ABCC8 p.Ile152Leu
| Predicted by SNAP2: | A: N (87%), C: N (82%), D: D (63%), E: D (59%), F: N (93%), G: D (53%), H: N (53%), K: D (53%), L: N (93%), M: N (87%), N: D (53%), P: D (53%), Q: N (53%), R: N (53%), S: N (72%), T: N (72%), V: N (97%), W: N (66%), Y: N (72%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D, |
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[hide] CFTR chloride channel drug discovery--inhibitors a... Curr Pharm Des. 2006;12(18):2235-47. Verkman AS, Lukacs GL, Galietta LJ
CFTR chloride channel drug discovery--inhibitors as antidiarrheals and activators for therapy of cystic fibrosis.
Curr Pharm Des. 2006;12(18):2235-47., [PMID:16787252]
Abstract [show]
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cAMP-activated chloride channel expressed in epithelia in the lung, intestine, pancreas, testis and other tissues, where it facilitates transepithelial fluid transport. In the intestine CFTR provides the major route for chloride secretion in certain diarrheas. Mutations in CFTR cause the hereditary disease cystic fibrosis, where chronic lung infection and deterioration in lung function cause early death. CFTR is a well-validated targeted for development of inhibitors for therapy of secretory diarrheas and activators for therapy in cystic fibrosis. Our lab has identified and optimized small molecule inhibitors of CFTR, as well as activators of DeltaF508-CFTR, the most common mutant CFTR causing cystic fibrosis. High-throughput screening of small molecule collections utilizing a cell-based fluorescence assay of halide transport yielded thiazolidinone and glycine hydrazide CFTR inhibitors that block enterotoxin-mediated secretory diarrhea in rodent models, including a class of non-absorbable inhibitors that target the CFTR pore at its external entrance. Benzothiophene, phenylglycine and sulfonamide potentiators were identified that correct the defective gating of DeltaF508-CFTR chloride channels, and other small molecules that correct its defective cellular processing. Small molecule modulators of CFTR function may be useful in the treatment of cystic fibrosis, secretory diarrhea and polycystic kidney disease.
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No. Sentence Comment
209 To identify correctors of defective ∆F508-CFTR gating ('potentiators`), we designed a high-throughput screen using stably transfected FRT cells that co-express ∆F508-CFTR and the halide sensitive indicator YFP-H148Q/I152L [16] (Fig. 6A,B).
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ABCC8 p.Ile152Leu 16787252:209:230
status: NEW232 Cells co-expressing ∆F508-CFTR and the halide-sensitive fluorescent protein YFP-H148Q/I152L were grown for 24 h at 27 o C (to allow plasma membrane ∆F508-CFTR expression).
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ABCC8 p.Ile152Leu 16787252:232:93
status: NEW233 After washing, test compounds (2.5 µM) and forskolin (20 µM) were added, and I- influx was assayed from the time course of YFP-H148Q/I152L fluorescence after adding Ito the external solution. B. Representative time courses of YFP-H148Q/I152L fluorescence in control wells (saline, negative control; 50 µM genistein, positive control) with examples of inactive and active test compounds.
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ABCC8 p.Ile152Leu 16787252:233:143
status: NEWX
ABCC8 p.Ile152Leu 16787252:233:246
status: NEW[hide] Cyanoquinolines with independent corrector and pot... Mol Pharmacol. 2011 Oct;80(4):683-93. Epub 2011 Jul 5. Phuan PW, Yang B, Knapp JM, Wood AB, Lukacs GL, Kurth MJ, Verkman AS
Cyanoquinolines with independent corrector and potentiator activities restore {delta}phe508-cystic fibrosis transmembrane conductance regulator chloride channel function in cystic fibrosis.
Mol Pharmacol. 2011 Oct;80(4):683-93. Epub 2011 Jul 5., [PMID:21730204]
Abstract [show]
The DeltaPhe508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein impairs its folding, stability, and chloride channel gating. Although small molecules that separately correct defective DeltaPhe508-CFTR folding/cellular processing ("correctors") or chloride channel gating ("potentiators") have been discovered and are in clinical trials, single compounds with bona fide dual corrector and potentiator activities have not been identified. Here, screening of approximately 110,000 small molecules not tested previously revealed a cyanoquinoline class of compounds with independent corrector and potentiator activities (termed CoPo). Analysis of 180 CoPo analogs revealed 6 compounds with dual corrector and potentiator activities and 13 compounds with only potentiator activity. N-(2-((3-Cyano-5,7-dimethylquinolin-2-yl)amino)ethyl)-3-methoxybenzamide (CoPo-22), which was synthesized in six steps in 52% overall yield, had low micromolar EC(50) for DeltaPhe508-CFTR corrector and potentiator activities by short-circuit current assay. Maximal corrector and potentiator activities were comparable with those conferred by the bithiazole Corr-4a and the flavone genistein, respectively. CoPo-22 also activated wild-type and G551D CFTR chloride conductance within minutes in a forskolin-dependent manner. Compounds with dual corrector and potentiator activities may be useful for single-drug treatment of cystic fibrosis caused by DeltaPhe508 mutation.
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No. Sentence Comment
38 Each of the CFTR-expressing cell lines (and the nontransfected parental cells) was also transfected with halide-sensitive green fluorescent protein YFP-H148Q/I152L/F46L.
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ABCC8 p.Ile152Leu 21730204:38:158
status: NEW[hide] Azithromycin fails to reduce inflammation in cysti... Eur J Pharmacol. 2012 Jan 5;674(1):1-6. doi: 10.1016/j.ejphar.2011.10.027. Epub 2011 Oct 26. Saint-Criq V, Ruffin M, Rebeyrol C, Guillot L, Jacquot J, Clement A, Tabary O
Azithromycin fails to reduce inflammation in cystic fibrosis airway epithelial cells.
Eur J Pharmacol. 2012 Jan 5;674(1):1-6. doi: 10.1016/j.ejphar.2011.10.027. Epub 2011 Oct 26., [PMID:22056837]
Abstract [show]
Cystic fibrosis is a hereditary disease caused by a mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene that encodes a chloride (Cl(-)) channel. Cystic fibrosis pulmonary pathophysiology is characterised by chronic inflammation and bacterial infections. Azithromycin, a macrolide antibiotic, has shown promising anti-inflammatory properties in some inflammatory pulmonary diseases. Moreover, all clinical studies have presented an improvement of the respiratory condition of cystic fibrosis patients, but the molecular and cellular mechanisms remain unknown. The aim of this study was to investigate, in bronchial epithelial cells, the effects of azithromycin on inflammatory pathways involved in cystic fibrosis. We have analysed the effects of azithromycin on cystic fibrosis and non-cystic fibrosis bronchial epithelial cell lines but also in non-immortalized non-cystic fibrosis human glandular cells. To create an inflammatory context, cells were treated with Tumor Necrosis Factor (TNF)-alpha or Interleukin (IL)1-beta. Activation of the NF-kappaB pathway was investigated by luciferase assay, western blotting, and by Forster Resonance Energy Transfer imaging, allowing the detection of the interaction between the transcription factor and its inhibitor in live cells. In all conditions tested, azithromycin did not have an anti-inflammatory effect on the cystic fibrosis human bronchial epithelial cells and on CFTR-inhibited primary human bronchial glandular cells. More, our data showed no effect of azithromycin on IL-1beta- or TNF-alpha-induced IL-8 secretion and NF-kappaB pathway activation. Taken together, these data show that azithromycin is unable to decrease in vitro inflammation in cystic fibrosis cells from airways.
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No. Sentence Comment
51 Iodide rather than chloride is used because of strong YFP-H148Q/I152L quenching by iodide, and because CFTR is permeable to iodide (Verkman and Galietta, 2009).
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ABCC8 p.Ile152Leu 22056837:51:64
status: NEW107 CFTR activities were determined by CFTR halide conductance in cells expressing an YFP-H148Q/ I152L/F46L indicator stimulated by CFTR agonist mixture of CFTR.
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ABCC8 p.Ile152Leu 22056837:107:93
status: NEW[hide] Proinflammatory cytokine secretion is suppressed b... Mol Biol Cell. 2012 Nov;23(21):4188-202. doi: 10.1091/mbc.E12-06-0424. Epub 2012 Sep 12. Veit G, Bossard F, Goepp J, Verkman AS, Galietta LJ, Hanrahan JW, Lukacs GL
Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia.
Mol Biol Cell. 2012 Nov;23(21):4188-202. doi: 10.1091/mbc.E12-06-0424. Epub 2012 Sep 12., [PMID:22973054]
Abstract [show]
Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air-liquid but not liquid-liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr(F508/F508) immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor-mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.
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No. Sentence Comment
187 Consistent results were obtained by measuring the halide conductance with cytosolic YFP-H148Q/I152L/F46L, a halide-sensitive fluorescent protein (Ferrera et al., 2009; Figure 7, E and F, and Supplemental Figure S5I).
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ABCC8 p.Ile152Leu 22973054:187:94
status: NEW355 Halide permeability determination of the PM The halide sensor YFP-F46L/H148Q/I152L (Namkung et al., 2010) was amplified by PCR and cloned into the XhoI/BamHI restriction sites of the pLVX-IRES-Hyg lentiviral vector (Clontech).
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ABCC8 p.Ile152Leu 22973054:355:77
status: NEW[hide] Synergy-based small-molecule screen using a human ... Mol Pharmacol. 2014 Jul;86(1):42-51. doi: 10.1124/mol.114.092478. Epub 2014 Apr 15. 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., [PMID:24737137]
Abstract [show]
The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, DeltaF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. 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-methy l-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation DeltaF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for DeltaF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 microM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a DeltaF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.
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No. Sentence Comment
49 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).
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ABCC8 p.Ile152Leu 24737137:49:278
status: NEW[hide] Potentiators of Defective DeltaF508-CFTR Gating th... Mol Pharmacol. 2015 Oct;88(4):791-9. doi: 10.1124/mol.115.099689. Epub 2015 Aug 5. Phuan PW, Veit G, Tan JA, Finkbeiner WE, Lukacs GL, Verkman AS
Potentiators of Defective DeltaF508-CFTR Gating that Do Not Interfere with Corrector Action.
Mol Pharmacol. 2015 Oct;88(4):791-9. doi: 10.1124/mol.115.099689. Epub 2015 Aug 5., [PMID:26245207]
Abstract [show]
Combination drug therapies under development for cystic fibrosis caused by the F508 mutation in cystic fibrosis transmembrane conductance regulator (CFTR) include a "corrector" to improve its cellular processing and a "potentiator" to improve its chloride channel function. Recently, it was reported that the approved potentiator N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (Ivacaftor) reduces F508-CFTR cellular stability and the efficacy of investigational correctors, including 3-(6-[([1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl) amino]-3-methyl-2-pyridinyl)-benzoic acid and 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-(1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2- (2-hydroxy-1,1-dimethylethyl)-1H-indol-5-yl), which might contribute to the modest reported efficacy of combination therapy in clinical trials. Here, we report the identification and characterization of potentiators that do not interfere with F508-CFTR stability or corrector action. High-throughput screening and structure-activity analysis identified several classes of potentiators that do not impair corrector action, including tetrahydrobenzothiophenes, thiooxoaminothiazoles, and pyrazole-pyrrole-isoxazoles. The most potent compounds have an EC(50) for F508-CFTR potentiation down to 18 nM and do not reduce corrector efficacy in heterologous F508-CFTR-expressing cells or primary cultures of F508/F508 human bronchial epithelia. The DeltaF508-CFTR potentiators also activated wild-type and G551D CFTR, albeit weakly. The efficacy of combination therapy for cystic fibrosis caused by the F508 mutation may be improved by replacement of Ivacaftor with a potentiator that does not interfere with corrector action.
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No. Sentence Comment
30 Fisher rat thyroid (FRT) epithelial cells stably expressing ƊF508, wild-type, or G551D CFTR, together with halide-sensitive green fluorescent protein yellow fluorescence protein (YFP)-H148Q/I152L/F46L (Galietta et al., 2001) were as reported (Ma et al., 2002; Pedemonte et al., 2005).
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ABCC8 p.Ile152Leu 26245207:30:195
status: NEW