Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel

JCF-01311; No of Pages 11

Journal of Cystic Fibrosis xx (2016) xxx – xxx www.elsevier.com/locate/jcf

Original Article

Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel Emanuela Pesce a , Giulia Gorrieri a , Francesco Sirci b , Francesco Napolitano b , Diego Carrella b , Emanuela Caci a , Valeria Tomati a , Olga Zegarra-Moran a , Diego di Bernardo b,⁎, Luis J.V. Galietta a,⁎ b

a Istituto Giannina Gaslini, via Gerolamo Gaslini 5, 16147 Genova, Italy Telethon Institute of Genetics and Medicine, Via Campi Flegrei 34, 80078 Pozzuoli, Italy

Received 14 July 2015; revised 21 January 2016; accepted 22 February 2016

Abstract Background: Mistrafficking of CFTR protein caused by F508del, the most frequent mutation in cystic fibrosis (CF), can be corrected by cell incubation at low temperature, an effect that may be mediated by altered expression of proteostasis genes. Methods: To identify small molecules mimicking low temperature, we compared gene expression profiles of cells kept at 27 °C with those previously generated from more than 1300 compounds. The resulting candidates were tested with a functional assay on a bronchial epithelial cell line. Results: We found that anti-inflammatory glucocorticoids, such as mometasone, budesonide, and fluticasone, increased mutant CFTR function. However, this activity was not confirmed in primary bronchial epithelial cells. Actually, glucocorticoids enhanced Na+ absorption, an effect that could further impair mucociliary clearance in CF airways. Conclusions: Our results suggest that rescue of F508del-CFTR by low temperature cannot be easily mimicked by small molecules and that compounds with closer transcriptional and functional effects need to be found. © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: CFTR; Chloride channel; F508del; Corrector; Systems biology

1. Introduction CFTR, a plasma membrane chloride/bicarbonate channel expressed in epithelial cells, is defective in cystic fibrosis (CF), a severe genetic disease that particularly affects the respiratory system and the gastrointestinal tract [1,2]. In CF airways, lack of CFTR-dependent anion secretion impairs mucociliary transport [3,4], mucus release [5–7], and bactericidal activity [8]. Such defects favor survival and proliferation of opportunistic bacteria like Pseudomonas aeruginosa that in turn trigger a severe inflammatory process that has destructive consequences on lung anatomy and respiratory function [2]. ⁎ Corresponding authors.

The most frequent mutation, F508del [2,9], decreases the folding efficiency and stability of CFTR protein [10]. Consequently, CFTR is degraded by multiple quality control mechanisms [11,12]. F508del mutation also causes a channel gating defect resulting in a reduced open channel probability [13]. Other types of CF mutations, like G551D, only cause a gating defect, although more severe than that of F508del [14]. Pharmacological rescue of mutant CFTR is a promising strategy [15]. Small molecules called potentiators, in particular VX-770, strongly stimulate the activity of CFTR mutants with a channel gating defect [16,17]. Unfortunately, there are no compounds that are similarly effective in targeting the trafficking defect associated with the F508del mutation. Such compounds, called correctors, show only a partial ability to save mutant CFTR from degradation when tested in vitro [18].

http://dx.doi.org/10.1016/j.jcf.2016.02.009 1569-1993© 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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One of the best correctors available, VX-809 [19], probably acts by direct binding to CFTR. Combination of VX-809 with other correctors generates additive/synergistic effects on F508del-CFTR rescue [19]. These other correctors may act as pharmacological chaperones, by binding to a second site in the CFTR protein [20], or by modifying the proteostasis environment [19,21,22]. Incubation of cells at low temperature is a well-known way to induce a marked rescue of F508del-CFTR [23]. This may in part be dependent on stabilization of mutant CFTR protein, but may also be favored by a profound change in proteostasis environment with altered expression of genes involved in protein biosynthesis/degradation and endoplasmic reticulum stress [24,25]. Therefore, a small molecule mimicking the pleiotropic effect of low temperature could behave as a very effective F508del corrector. Recently we developed a novel systems biology approach to reposition drugs for novel therapeutic applications by analyzing the transcriptional profiles that they induce in treated cells [26]. The approach, named MANTRA for Mode of Action by NeTwoRk Analysis, relies on the identification of “drug networks” consisting of drugs inducing similar transcriptional responses. We used MANTRA to identify compounds inducing transcriptional profiles similar to those of hypothermia. A similar approach has previously been used [25]. Compounds were tested on cells to assess their ability to rescue mutant CFTR function.

2. Results In a previous study, we reported the gene expression profiles (GEPs) of CFBE41o− and primary bronchial epithelial cells treated at 27 °C for 24 h, a condition that leads to a marked rescue of F508del-CFTR trafficking and function [24]. GEPs were determined by microarrays (Affymetrix GeneChip HG133A2) and processed using standard procedures (GEO Access Number: GSE70442). In this way, we found that hypothermia has a very broad pleiotropic effect at the gene expression level [24]. To check that for our new study CFBE41o − cells were still responding to low temperature as described previously, we selected a panel of the most upregulated genes and determined their expression by real time PCR. We found that such genes were still upregulated by treatment at 27 °C (Supplementary Fig. 1A). The gene expression profiles associated with low temperature have now been analyzed with MANTRA. MANTRA (http:// mantra.tigem.it) is a systems biology approach to identify drugs having a similar mode of action at the gene expression level [26,27]. MANTRA is based on the Connectivity Map (cMAP) database [28] containing 6100 genome-wide expression profiles obtained by treatment of five different human cell lines at different dosages with a set of 1309 different molecules. In MANTRA, drugs are represented as nodes and connected by a line if they induce similar transcriptional responses. Analysis by MANTRA identified a set of 46 small molecules with high similarity (short distance) to low temperature (Fig. 1 and Supplementary Table 1).

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. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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Among these compounds, we found several proteasome inhibitors, protein synthesis inhibitors and cell cycle modulators, consistent with the known effects of low temperature on cell cycle arrest and protein stabilization. Interestingly, this list included molecules that were previously reported to rescue F508del-CFTR such as thapsigargin [29] or cardiac glycosides, namely ouabain, digitoxin, and digoxin [25]. We also used a 3D Molecular Interaction Field method to compute similarity in chemical structure among all the pairs of compounds in the set of 46 drugs, in order to check for a common chemical structure. Fig. 1 shows the transcriptional and chemical similarities of 46 drugs inducing a transcriptional profile similar to low temperature. Interestingly, two broad clusters of drugs can be identified. Each cluster is made up of drugs that have a similar chemical structure and induce a similar transcriptional response in treated cells. One

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cluster is composed mainly of cardiac glycosides plus mometasone, a glucorticosteroid. The second one is composed of proteasome modulators, protein synthesis inhibitors and drugs from different pharmacologically classes. As a further validation step, we verified by real time PCR the expression of genes that, according to MANTRA, were upregulated by both low temperature and mometasone (Supplementary Fig. 1B). We selected 20 out of 46 compounds that were available at the time of the study for testing on CFBE41o − cells as correctors. Cells were treated with compounds for 24 h at three different concentrations: 2, 10, and 25 μM (Fig. 2A). For some compounds, showing evident toxicity at the highest concentrations, we performed the tests with more diluted solutions. After treatment, F508del-CFTR activity in the plasma membrane was assessed with the halide-sensitive yellow fluorescent protein

Fig. 2. Functional evaluation of compounds related to low temperature gene expression signatures. Compounds were tested by treating CFBE41o− cells for 24 h with a high, a medium, and a low concentration, in the absence (A) and in the presence (B) of VX-809 at 1 μM. For most compounds, the three concentrations were 25, 10, and 2 μM. Ouabain, ionomycin, and thapsigargin were tested at 10, 2, and 0.4 μM. Digitoxigen was tested at 5, 1, and 0.2 μM. 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. Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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(HS-YFP) assay [24]. As positive control, we used the corrector VX-809 (1 μM) that caused a nearly three-fold increase in anion transport compared to vehicle-treated cells. Among the compounds with gene expression profile similar to that of low temperature, only digitoxigenin, a cardenolide with structure similarity to digitoxin, and mometasone, a glucocorticoid used to treat asthma and allergic rhinitis, increased anion transport (Fig. 2A). In particular, digitoxigenin was effective at 1 μM but not at higher or lower concentrations. Mometasone was effective at 2 μM but not at 10 and 25 μM. We also tested all compounds in combination with VX-809 to detect possible additive or synergic effects (Fig. 2B). Under this condition, mometasone and ouabain (both at 2 μM) significantly increased anion transport with respect to VX-809 alone. Many other compounds, particularly at the highest concentrations, significantly reduced VX-809 rescue. This negative effect may result from general or mechanism-specific citotoxicity. We further investigated the effects of digitoxigenin and mometasone. We tested digitoxigenin at different concentrations in the range between 0.05 and 25 μM (Fig. 3A). The response of F508del-CFTR function to digitoxigenin showed a narrow bell-shaped relationship. A significant increase was observed at 1 μM but higher doses were ineffective or even inhibitory. This behavior was even more evident when digitoxigenin was combined with VX-809 (Fig. 3A). At 0.2–

1 μM, digitoxigenin increased anion transport above the level elicited by VX-809 alone. However, at higher concentrations digitoxigenin markedly antagonized VX-809 activity. In a previous study, we found that agents that increase CFTR mRNA levels such as histone deacetylase inhibitors (e.g. SAHA) appear as correctors, probably because, by increasing F508del-CFTR biosynthesis, they allow more mutant protein to move to the plasma membrane by mass action [24]. We asked whether digitoxigenin has a similar effect. CFTR expression was evaluated by real time RT-PCR. After 24 h treatment with digitoxigenin, CFTR mRNA levels were increased nearly 12-fold compared to control cells (Fig. 3B). In contrast, VX-809 had no significant effect. The effect of digitoxigenin on F508del-CFTR maturation was studied in western blot experiments (Fig. 3C). Treatment with digitoxigenin increased by nearly two fold the intensity of band B (the partially-glycosylated immature form of the protein) without affecting band C (the mature form). Instead, VX-809 improved mutant CFTR maturation as indicated by the appearance of band C (Fig. 3C). Combination of the two compounds did not increase the maturation above the level achieved with VX-809 alone. We also tested ouabain, which was found to rescue F508del-CFTR in a previous study [25]. Surprisingly, ouabain consistently decreased global CFTR protein levels (Fig. 3C). To further evaluate digitoxigenin as a F508del-CFTR corrector, we carried out experiments on primary

Fig. 3. Analysis of digitoxigenin activity. (A) Dose response of digitoxigenin in the absence (top) and presence (bottom) of VX-809 (**, p b 0.01 vs. untreated cells). (B) CFTR mRNA levels measured by real time RT-PCR. Digitoxigenin strongly upregulated CFTR expression (*, p b 0.05 vs. untreated cells). (C) Western blot analysis of CFTR protein maturation. Digitoxigenin or ouabain (1 μM) 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 (10 μM) to block the epithelial Na+ channel ENaC, an activating cocktail (AC) consisting of CPT-cAMP (100 μM) and genistein (50 μM) to maximally stimulate F508del-CFTR, the CFTR inhibitor-172 (10 μM). The bar graph reports the amplitude of the current drop induced by the inhibitor (**, p b 0.01 vs. untreated cells). Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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human bronchial epithelial cells from F508del-CFTR homozygous patients. Treatment of cells with VX-809 evoked a nearly 3-fold increase in CFTR-dependent chloride secretion (Fig. 3D). In contrast, digitoxigenin, with or without VX-809, was ineffective. We tested mometasone as the second active agent derived from our bioinformatics analysis. Mometasone was also interesting because other glucocorticoids have been previously shown to affect CFTR [30–34]. Therefore, we also tested additional drugs of this class. CFBE41o − cells were treated for 24 h with mometasone, fluticasone, budesonide, and hydrocortisone at various concentrations in the 1 pM to 1 μM range (Fig. 4A). For comparison, we also treated cells with 1 μM VX-809. The treatments resulted in a significant increase in anion transport as indicated by the accelerated quenching of HS-YFP fluorescence (Fig. 4A). Interestingly, mometasone, fluticasone, and budesonide were effective at very low concentrations. By calculating the half effective concentrations, we found values of 50 pM, 120 pM, and 190 pM, respectively (Fig. 4B). These values are consistent with the high affinity of these drugs for the glucocorticoid receptor. The maximal effect, which was reached at 1 nM and maintained up to 1 μM, was 40–50% of that elicited by VX-809 (see Fig. 4A). Hydrocortisone was also effective although, as expected, with a much lower potency, the half effective concentration being 20 nM (Fig. 4B). Importantly, in contrast to digitoxigenin, mometasone did not upregulate CFTR mRNA levels in real time RT-PCR experiments (Fig. 3B). We tested the combination of glucocorticoids and VX-809. The activity resulting from the treatment of mometasone, fluticasone, or budesonide (100 nM) together with VX-809 was significantly higher than that measured with VX-809 alone

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(Fig. 5A). Actually, the combination of compounds appeared to generate a synergistic effect since it was larger than the sum of single compound activity. To confirm the mechanism of action of glucocorticoids, we used two molecules, spironolactone and mifepristone, which are antagonists of the mineralcorticoid and gluococorticoid receptors, respectively. The effect of mometasone, fluticasone, and budesonide were decreased by mifepristone but not by spironolactone (Fig. 5B). We tested the effect of mometasone on F508del-CFTR maturation. The glucocorticoid agonist increased the intensity of band B by nearly two-fold (Fig. 5C). In combination with VX-809, mometasone also increased the expression of band C compared to cells treated with VX-809 alone (Fig. 5C). However, the electrophoretic mobility in some conditions (see lane 8) was not identical to that of wild type CFTR thus suggesting a different degree of glycosilation. To demonstrate the involvement of CFTR in the effect of glucocorticoids, we used the selective inhibitor CFTRinh-172. The anion transport increases elicited by mometasone, fluticasone, and budesonide, as well as that of VX-809, were markedly reduced by the CFTR inhibitor (Supplementary Fig. 2A). To further confirm the involvement of CFTR, we transfected CFBE41o− cells with specific DSiRNAs. Mometasone effect was significantly decreased by silencing F508del-CFTR expression (Supplementary Fig. 2B). Interestingly, the anion transport measured in untreated cells was also affected by treatment with CFTRinh-172 or by transfection with anti-CFTR DsiRNAs (Supplementary Fig. 2A,B). These results suggest that a small but detectable amount of F508del-CFTR reaches cell surface. Importantly, in parental CFBE41o − cells (with undetectable CFTR expression, see

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 (100 nM) or with VX-809 (1 μM) for 24 h 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. Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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Fig. 5. Analysis of glucocorticoid mechanism of action. (A) Synergistic effects generated by combination of glucocorticoids (100 nM) with VX-809 (1 μM). Anion transport in CFBE41o− cells with F508del-CFTR was measured with the HS-YFP assay (n = 6–8 per condition; **, p b 0.01 vs. untreated cells; ##, p b 0.01 vs. cells treated with VX-809 alone). (B) Evaluation of receptor antagonists: mifepristone (1 μM) for glucocorticoid receptor and spironolactone (1 μM) for mineralcorticoid receptor. Mifepristone, but not spironolactone, significantly inhibited the effect of the glucocorticoid (GC) mometasone or fluticasone, both used at 100 nM (n = 8 per condition; **, p b 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 (1 μM), mometasone (0.5 and 2 μM) plus/minus VX-809, or incubated at low temperature. For comparison, lysates from parental CFBE41o− or CFBE41o− with wild type CFTR are also included.

Fig. 5C, first lane), glucocorticoids were ineffective (Supplementary Fig. 2C). We also evaluated the effect of mometasone in electrophysiological assays, namely patch-clamp and short-circuit current recordings. In patch-clamp experiments, cells were acutely stimulated with forskolin plus genistein to fully activate F508del-CFTR and then exposed to CFTRinh-172. The membrane current drop caused by CFTRinh-172 was used to estimate the amount of F508del-CFTR in the plasma membrane. Treatment for 24 h with 1 μM VX-809 increased the amplitude of whole-cell membrane currents (Supplementary Fig. 3A). Interestingly, in VX-809 treated cells, we noted relatively large currents even under resting conditions, i.e. before stimulation with the CFTR-activating cocktail. The currents were further increased by forskolin plus genistein and then abolished by CFTRinh-172 (Supplementary Fig. 3A). The amplitude of the inhibitor-sensitive current (at + 100 mV) was 7.9 ± 2.4 and 39.9 ± 9.9 pA/pF in vehicle and VX-809 treated cells, respectively (n = 15 for both conditions, p b 0.01, Supplementary Fig. 3B). In contrast, after treatment with mometasone (100 nM) the inhibitor-sensitive current was 12.4 ± 5.9 pA/pF (n = 14), not significantly different from vehicle-treated cells (Supplementary Fig. 3B). We saw no effect of mometasone even if combined with VX-809. Under this condition, the CFTR-dependent current was 44.5 ± 14.2 pA/pF (n = 10; Supplementary Fig. 3A,B).

We obtained similar findings also by measuring CFTR function in short-circuit current recordings. CFBE41o − cells were seeded on porous membranes to form a tight epithelium and then mounted in Ussing chambers to measure transepithelial ion transport. F508del-CFTR was stimulated with a cAMP analog plus the VX-770 potentiator and then blocked with CFTRinh-172. The amplitude of the current drop caused by the inhibitor was taken to estimate maximal CFTR activity. This current was significantly increased by incubation for 24 h with VX-809 (Fig. 6A,B). In contrast to HS-YFP experiments, the combination of VX-809 with mometasone or fluticasone did not cause an increase with respect to VX-809 alone (Fig. 6A,B). However, we noted that the baseline current, before stimulation with VX-770 and CPT-cAMP, was markedly higher in cells treated with glucocorticoids. The fraction of CFTR active before stimulation was 80–90% with mometasone or fluticasone plus VX-809 and only 20–30% in cells treated with VX-809 alone (Fig. 6C). To further investigate this effect, we repeated the experiments with the HS-YFP assay in the absence or presence of stimulation. We found that glucocorticoids indeed enhance basal CFTR activity (Supplementary Fig. 4). To finally evaluate the effect of glucocorticoids on F508del-CFTR, we tested mometasone in primary bronchial epithelial cells. Differentiated epithelia were mounted in Ussing chambers to measure transepithelial ion transport. At the

Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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Fig. 6. Transepithelial currents measured in CFBE41o− cells. (A) Representative short-circuit recordings from CFBE41o− cells treated for 24 h with indicated compounds. During recordings, cells were acutely treated with CPT-cAMP (100 μM), VX-770 (1 μM), CFTRinh-172 (10 μM). (B) Summary of the current inhibited by CFTRinh-172 in cells exposed to the indicated treatments (n = 8–10; *, p b 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 b 0.01 vs. VX-809).

beginning of recordings, we added amiloride to block the epithelial Na+ channel, ENaC, and then stimulated CFTR with CPT-cAMP plus VX-770 (Fig. 7A). While treatment with VX-809 (24 h, 1 μM) increased F508del-CFTR activity, as shown by the larger amplitude of the current blocked by CFTRinh-172, mometasone was without effect (Fig. 7A,C). The increase in basal CFTR activity observed for mometasone in CFBE41o − cells (Fig. 6) was not detected in primary cells. Importantly, mometasone increased the amplitude of the amiloride-sensitive current. This effect was blocked when mometasone was combined with the glucocorticoid receptor antagonist mifepristone (Fig. 7A,B). We also measured transepithelial electrical resistance for the various conditions: control, 1229 ± 277 Ω · cm2; VX-809, 1177 ± 210 Ω · cm2; mometasone, 951 ± 124 Ω · cm2; mifepristone, 1158 ± 109 Ω · cm2; mifepristone plus mometasone, 1056 ± 87 Ω · cm2. In order to better understand which could be the potential mechanisms implicated in the failure of digitoxigenin and mometasone in mimicking the effect of low temperature, we performed a Gene Set Enrichment Analysis (GSEA) [35] on the list of differentially expressed genes for digitoxigenin, mometasone and low temperature present in MANTRA. The results of GSEA clearly show that the low temperature treatment significantly affects the expression of the proteasome complex genes (Supplementary Table 2), unlike mometasone

and digitoxigenin. According to GSEA, mometasone instead affects expression of genes involved in endoplasmatic reticulum (ER) and ER to Golgi transport (Supplementary Table 2), whereas digitoxigenin seems to modulate the expression of genes involved in DNA repair, cell cycle and transcription (Supplementary Table 2). 3. Discussion Incubation of cells at low temperature (i.e. 27–32 °C) is a very effective way to rescue CFTR protein with the F508del mutation [23,24]. Our goal was to find small molecules that mimic the effect of low temperature, an approach that has been already attempted in a previous study [25]. Using the MANTRA tool, we compared the gene expression profile associated with exposure of cells to low temperature [24] with those of 1309 small molecules available in databases. This analysis generated a list of drugs and other compounds that also included molecules that had been previously described as F508del correctors, i.e. thapsigargin and cardiac glycosides. Thapsigargin is an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) that was found to rescue F508del-CFTR protein in cell lines and in a mouse model [29]. Cardiac glycosides, particularly ouabain, were identified as molecules reproducing the effect of low temperature on F508del-CFTR in cell lines and in mice [25]. The list of possible correctors also

Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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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 (1 μM VX-809, 100 nM mometasone, 1 μM mifepristone) for 24 h. During recordings, the cells were acutely treated with amiloride (10 μM), CPT-cAMP (100 μM), CFTRinh-172 (10 μM), and UTP (100 μM). (B) Amplitude of currents blocked by amiloride indicating ENaC activity (n = 17–18; **, p b 0.01 vs. control; #, p b 0.05 vs. mometasone). (C) Amplitude of currents blocked by CFTRinh-172 indicating F508del-CFTR activity (n = 17–18; **, p b 0.01 vs. control). Data for ENaC and F508del-CFTR were obtained from cells of two F508del homozygous patients.

included mometasone, an inhalatory glucocorticoid. Although mometasone has not been tested on CFTR, other glucocorticoids used as anti-inflammatory agents were found to affect wild type and mutant CFTR expression. In a bronchial cell line, dexamethasone increased the amount of wild type CFTR in the plasma membrane with a mechanism involving SGK1 [30] and mediated by interaction with Shank2E protein [32]. Dexamethasone also increased wild type CFTR protein levels in Calu-3, a cell line with characteristics of airway submucosal glands [33]. In a pancreatic cell line, CFPAC-1, dexamethasone reduced the

degradation of F508del-CFTR by modulating the activity of Nedd4-2 [31]. This effect resulted in enhanced anion transport. In another study, a bronchial cell line responded to dexamethasone or hydrocortisone with increased expression of F508del-CFTR protein, although without evidence of a concomitant improvement in anion transport [34]. We tested the different compounds postulated by MANTRA analysis as possible correctors of F508del-CFTR, by themselves or in combination with VX-809. Most compounds were ineffective including ouabain and thapsigargin. Instead,

Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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digitoxigenin, a cardenolide derived from digitoxin, and mometasone, an anti-inflammatory glucocorticoid, induced F508del-CFTR rescue. We investigated the efficacy and mechanism of action of these two active compounds. Regarding digitoxigenin, we found that it has a very narrow bell-shaped dose–response relationship. This is probably due to cytotoxic/undesired effects generated by the drug at relatively high concentrations. It should be noted that ouabain and other related molecules may cause a variety of negative effects including block of protein synthesis [36–38] and inhibition of topoisomerase I [39]. Furthermore, GSEA revealed that digitoxigenin affects genes involved in DNA repair, cell cycle and transcription. On the other hand, we found that digitoxigenin causes a marked increase in CFTR mRNA levels when tested in CFBE41o − cells. This could be explained with an effect on the viral promoter that drives F508del-CFTR expression in recombinant CFBE41o − cells, similarly to other putative correctors studied before [24]. Therefore, it is probable that part of the increase in F508del-CFTR function elicited by digitoxigenin, as well by other molecules of this class, is due to protein hyperexpression and exit from the endoplasmic reticulum by mass action. In agreement with this interpretation, digitoxigenin was ineffective in primary bronchial epithelial cells in which CFTR expression is controlled by the endogenous promoter. Summarizing, the cell response to digitoxigenin and other molecules with steroid-like structure may involve a complex pattern of mechanisms that are in part favorable and in part detrimental for F508del-CFTR biosynthesis/maturation and cell viability. The consequence is a very narrow corrective range that may be strongly dependent on cell type and experimental conditions. In contrast to digitoxigenin, mometasone caused no effect on CFTR mRNA in CFBE41o − cells. In addition to mometasone, other glucocorticoids such as budesonide, fluticasone, and hydrocortisone, were also effective, with a mechanism that probably involves the glucocorticoid receptor, as indicated by the picomolar affinity and the response to mifepristone. Specific rescue of F508del-CFTR function by glucocorticoids was demonstrated by blocking anion transport with a selective CFTR inhibitor, by knocking down CFTR expression with short interfering RNAs, and by using parental CFBE41o − cells with undetectable CFTR expression. Surprisingly, despite the positive results obtained with the HS-YFP assay, we could not confirm F508del-CFTR correction by patch-clamp and Ussing chamber, two techniques that measure electrogenic ion transport. These results could be interpreted by assuming that CFTR, under some particular conditions, performs an electroneutral transport, a type of activity that has never been described before. It should be noted that the only previous functional evidence of F508del-CFTR rescue by a glucocorticoid was obtained in a pancreatic cell line with a fluorescence assay based on the MQAE probe [31], a technique that, similarly to HS-YFP assay, cannot distinguish between electroneutral and electrogenic transport. Intriguingly, in Ussing chamber and HS-YFP experiments on CFBE41o− cells, the basal F508del-CFTR activity measured in the absence of cAMP-dependent stimulation, was markedly enhanced by glucocorticoids. Further studies are required to understand if this

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phenomenon is in some way related to apparent F508del-CFTR rescue. The results of our study lead to two conclusions. The first conclusion is that the correction of F508del-CFTR protein by low temperature incubation cannot be easily reproduced by a single small molecule. This approach has previously been attempted by others [25] with the identification of ouabain and other cardiac glycosides. In the present study, we have used MANTRA, a tool that was successful in the repositioning of Fasudil (a Rho-kinase inhibitor) as an enhancer of cellular autophagy [26]. However, compounds detected by MANTRA were not effective in primary bronchial epithelial cells, a cell model that is essential to confirm the activity of F508del correctors [19]. Such negative results suggest that a significant fraction of CFTR rescue by low temperature is more related to direct stabilization of the mutant CFTR protein than to alteration of proteostasis environment by gene expression changes. This would imply that rescue by low temperature may be better mimicked by a pharmacological chaperone rather than by a proteostasis regulator. However, it may be speculated that some of the compounds tested in our study are indeed transcriptional “neighbors” of low temperature but that their corrective effect on F508del-CFTR is masked by additional undesired effect on other cellular targets and pathways. It can be hypothesized that future investigation of additional small molecules, not included in the cMAP [28], will reveal correctors with higher similarity (shorter transcriptional distance) to hypothermia and reduced side effects. These studies may include cells in which mutant CFTR expression is under the control of the native promoter in order to detect positive effects of compounds on endogenous CFTR expression. Furthermore, initiatives like cMAP are based on the use of a limited set of fixed concentrations that are often far from those at which compounds work on their main biological target. This is a limitation that should be considered in future projects. The second conclusion regards inhaled glucocorticoids. These potent drugs are used as anti-inflammatory agents to treat various respiratory diseases including asthma and CF. The use of glucocorticoids in CF patients carrying the F508del mutation could be further supported by the finding that these molecules positively modulate CFTR expression and trafficking [30–34]. However, our findings do not confirm the activity of glucocorticoids as correctors in primary bronchial epithelial cells. Rather, we found that glucocorticoids stimulate ENaC activity. By failing to rescue Cl− secretion and by stimulating Na+ absorption, glucocorticoids may actually produce a decreased airway hydration, an effect that could be detrimental for CF patients despite their potent anti-inflammatory activity. 4. Methods 4.1. Cell culture conditions The bronchial epithelial cell line CFBE41o − with and without stable co-expression of F508del-CFTR or wild type CFTR was cultured with MEM medium supplemented with

Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009

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10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The generation of CFBE41o − cells co-expressing F508del-CFTR and the halide-sensitive yellow fluorescent protein (HS-YFP) was previously described [24]. The culture conditions for primary bronchial epithelial cells were also previously described [40]. 4.2. Fluorescence assay for CFTR activity CFBE41o − cells with co-expression of F508del-CFTR and HS-YFP were treated for 24 h with test compounds and/or VX-809. After treatments, cells were stimulated for 30 min with forskolin (20 μM) and genistein (50 μM) and F508del-CFTR activity in the plasma membrane was determined by measuring the rate of HS-YFP quenching due to I− influx. In experiments shown for Fig. 1S, cells were also left in resting conditions, without acute stimulation. 4.3. Patch-clamp recordings Whole-cell membrane currents were recorded in CFBE41o − cells with stable expression of F508del-CFTR. The extracellular (bath) solution had the following composition: 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM mannitol, 10 mM Na-HEPES (pH = 7.4). The pipette (intracellular) solution contained 120 mM CsCl, 10 mM TEA-Cl, 0.5 mM EGTA, 1 mM MgCl2, 10 mM HEPES, 40 mM mannitol, 1 mM ATP (pH 7.4). 4.4. Western blot detection of CFTR protein CFTR protein was detected with the mouse monoclonal anti-CFTR antibody (596, Cystic Fibrosis Foundation Therapeutics and University of North Carolina, Chapel Hill) diluted 1:5000. The Na+/K+-ATPase β-1 protein was detected with the mouse monoclonal antibody C464.8 (Merck Millipore) diluted 1:6000. 4.5. Short-circuit current recordings CFBE41o − and primary bronchial epithelial cells were plated at high density (500,000 cells/cm2) on porous membranes (Snapwell inserts, Corning 3801). Bronchial epithelial cells were obtained from two CF patients homozygous for F508del mutation (BE37 and BE43). Data obtained from these cells were similar and therefore were combined together (Figs. 3 and 7). As described previously, a differentiating medium was used for primary bronchial epithelial cells 24 h after plating [40]. Experiments were done after 5–6 days for CFBE41o − and after 12–14 days for primary cells. Snapwell inserts were mounted in a self-contained Ussing chamber system. For primary cells, both apical and basolateral chambers were filled with the same solution containing (in mM): 126 NaCl, 0.38 KH2PO4, 2.1 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, and 10 glucose. For CFBE41o − cells, a transepithelial Cl− gradient was applied by using a modified apical solution in which 63 mM NaCl was replaced with an equimolar amount of sodium gluconate.

4.6. The MANTRA network for drug repositioning Gene expression profiles (GEPs) induced by incubating cells at 27 °C (Sondo et al. [24]) were compared to those in the Connectivity Map database (cMAP) that includes GEPs following treatment of five different cell lines with 1309 compounds [28]. We then computed the similarity between transcriptional responses induced by low temperature treatment and each one of the 1309 compounds using a computational approach named MANTRA that we previously described [26]. Briefly, given the transcriptional response to two drugs, MANTRA checks how similar they are, by evaluating whether the genes that are upregulated by one drug are also upregulated by the other drug, and similarly for downregulated genes. The result is a score (distance) that is greater or equal to 0, with 0 implying identical transcriptional responses (see Supplementary material for more details). For more details on all methods, see online Supplementary material. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgments This work was supported by grants from Ministero della Salute (Ricerca Finalizzata: GR-2009-1596824; Ricerca Corrente: Cinque per mille) to LJVG and DdB and by a Fondazione Telethon Grant (TGM11SB1) to DdB. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jcf.2016.02.009. References [1] Gelfond D, Borowitz D. Gastrointestinal complications of cystic fibrosis. Clin Gastroenterol Hepatol 2013;11:333–42. [2] Proesmans M, Vermeulen F, De Boeck K. What's new in cystic fibrosis? From treating symptoms to correction of the basic defect. Eur J Pediatr 2008;167:839–49. [3] Matsui H, Randell SH, Peretti SW, Davis CW, Boucher RC. Coordinated clearance of periciliary liquid and mucus from airway surfaces. J Clin Invest 1998;102:1125–31. [4] Button B, Cai LH, Ehre C, Kesimer M, Hill DB, Sheehan JK, et al. A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 2012;337:937–41. [5] Garcia MA, Yang N, Quinton PM. Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator-dependent bicarbonate secretion. J Clin Invest 2009;119:2613–22. [6] Gustafsson JK, Ermund A, Ambort D, Johansson ME, Nilsson HE, Thorell K, et al. Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype. J Exp Med 2012;209:1263–72. [7] Hoegger MJ, Fischer AJ, McMenimen JD, Ostedgaard LS, Tucker AJ, Awadalla MA, et al. Cystic fibrosis. Impaired mucus detachment disrupts

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Please cite this article as: Pesce E, et al, Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel, J Cyst Fibros (2016), http://dx.doi.org/10.1016/j.jcf.2016.02.009