title: CCR4 expressing cells cultured adherently on a capillary wall and formaldehyde fixed as the stationary phase for ligand screening by ACE.
abstract: We have developed an on-line screening method for CC chemokine receptor 4 (CCR4) ligands, in which the whole cells expressed with CCR4 were cultured adherently and immobilized on the inner wall of the capillary as the stationary phase for the first time. Moreover, in this method it is unnecessary to isolate and purify the target receptors from cell membranes. Therefore, it is possible to almost completely preserve the native conformation of the target receptors. The binding activities of the immobilized CCR4 did not change. A known antagonist of CCR4, compound A, was employed to validate the bioactivity of the cell layer and stability of this method. The intraday, interday, and batch-to-batch reproducibilities were investigated (RSD ≤ 13.9%). Nonlinear chromatography was used to calculate the binding constant between the compound A and CCR4 (6.4 × 10(4)/M, RSD = 4.96%). Using this method, the qualitative and quantitative characterizations of 23 computer-aided drug design compounds were achieved and the kinetic parameters (K, k(a), k(d), and k') were obtained by nonlinear chromatography. Three active compounds were screened out, which also showed activity in chemotaxis inhibition assay. The experimental results show that this method is simple, sensitive, and efficient for drug screening. Moreover, it offered a novel way to detect the nonspecific interactions between ligands and cell membrane.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs and Department of Pharmaceutical Analysis, School of Pharmaceutical Sciences, Peking University, Beijing, PR China.
authors.forename: Pazilaiti
authors.initials: P
authors.lastname: Yakufu
authors.forename: Hui
authors.initials: H
authors.lastname: Qi
authors.forename: Meina
authors.initials: M
authors.lastname: Li
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Ling
authors.forename: Wenjing
authors.initials: W
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
mesh_terms: D002448:Cell Adhesion / Q000502:physiology; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D018914:Cells, Immobilized / Q000166:cytology* / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D054398:Receptors, CCR4 / Q000096:biosynthesis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: C515354:CCR4 protein, human; D008024:Ligands; D054398:Receptors, CCR4; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins