Development of new chromatographic tools based on A_(2A) adenosine receptor subtype for ligand characterization and screening by FAC-MS

摘要

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing A_(2A) adenosine receptor (A_(2A)AR) is firstly described. Cellular membranes from CHO cells stably transfected with human A_(2A)AR vector (A_(2A)(+)) and from the same cell line transfected with the corresponding empty vector (A_(2A)(-)) were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6 mm I.D. glass columns to create A_(2A)(+)-IAM and A_(2A)(-)-IAM stationary phases. Frontal chromatography experiments on both A 2A(+)-IAM and A_(2A)(-)-IAM columns demonstrated the presence of a low specific interaction with the receptor. However, immobilized A 2A retained its ability to specifically bind known ligands as demonstrated by the agreement of the calculated K d values with two different chromatographic protocols in comparison to previously reported data. In order to maximize the specific interaction, the same cellular membranes were immobilized on the inner surface of a silica capillary (40 cm∈×∈ 100 μm I.D.) by non-covalent interactions using the avidin-biotin coupling system to create two open tubular columns A_(2A)(+)-OT and A 2A(-)-OT. The open tubular system was characterized by ranking experiments for affinity studies in mixture useful for the selection of new potential candidates.