9 Cells; D054852:Small Molecule Libraries / Q000378:metabolism* / Q000494:pharmacology; D018411:Spodoptera; D020349:Surface Plasmon Resonance / Q000379:methods
D058917:Adenosine A2 Receptor Antagonists; D055767:Immobilized Proteins; D008075:Lipoproteins, HDL; D043705:Receptor, Adenosine A2A; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D054852:Small Molecule Libraries
G protein-coupled receptor; Lipid disc; Residence time; Surface plasmon resonance