Figure 2
Cell-based and electrophysiological assays demonstrate the ability of glafenine derivatives to correct F508del-CFTR. (A) Cell-based HTS assay measuring surface F508del-CFTR in BHK cells after 24 h of treatment with glafenine and its analogs at 10 μM. Glafenine (compound 37) is marked in green, most analogs in black and the best responding analogs in red (n = 4). (B) FMP assay (FMP) that monitors membrane depolarization induced by forskolin + genistein when cells are pretreated for 24 h with glafenine and its derivatives (10 μM) performed in BHK cells expressing F508del-CFTR (n = 4). (C) F508del-CFTR functional expression in well-differentiated CFBE41o- cell epithelial cells determined from the increase in Isc stimulated by acute addition of forskolin + genistein (ΔIsc). The basolateral membrane was permeabilized using nystatin, and an apical-to-basolateral chloride gradient was imposed. All compounds tested at 10 µM for 24 h (n = 4). (D) Immunoblot of F508del-CFTR in BHK cells after 24 h of treatment with VX-809 (1 µM) glafenine and selected derivatives (compounds 49, 53, 54, 55, 56) (10 μM) and with BHK cells expressing wild-type CFTR (n = 4). (E) Relative intensity of bands B and band C in each lane in (D). Data in panels E is presented as the means ± SEM, n = 4.
