Lumacaftor (VX-809)

3-(6-(1-(2, 2-difluorobenzo[d][1, 3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid

Dissolved in DMSO, final concentrations ca.0.1 mM

More specific and efficacious than previously reported CFTR defect drugs

VX-809 acts at the level of the ER to allow a fraction of the F508del-CFTR to adopt a properly folded form, to exit the ER and mobilize to the cell surface for normal functioning. In Fischer rat thyroid (FRT) cells expressing F508del-CFTR, VX-809 treatment significantly improves F508del-CFTR maturation by 7.1 fold with an EC50 of 0.1 uM, and enhances F508del-CFTR-mediated chloride transport by approximately 5 fold with EC50 of 0.5 uM, while VRT-768 has higher EC50 values of 7.9 uM and 16 uM, respectively. In HEK-293 cells expressing F508del-CFTR, VX-809 (3 uM) treatment increases F508del-CFTR exit from the ER by 6 fold, reaching levels comparable to 34% of CFTR. In primary human bronchial epithelial (HBE) cells with F508del-CFTR mutation, VX-809 increases CFTR maturation and enhances chloride secretion with EC50 of 350 nM and 81 nM, respectively, more efficacious than Corr-4a and VRT-325. F508del-CFTR corrected by VX-809 exhibits single-channel open probability of 0.39 similar to normal CFTR of 0.40. Unlike VX-770, VX-809 is not a CFTR potentiator, as acute addition of VX-809 has no effect on F508del-CFTR function. In contrast to VRT-325 and Corr-4a, VX-809 does not improve the processing of the normal or mutant forms of hERG or P-gp, as well as other disease-causing mislocalized proteins, including alpha1-antitrypsin Z mutant (E342K-alpha1-AT) or N370S-beta-glucosidase, suggesting that VX-809 is specific for CFTR. VX-809 in combination with VRT-325 or Corr-4a has additive effect on CFTR-mediated chloride transport in cultured F508del-HBE. [1]

F508del-CFTR maturation, 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 ug total protein is heated in Laemmli buffer with 5% beta-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.

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, , Xuehong Liu of Oregon Health, Science University, VX-809purchased from Selleck, VX809 inceases the expression of DF508 CFTR in oocytes. Oocytes injected with DF508 CFTR and b-adrenergic receptor RNAs were incubated at 17C for 3 days with and without 5 M VX809. The conductance at the reversal potential (gCl at Vm = Erev) was assayed using two-electrode-voltage clamp. Oocytes were placed in experimental chambers and perfused with standard Frog Ringer', s at room temperature where the conductance was monitored as a function of time. The conductance was near zero before stimulation using a cocktail containing 10 M isoproterenol and 1 M 3-isobutyl-1-methylxanthine (hatched bar). At steady state, conductance of oocytes treated with 5 M VX809 (x symbols in panel A and white bar in panel B) was significantly larger than that of untreated oocytes (+ symbols in panel A and black bar in panel B). Towards the end of the experiments, oocytes were exposed to 10 M CFTR-172 to verify that the conductance was due to CFTR chloride channels.

2 years -20CPowder, 2 weeks4Cin DMSO, 2 months-80Cin DMSO