Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry |
| |
Authors: | Temporini Caterina Ceruti Stefania Calleri Enrica Ferrario Silvia Moaddel Ruin Abbracchio Maria P Massolini Gabriella |
| |
Affiliation: | a Department of Pharmaceutical Chemistry, University of Pavia, 27100 Pavia, Italy b Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological Sciences, University of Milan, 20133 Milan, Italy c Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA |
| |
Abstract: | 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 (Kd) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y12/P2Y13 antagonist), MRS2179 (a P2Y1 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 Kd 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. |
| |
Keywords: | FAC-MS GPR17 receptor Binding assay Immobilized artificial membranes (IAM) |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|