title: Construction of drug screening cell model and application to new compounds inhibiting FITC-fibrinogen binding to CHO cells expressing human alphaIIbbeta3.
abstract: To identify potential candidates for antiplatelet drugs, human alphaIIbbeta3 (GPIIb/IIIa) was expressed in Chinese hamster ovary (CHO) cells, which was validated by tetrapeptide RGDS (Arg-Gly-Asp-Ser) with IC(50) of 0.057 mM, supported by Basani's results [Basani, R. B., French, D. L., Vilaire, G., Brown, D. L., Chen, F., Coller, B. S., Derrick, J. M., Gartner, T. K., Bennett, J. S., Poncz, M., 2000. A naturally occurring mutation near the amino terminus of alpha IIb defines a new region involved in ligand binding to alpha IIbbeta 3. Blood 95, 180-188]. The ability of 2-(4-substituted-piperazin-1-ylacetyl)-1,2,3,4-tetrahydroisoquinoline derivatives to inhibit fibrinogen binding to alphaIIbbeta3 based on the CHO cell model was measured by flow cytometry using GPIIb/IIIa assay, and the IC(50) values of compounds 1-6 were 0.166, 0.037, 0.311, 0.025, 0.034, and 0.184 mM, respectively. Our research results indicated that the compounds with phenylsulfonyl (compounds 1 and 2) and benzoyl groups (compounds 4 and 5) at position 4 of piperazine showed higher IC(50) values of inhibiting ADP-induced human platelet aggregation. Particularly compound 4 possessed IC(50) value of approximately 6.84 nM. Additionally, a complex model of alphaIIbbeta3 with compound 4 revealed that the pharmacophore of compound 4, including m-nitro group of 4-benzene-piperazine, the nitrogen atom in the piperazine group, and 2-nitrogen of 1,2,3,4-tetrahydroisoquinoline nucleus, interacted with the hydroxyl groups of Thr125 of beta3 and Tyr166 of alpha2b by hydrogen bonds and the carboxyl group at side chain of Asp179 of alpha2b in the fashion of electrostatic interaction. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays showed that compounds 4 and 5 possess potential anti-cancer activities, suggesting a potential role of integrin-guided signal pathway in cancer therapy. Further evaluation is under investigation.
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, College of Life Sciences, Nanjing University, Nanjing, PR China. luckyjyj@sina.com.cn
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Jia
authors.initials: J
authors.lastname: Yao
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.forename: Xiao-Ning
authors.initials: XN
authors.lastname: Wang
authors.forename: Tong-Yang
authors.initials: TY
authors.lastname: Zhu
authors.forename: Li-Li
authors.initials: LL
authors.lastname: Chen
authors.forename: Ping
authors.initials: P
authors.lastname: Chu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000378:metabolism*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D020128:Inhibitory Concentration 50; D008297:Male; D051379:Mice; D008954:Models, Biological; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000494:pharmacology* / Q000633:toxicity; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000032:analysis / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D044005:Tetrahydroisoquinolines / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity
chemical_list: D000906:Antibodies; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D044005:Tetrahydroisoquinolines; D005340:Fibrinogen; D016650:Fluorescein-5-isothiocyanate