ABCMdb

PMID: 24737137

Phuan PW, Veit G, Tan J, Roldan A, Finkbeiner WE, Lukacs GL, Verkman AS
Synergy-based small-molecule screen using a human lung epithelial cell line yields DeltaF508-CFTR correctors that augment VX-809 maximal efficacy.
Mol Pharmacol. 2014 Jul;86(1):42-51. doi: 10.1124/mol.114.092478. Epub 2014 Apr 15., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Arg1070Trp The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2- difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Login to comment 4 ABCC7 p.Arg1070Trp We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. Login to comment 5 ABCC7 p.Arg1070Trp A biochemical screen for DF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o2 ) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Login to comment 25 ABCC7 p.Gly551Asp The CFTR "potentiator" VX-770 [N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide; Ivacaftor], which corrects defective channel gating of some CFTR mutants, has been approved for CF therapy caused by the defective channel gating but unimpaired cellular processing and plasma membrane targeting of the G551D-CFTR mutation (Van Goor et al., 2009; Accurso et al., 2010). Login to comment 35 ABCC7 p.Arg1070Trp Therefore, novel corrector molecules targeting either the NBD1 and/or its interface defects should be preferentially identified by screening in the background of VX-809 or the interface-stabilizing mutation R1070W. Login to comment 43 ABCC7 p.Arg1070Trp ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg The cloning and characterization of 3HA-tagged variants of ƊF508-CFTR, R1070W- ƊF508-CFTR, and 3S-ƊF508-CFTR (containing the F494N, Q637R, and F429S NBD1 suppressor mutations) were described (Okiyoneda et al., 2013). Login to comment 49 ABCC8 p.Ile152Leu A549 lung epithelial cells (ATCC CCL-185; American Type Culture Collection, Manassas, VA) stably expressing ƊF508-CFTR (Pedemonte et al., 2010) were provided by Dr. Luis Galietta (Genoa, Italy) and cotransfected with halide-sensitive yellow fluorescent protein (YFP)-H148Q/I152L/F46L (Galietta et al., 2001). Login to comment 58 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp In one set of assays, R1070W-ƊF508-CFTR-HRP (R1070W-HRP)-expressing CFBE41o2 cells were incubated with 100 ml medium containing 25 mM test compounds and 0.5 mg/ml doxycycline for 24 hours at 37&#b0;C. Login to comment 81 ABCC7 p.Phe494Asn Isolation of recombinant human NBD1 containing a single suppressor mutation (1S; F494N) and melting temperature measurement were performed as described (Rabeh et al., 2012). Login to comment 87 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp A second screen ("R1070W screen") (Fig. 1B) used CFBE41o2 cells transfected with DF508-CFTR-HRP containing a R1070W mutation (R1070W-HRP CFBE41o2 ). Login to comment 90 ABCC7 p.Arg1070Trp For the ƊF508 screen, cells were incubated with test compounds (at 25 mM) together with 2 mM VX-809; for the R1070W screen, cells were incubated with test compounds (at 25 mM) alone. Login to comment 96 ABCC7 p.Arg1070Trp Similarly, with the R1070W-HRP CFBE41o2 cells, VX-809 increased the signal maximally to approximately 220 a.u. over the DMSO control baseline of approximately 85 a.u., representing an approximately 2.5-fold signal increase (bar graphs in Fig. 1). Login to comment 98 ABCC7 p.Arg1070Trp Low-temperature rescue (27&#b0;C) of ƊF508-CFTR increased the HRP luminescence signal by approximately 2-fold (compared with 37&#b0;C) in ƊF508-HRP CFBE41o2 cells and approximately 3-fold in R1070W-HRP CFBE41o2 cells. Login to comment 100 ABCC7 p.Arg1070Trp EC50 values were 30 and 78 nM in the low temperature-rescued ƊF508-HRP and R1070W-HRP CFBE41o2 cells, respectively. Login to comment 101 ABCC7 p.Arg1070Trp ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Preferential correction of DF508-CFTR-3HA with the NBD1 stabilizing 3S mutations (F494N, Q637R, and F429S) compared with CFTR carrying the R1070W interface-stabilizing mutation has been taken as evidence that VX-809 preferentially stabilizes the interface between NBD1 and MSDs but not the NBD1 folding defect CFTR (Okiyoneda et al., 2013). Login to comment 103 ABCC7 p.Arg1070Trp The relative insensitivity of R1070W-HRP to VX-809 was used to identify correctors that act in synergy with VX-809. Login to comment 105 ABCC7 p.Arg1070Trp A total of 110,240 drug-like small synthetic molecules were tested in the ƊF508 and R1070W screens. Login to comment 108 ABCC7 p.Arg1070Trp For the R1070W screen, 25 active compounds were identified based on a .50% increase in the luminescence signal over that of DMSO. Login to comment 109 ABCC7 p.Arg1070Trp After retesting, nine compounds, grouped into six classes, were confirmed from the R1070W screen. Figure 2D shows structures of the six most active compounds (A-01, B-01, C-01, D-01, E-01, and F-01). Login to comment 110 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp Because different small-molecule collections were used for the ƊF508 and R1070W screens, we cross-tested all active correctors in both the ƊF508-HRP and R1070W-HRP CFBE41o2 cell lines (Supplemental Fig. 2). Login to comment 112 ABCC7 p.Arg1070Trp However, compounds D-01, E-01, and F-01, discovered from the R1070W screen, were not active in ƊF508-HRP CFBE41o2 cells. Login to comment 113 ABCC7 p.Arg1070Trp J-01, discovered from the ƊF508 screen, was not active in R1070W-HRP CFBE41o2 cells. Login to comment 117 ABCC7 p.Arg1070Trp We also measured the concentration-dependent activities of A-01, B-01, D-01, H-01, and K-01 (Fig. 3B) in R1070W-CFBE41o2 cells and found that D-01 is the most potent corrector, with an EC50 value of approximately 1.2 mM and a maximal signal that is approximately 65% of that produced by 2 mM VX-809. Login to comment 126 ABCC7 p.Arg1070Trp Similar compound potency and efficacy were found for class D analogs in R1070W-HRP CFBE41o2 cells (Fig. 4C). Login to comment 141 ABCC7 p.Arg1070Trp (B) Screening assay for R1070W-ƊF508-CFTR-HRP CFBE41o cells (top) showing incubation with 25 mM test compounds for 24 hours at 37&#b0;C. Login to comment 163 ABCC7 p.Arg1070Trp (C) Summary of primary findings of the R1070W-DF508-CFTR screen. Login to comment 170 ABCC7 p.Arg1070Trp To investigate whether any of the correctors identified here may target the NBD2 interface, their synergy with C4 was evaluated in R1070W-HRP CFBE41o2 cells using the HRP luminescence assay. Login to comment 191 ABCC7 p.Arg1070Trp (B) Concentration-dependence data of A-01, B-01, D-01, H-01, and K-01 in R1070W-ƊF508-CFTR-HRP CFBE41o2 cells (S.E., n = 3). Login to comment 197 ABCC7 p.Arg1070Trp (A) Concentration-dependence data of class D analogs in R1070W-ƊF508-CFTR-HRP CFBE41o2 cells (S.E., n = 3). Login to comment 212 ABCC7 p.Arg1070Trp ABCC7 p.Arg1070Trp A second screen was done in cells expressing R1070W-ƊF508-CFTR (in the absence of VX-809), since in the background of genetically stabilized ƊF508-NBD1, the R1070W mutation was necessary and sufficient to restore robust CFTR domain assembly and cell surface expression (Thibodeau et al., 2010; Mendoza et al., 2012; Rabeh et al., 2012). Login to comment 213 ABCC7 p.Arg1070Trp Screening was performed using a human lung epithelium-derived cell line (CFBE41o2 ) that was stably transfected with HRP-tagged ƊF508-CFTR or R1070W-ƊF508-CFTR. Login to comment 227 ABCC7 p.Arg1070Trp (C) Relative effect of corrector 4 (10 mM, 24 hours, 37&#b0;C) on the PM density of R1070W-HRP CFBE41o2 cells treated with A-01, B-01, C-01, D-01, H-01, J-01, K-01, or VX-809 at the indicated concentrations. Login to comment