Lumacaftor (VX-809)
VX-809 (Lumacaftor, VRT 826809) acts to correct CFTR mutations common in cystic fibrosis by increasing mutant CFTR (F508del-CFTR) maturation,EC50 of 0.1 μM in fisher rat thyroid cells.
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Proc Natl Acad Sci U S A,2011 Nov 15;108(46):18843-8.
Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.[Pubmed:
21976485]
Fischer rat thyroid (FRT) cells stably expressing F508del-CFTR are treated with increasing concentrations of VX-809 for 48 hours. After incubation, cells are harvested in ice-cold D-PBS solution (without calcium and magnesium) and pelleted at 1,000 × g at 4 °C. Cell pellets are lysed in 1% Nonidet P-40, 0.5% sodium deoxycholate, 200 mM NaCl, 10 mM Tris, pH 7.8, and 1 mM EDTA plus protease inhibitor mixture (1:250) for 30 minutes on ice. Lysates are spun for 10 minutes at 10,000 × g at 4 °C to pellet nuclei and insoluble material. Approximately 12 μg total protein is heated in Laemmli buffer with 5% β-mercaptoethanol at 37 °C for 5 minutes and loaded onto a 3% to 8% Tris-acetate gel. The gel is transferred to nitrocellulose and processed for Western blotting by using monoclonal CFTR antibody or polyclonal to GAPDH. Blots are developed by enhanced chemiluminescence. Quantification of the relative amounts of bands C and GAPDH is performed by using NIH ImageJ analysis of scanned films.
Proc Natl Acad Sci U S A,2011 Nov 15;108(46):18843-8.
Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.[Pubmed:
21976485]
Cell lines:FRT (CFTR or F508del-CFTR), HEK-293 (CFTT or F508del-CFTR) , and HBE cells
Concentrations: Dissolved in DMSO, final concentrations ~0.1 mM
Incubation Time: 24 or 48 hours
Method:
Cells are exposed to various concentrations of VX-809 for 24 or 48 hours. Ussing chamber techniques are used to record the transepithelial current (IT) resulting from CFTR-mediated chloride transport. The single-channel activity of CFTR is measured by using excised inside-out membrane patch recordings. Immunoblot techniques using themonoclonal CFTR antibody are used to measure CFTR maturation in FRT, HEK-293, or HBE cells expressing CFTR or F508del-CFTR.
