Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel  Emanuela Pesce, Giulia Gorrieri, Francesco Sirci, Francesco Napolitano, Diego Carrella, Emanuela Caci, Valeria Tomati, Olga Zegarra- Moran, Diego di Bernardo, Luis J.V. Galietta  Journal of Cystic Fibrosis  Volume 15, Issue 4, Pages 425-435 (July 2016) DOI: 10.1016/j.jcf.2016.02.009 Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

Journal of Cystic Fibrosis 2016 15, 425-435DOI: (10. 1016/j. jcf. 2016 Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

Fig. 1 Transcriptional and structural similarity of the 46 drugs inducing a transcriptional response similar to that of low temperature. Each node represents a drug. Green and red lines connecting two nodes represent a significant transcriptional and structural similarity, respectively. Dark blue lines connecting two nodes represent both a significant transcriptional AND structural similarity. We selected all the drugs that were below a transcriptional distance threshold of 0.85 from the low temperature treatment. We then selected as significantly similar, from the chemical structure point of view, those drug-pairs with a structural distance below 0.7. Journal of Cystic Fibrosis 2016 15, 425-435DOI: (10.1016/j.jcf.2016.02.009) Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

Fig. 2 Functional evaluation of compounds related to low temperature gene expression signatures. Compounds were tested by treating CFBE41o− cells for 24h with a high, a medium, and a low concentration, in the absence (A) and in the presence (B) of VX-809 at 1μM. For most compounds, the three concentrations were 25, 10, and 2μM. Ouabain, ionomycin, and thapsigargin were tested at 10, 2, and 0.4μM. Digitoxigen was tested at 5, 1, and 0.2μM. The bars report anion transport (quenching rate) as measured with the halide-sensitive yellow fluorescent protein. Absence of bars (i.e. proscillaridin at all concentrations, irinotecan and cephaeline at the highest concentrations) indicates that the compound was markedly cytotoxic and therefore that anion transport could not be determined. Dotted and dashed lines indicate activity measured in cells treated with vehicle and VX-809, respectively. Journal of Cystic Fibrosis 2016 15, 425-435DOI: (10.1016/j.jcf.2016.02.009) Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

Fig. 3 Analysis of digitoxigenin activity. (A) Dose response of digitoxigenin in the absence (top) and presence (bottom) of VX-809 (**, p<0.01 vs. untreated cells). (B) CFTR mRNA levels measured by real time RT-PCR. Digitoxigenin strongly upregulated CFTR expression (*, p<0.05 vs. untreated cells). (C) Western blot analysis of CFTR protein maturation. Digitoxigenin or ouabain (1μM) was tested with and without VX-809. P: parental CFBE41o− cells (undetectable endogenous CFTR expression). WT: CFBE41o− cells with wild type CFTR protein expression. The results are representative of three similar experiments. (D) Evaluation of digitoxigenin in primary bronchial epithelial cells. Representative traces show short-circuit recordings in which cells sequentially received: apical amiloride (10μM) to block the epithelial Na+ channel ENaC, an activating cocktail (AC) consisting of CPT-cAMP (100μM) and genistein (50μM) to maximally stimulate F508del-CFTR, the CFTR inhibitor-172 (10μM). The bar graph reports the amplitude of the current drop induced by the inhibitor (**, p<0.01 vs. untreated cells). Journal of Cystic Fibrosis 2016 15, 425-435DOI: (10.1016/j.jcf.2016.02.009) Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

Fig. 4 Anion transport induced by treatment of CFBE41o− cells with glucocorticoids. (A) Representative traces showing cell fluorescence changes in CFBE41o− cells coexpressing F508del-CFTR and the halide-sensitive yellow fluorescent protein HS-YFP. The arrow indicates the moment of addition of iodide-rich solution. Treatment of cells with various glucocorticoids (100nM) or with VX-809 (1μM) for 24h resulted in increased anion transport as indicated by faster fluorescence quenching by iodide influx. (B) Dose-dependence of gluococorticoid effect. The maximal quenching rate derived from HS-YFP experiments is plotted vs. the glucocorticoid concentration. Each point is the mean±SEM of 8 experiments. Data are fitted with the Hill equation. Journal of Cystic Fibrosis 2016 15, 425-435DOI: (10.1016/j.jcf.2016.02.009) Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

Fig. 5 Analysis of glucocorticoid mechanism of action. (A) Synergistic effects generated by combination of glucocorticoids (100nM) with VX-809 (1μM). Anion transport in CFBE41o− cells with F508del-CFTR was measured with the HS-YFP assay (n=6–8 per condition; **, p<0.01 vs. untreated cells; ##, p<0.01 vs. cells treated with VX-809 alone). (B) Evaluation of receptor antagonists: mifepristone (1μM) for glucocorticoid receptor and spironolactone (1μM) for mineralcorticoid receptor. Mifepristone, but not spironolactone, significantly inhibited the effect of the glucocorticoid (GC) mometasone or fluticasone, both used at 100nM (n=8 per condition; **, p<0.01 vs. GC alone). (C) Western blot analysis of CFTR protein maturation. CFBE41o− cells with expression of F508del-CFTR cells were treated with VX-809 (1μM), mometasone (0.5 and 2μM) plus/minus VX-809, or incubated at low temperature. For comparison, lysates from parental CFBE41o− or CFBE41o− with wild type CFTR are also included. Journal of Cystic Fibrosis 2016 15, 425-435DOI: (10.1016/j.jcf.2016.02.009) Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

Fig. 6 Transepithelial currents measured in CFBE41o− cells. (A) Representative short-circuit recordings from CFBE41o− cells treated for 24h with indicated compounds. During recordings, cells were acutely treated with CPT-cAMP (100μM), VX-770 (1μM), CFTRinh-172 (10μM). (B) Summary of the current inhibited by CFTRinh-172 in cells exposed to the indicated treatments (n=8–10; *, p<0.05 vs. control). (C) Bar graph reporting the fraction of total CFTR current that was active before stimulation with CPT-cAMP and VX-770 (n=8–10; **, p<0.01 vs. VX-809). Journal of Cystic Fibrosis 2016 15, 425-435DOI: (10.1016/j.jcf.2016.02.009) Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

Fig. 7 Transepithelial currents in primary CF bronchial epithelial cells. (A) Representative short-circuit current recordings from bronchial epithelial cells from F508del homozygous CF patients. Cells were treated with the indicated compounds (1μM VX-809, 100nM mometasone, 1μM mifepristone) for 24h. During recordings, the cells were acutely treated with amiloride (10μM), CPT-cAMP (100μM), CFTRinh-172 (10μM), and UTP (100μM). (B) Amplitude of currents blocked by amiloride indicating ENaC activity (n=17–18; **, p<0.01 vs. control; #, p<0.05 vs. mometasone). (C) Amplitude of currents blocked by CFTRinh-172 indicating F508del-CFTR activity (n=17–18; **, p<0.01 vs. control). Data for ENaC and F508del-CFTR were obtained from cells of two F508del homozygous patients. Journal of Cystic Fibrosis 2016 15, 425-435DOI: (10.1016/j.jcf.2016.02.009) Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions