Construction of drug screening cell model and application to new compounds inhibiting FITC-fibrinogen binding to CHO cells expressing human αIIbβ3

To identify potential candidates for antiplatelet drugs, human αIIbβ3 (GPIIb/IIIa) was expressed in Chinese hamster ovary (CHO) cells, which was validated by tetrapeptide RGDS (Arg-Gly-Asp-Ser) with IC50 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 αIIbβ3 based on the CHO cell model was measured by flow cytometry using GPIIb/IIIa assay, and the IC50 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 IC50 values of inhibiting ADP-induced human platelet aggregation. Particularly compound 4 possessed IC50 value of approximately 6.84 nM. Additionally, a complex model of αIIbβ3 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 β3 and Tyr166 of α2b by hydrogen bonds and the carboxyl group at side chain of Asp179 of α2b 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.