Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry.
A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17+] and from the same cell line transfected with the corresponding empty vector [GPR17(-)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)-IAM and GPR17(-)-IAM stationary phases. Frontal chromatography experiments on both GPR17(+)-IAM and GPR17(-)-IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)-IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants (K(d)) of three ligands-the antagonist cangrelor (formerly AR-C69931MX, a P2Y(12)/P2Y(13) antagonist), MRS2179 (a P2Y(1) receptor antagonist), and the agonist UDP-all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K(d) values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)-IAM in ranking affinity studies for the selection of new potential candidates.
Department of Pharmaceutical Chemistry, University of Pavia, 27100 Pavia, Italy.
Caterina
C
Temporini
Stefania
S
Ceruti
Enrica
E
Calleri
Silvia
S
Ferrario
Ruin
R
Moaddel
Maria P
MP
Abbracchio
Gabriella
G
Massolini
D001665:Binding Sites; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods*; D008567:Membranes, Artificial; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
C121670:GPR17 protein, human; D055767:Immobilized Proteins; D008024:Ligands; D008567:Membranes, Artificial; D043562:Receptors, G-Protein-Coupled