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Title:
KINASE AND UBIQUITIN LIGASE INHIBITORS AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2017/103205
Kind Code:
A1
Abstract:
The present invention refers to a suppressor or inhibitor of expression and/or function of at least one gene, preferably a kinase or ubiquitin ligase, for use in the treatment of a protein conformational disorder.

Inventors:
LUINI, Alberto (ALDA S.r.l, Via Luigia Sanfelice 89, Napoli, 80127, IT)
BECCARI, Andrea, Rosario (ALDA S.r.l, Via Luigia Sanfelice 89, Napoli, 80127, IT)
HEGDE, Ramanath, Narayana (ALDA S.r.l, Via Luigia Sanfelice 89, Napoli, 80127, IT)
PARASHURAMAN, Seetaraman (ALDA S.r.l, Via Luigia Sanfelice 89, Napoli, 80127, IT)
Application Number:
EP2016/081578
Publication Date:
June 22, 2017
Filing Date:
December 16, 2016
Export Citation:
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Assignee:
ALDA S.R.L. (Via Luigia Sanfelice 89, Napoli, 80127, IT)
International Classes:
C12N15/113; A61K31/00; A61K31/713
Domestic Patent References:
WO2012158913A22012-11-22
Other References:
RAMANATH NARAYANA HEGDE ET AL: "Unravelling druggable signalling networks that control F508del-CFTR proteostasis", ELIFE, vol. 154, 23 December 2015 (2015-12-23), pages 609, XP055299026, DOI: 10.1016/j.cell.2013.06.038
GIANCARLO CHESI ET AL: "Identification of p38 MAPK and JNK as new targets for correction of Wilson disease-causing ATP7B mutants", HEPATOLOGY, vol. 63, no. 6, 25 June 2016 (2016-06-25), pages 1842 - 1859, XP055299416, ISSN: 0270-9139, DOI: 10.1002/hep.28398
AGATA M TRZCISKA-DANELUTI ET AL: "Use of Kinase Inhibitors to Correct deltaF508- CFTR Function", 1 January 2012 (2012-01-01), pages 745 - 757, XP055299327, Retrieved from the Internet [retrieved on 20160901]
AGATA M TRZCINSKA-DANELUTI ET AL: "RNA Interference Screen to Identify Kinases That Suppress Rescue of deltaF508-CFTR", MOLECULAR & CELLULAR PROTEOMICS, vol. 14046375, no. 10, 29 March 2015 (2015-03-29), US, pages 1569 - 1583, XP055299300, ISSN: 1535-9476, DOI: 10.1074/mcp
NICOLETTA PEDEMONTE ET AL: "Pharmacological Correctors of Mutant CFTR Mistrafficking", FRONTIERS IN PHARMACOLOGY, vol. 3, 1 January 2012 (2012-01-01), CH, XP055299029, ISSN: 1663-9812, DOI: 10.3389/fphar.2012.00175
C NOREZ ET AL: "Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism", BRITISH JOURNAL OF PHARMACOLOGY, vol. 171, no. 21, 24 October 2014 (2014-10-24), BASINGSTOKE, HANTS; GB, pages 4831 - 4849, XP055299082, ISSN: 0007-1188, DOI: 10.1111/bph.12859
Attorney, Agent or Firm:
CAPASSO, Olga et al. (Via Vincenzo Bellini 20, Rome, 00198, IT)
Download PDF:
Claims:
CLAIMS

1. A molecule which suppresses or inhibits the expression and/or function of at least one of the following genes:

JNK2/MAPK9, CAMK1, CDC42, HPK1/MAP4K1, PRKAA1 (AMPK), PRKAA2(AMPK), RAC2, TGFBR- 2, MAPK11, MAPK14, MAPK8/JNK1 , CALML5, ITPR2, RNF215, UBOX5, SART1, PDGFRB, CD2BP2, CKII/CSNK2A1, ASB8, STAG2, FBX07, PIK3CB, MLK3/MAP3K11 , CTDSP1, VEGFR2/KDR, GTSE1 , PRPF8, MEDl, OSMR, DSNl, NFKB2, SENP6, PDGFRA, MKK7/MAP2K7, PIK3CG, MAPK15, NUP50, CAMKK2, MIS18BP1/C14orfl06, YWHAH, VEGFR1/FLT1, TEP1, MEDl 3, PROKR1

for use in the treatment of a protein conformational disorder,

with the proviso that said molecule is not oxozeanol, SU5402 and SU6668.

2. The molecule for use according to claim 1, wherein said molecule doesn't suppress or inhibit the expression and/or function of at least one of the following genes:

FGFBP1, DCLK1, DNAJC2, S100A7, MKK1/MAP2K1, BIN2, RBM7, ERBB4, MKI67, MKK2/MAP2K2, PIK3CD, MKK3/MAP2K3, MKK4/MAP2K4, AKAP8, CYC1.

3. The molecule for use according to claim 1 or 2, wherein the molecule:

a) selectively suppresses or inhibits the expression and/or function of at least one:

i) of the kinases or of the kinase regulators selected from the group consisting of: JNK2/MAPK9, CAMK1, CAMKK2, CDC42, CKII/CSNK2A1, HPK1/MAP4K1, MAPK15, MKK7/MAP2K7, MLK3/MAP3K11 , PDGFRA, PDGFRB, PIK3CB, PIK3CG, PRKAAl (AMPK), PRKAA2(AMPK), RAC2, TGFBR-2, VEGFR1/FLT1, VEGFR2/KDR, MAPK11, MAPK14, MAPK8/JNK1, CALML5,

ITPR2 or

ii) of ubiquitin ligases selected from the group consisting of: RNF215, UBX05, ASB8, FBX07 and

b) doesn't suppress or inhibit the expression and/or function of at least one of the kinases selected from the group consisting of: ERBB4, MKK1/MAP2K1, MKK2/MAP2K2, MKK3/MAP2K3, MKK4/MAP2K4,

PIK3CD.

4. The molecule for use according to any one of the previous claims, wherein the protein conformational disorder is selected from cystic fibrosis or Wilson disease.

5. The molecule for use according to any one of the previous claims,

wherein the molecule selectively suppresses or inhibits the expression and/or function of at least one of the following combinations of kinases MLK3/MAP3K11 and CAMKK2, MLK3/MAP3K11 and CKII/CSNK2A1, MLK3/MAP3K11 and RNF215, CAMKK2 and CKII/CSNK2A1.

6. The molecule for use according to any one of the claims 1 -5,

wherein the molecule selectively suppresses or inhibits the expression and/or function of at least one of: JNK2/MAPK9, CAMK1, CAMKK2, CDC42, CKII/CSNK2A1, HPK1/MAP4K1, MAPK15, MKK7/MAP2K7, MLK3/MAP3K11, PDGFRA, PDGFRB, PIK3CB, PIK3CG, PRKAAl (AMPK), PRKAA2(AMPK), RAC2, TGFBR-2, VEGFRl/FLTl and VEGFR2/KDR, or any combination thereof and wherein the protein conformational disorder is cystic fibrosis.

7. The molecule for use according to any one of the claims 1 -5,

wherein the molecule selectively suppresses or inhibits the expression and/or function of at least one of: MLK3/MAP3K11 , MAPK8 (JNKI), MAPKl l (ρ38β) and MAPK14 (p38a), or any combination thereof and wherein the protein conformational disorder is Wilson disease.

8. The molecule for use according to any one of the previous claims, being selected from the group consisting of:

a) a polypeptide;

b) a polynucleotide coding for said polypeptide;

c) a polynucleotide able to inhibit the expression of said gene;

d) a vector comprising or expressing the polynucleotide as defined in b-c);

e) a host cell genetically engineered expressing said polypeptide or said polynucleotide; and f) a small molecule.

9. The molecule for use according to any one of the previous claims being selected from the group consisting of: JNKi IX, SP600125/JNKi II, BIRB-796, VX-745, JNKi XI, SB202190, Pazopanib, Dovitinib lactate, Bexarotene, Flunarizine, Cannabidiol, CPI-1189 and ENMD-2076.

10. The molecule for use according to any one of the previous claims being selected from the group consisting of: JNKi IX, SP600125/JNKi II, JNKi XI, Pazopanib, Dovitinib lactate, Bexarotene, wherein the protein conformational disorder is cystic fibrosis.

11. The molecule for use according to claim 8, being selected from the group consisting of:

VX-745, BIRB-796, JNKi II, SB202190, Bexarotene, Cannabidiol, CPI-1189 and ENMD-2076 wherein the protein conformational disorder is Wilson disease.

12. The molecule for use according to any one of claims 1-8, wherein said polynucleotide able to inhibit the expression of said gene is at least one RNAi agent targeting said gene.

13. The molecule for use according to any one of previous claims, in combination with a therapeutic agent.

14. The molecule for use according to claim 13, wherein the therapeutic agent is the pharmacochaperone VX-809 and the protein conformational disorder is cystic fibrosis.

15. A pharmaceutical composition comprising at least one molecule as defined in any one of claims 1 -14 and at least one pharmaceutically acceptable carrier.

16. A method of treating and/or preventing a protein conformational disorder comprising administering to a patient in need thereof a therapeutically effective amount of at least one molecule as defined in any one of claims 1-14.

Description:
KINASE AND UBIQUITIN LIGASE INHIBITORS AND USES THEREOF FIELD OF THE INVENTION

The present invention refers to a suppressor or inhibitor of expression and/or function of at least one gene, preferably a kinase, a kinase regulators or a ubiquitin ligase, for use in the treatment of a protein conformational disorder.

BACKGROUND ART

Protein conformational disorders are a group of proteostasis (protein homeostasis) disorders resulting from mutations that lead to misfolding of a protein (Balch et al., 2008; Calamini and Morimoto, 2012; Gregersen et al., 2006). This impaired folding results generally results in loss-of- function of the mutant protein. Examples of protein conformational disorders are the Wilson's disease, Cystic Fibrosis, the Niemann Pick disease, retinitis pigmentosa, alpha- 1 antitrypsin deficiency, familial intrahepatic cholestasis, Stargardt disease, Tangier disease, Dubin- Johnson syndrome, progressive familial cholestasis 2, intrahepatic cholestasis of pregnancy etc. Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene (Gene ID: 1080, NCBI Reference Sequence: NM 000492.3, NP 000483.3) that encodes a chloride channel localized to the apical membrane of several epithelial cells. Mutations that cause CFTR loss of function impair the transepithelial movement of salts at the cell surface, resulting in pleiotropic organ pathology and, in the lungs, in chronic bacterial infections that eventually lead to organ fibrosis and failure (Riordan 2008). The CFTR protein comprises two membrane-spanning domains, two cytosolic nucleotide- binding domains, and a regulatory domain, folded together into a channel (Riordan 2008). Folding occurs in the endoplasmic reticulum (ER) through the sequential action of multiple chaperone complexes (Rosser et al. 2008, Meacham et al. 1999, Loo et al. 1998) and is followed by export out of the ER and glycosylation in the Golgi before arrival at the plasma membrane (PM), where CFTR undergoes several cycles of endocytosis before degradation in the lysosomes (Gentzsch et al. 2004). The most frequent mutant, which is present in -90% of the CF patients, misses a phenylalanine at position 508 (F508del-CFTR) and folds in a kinetically and thermodynamically impaired fashion into a conformation that is recognized as defective by the ER quality control (ERQC) system. It is thus retained in the ER and targeted for ER-associated degradation (ERAD) by the ubiquitin-proteasome machinery (Jensen et al. 1995, Ward, Omura, and Kopito 1995). A small fraction of F508del-CFTR may escape degradation in the ER and reach the PM, where it can function as a channel. This might have therapeutic relevance because patients that express even low levels of functional channel have milder symptoms (Amaral 2005). However, at the PM, F508del-CFTR is recognized by the peripheral (or PM-associated) quality control (PQC) system and is rapidly degraded in the lysosomes (Okiyoneda et al. 2010). n previous studies, inventors have shown that constitutive intracellular trafficking is potently controlled by regulatory cascades triggered by both extra- and intra-cellular signals (Cancino et al. 2014, Giannotta et al. 2012, Pulvirenti et al. 2008) suggesting the presence of control systems that optimize the proteostatic capacity of the cell. However, systematic exploration of the signaling pathways that regulate the initial stages of the proteostasis viz. the folding and degradation of proteins is lacking. Several compounds have been identified over the years that enhance the ability of F508del-CFTR to reach the PM, largely through screening campaigns (Carlile et al. 2012, Kalid et al. 2010, Odolczyk et al. 2013, Pedemonte et al. 2005, Phuan et al. 2014, Van Goor et al. 2006). These 'correctors' of the F508del-CFTR defect, act either by binding to F508del-CFTR and inducing conformational changes that help this mutant to fold (pharmacochaperones) (Calamini et al. 2012, Sampson et al. 2011, Wang et al. 2007), or by altering the proteostatic environment of the cell, thereby increasing the probability that the F508del-CFTR mutant escapes the ER and accumulates at the PM (proteostasis regulators). The latter group (proteostasis regulators) include representatives of diverse pharmacological classes such as the histone deacetylase inhibitors (Hurt et al. 2010), poly(ADP-ribose) polymerase inhibitors (Carlile et al. 2012, Anjos et al. 2012), hormone receptor activators (Caohuy, Jozwik, and Pollard 2009), cardiac glycosides (Zhang et al. 2012), and others. Unfortunately, the effects of the available proteostasis correctors are too weak to be of clinical interest, and the molecular mechanism(s) by which they influence F508del- CFTR proteostasis remains unknown. The analysis of the mechanisms of action (MOAs) of these correctors can in principle be addressed by deconvolving the transcriptional effects of these agents. Changes in gene expression are significant components of the MOAs of many drugs (Santagata et al. 2013, Popescu 2003), and the analysis of transcriptional MOAs is a growing research area (Iorio et al. 2010, Iskar et al. 2013). A difficulty here is that the effects of the available F508del-CFTR correctors are most probably not mediated by the heterogeneous principal MOAs of these drugs, but by some unknown weak secondary MOAs ('side effects') that these drugs share. The challenge is therefore to tease out the transcriptional changes that are correction-related from those that are due to the (correction-irrelevant) principal MOAs of the corrector drugs.

A conformational disease that has many features in common with cystic fibrosis as caused by the F508del- CFTR mutant is the Wilson disease (WD), a rare inherited autosomal recessive disorder that is due to a mutation in the ATP7B gene (1 in 50.000 newborns) (Gene ID: 540, NCBI RefSeqGene NG 008806.1 ) and causes too much copper to accumulate in liver, brain and other vital organs. This is because CFTR and ATP7B share a similar structure with two sets of membrane spanning domains connected by a nucleotide- binding domain, and their main mutations lead to similar folding and trafficking defects. The ATP7B gene encodes a multi-transmembrane domain ATPase that traffics from the trans-Golgi network (TGN) to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. WD treatment is currently approached with zinc salts and Cu-chelating agents. However, these treatments have serious toxicities. Moreover about one-third of WD patients respond neither to Zn nor to Cu chelators. Thus, all considered, developing novel WD treatment strategies has become an important task. When approaching therapy solutions, properties of WD-causing mutants should be carefully considered. The most frequent ATP7B mutations, H1069Q (40%-75% in the white patient population) and R778L (10%-40% of the Asian patients), result in ATP7B proteins with significant residual transporter activities, however, they are strongly retained in the endoplasmic reticulum (ER). Moreover, many other WD-causing ATP7B mutants with substantial Cu-translocating activity undergo complete or partial arrest in the ER. Thus, although potentially able to transport Cu, these ATP7B mutants cannot reach the Cu excretion sites to remove excess Cu from hepatocytes. ER retention of such ATP7B mutants occurs due to their mis-folding and increased aggregation, and hence due to their failure to fulfill the requirements of the ER quality control machinery. As a result, the cellular proteostatic network recognizes ATP7B mutants as defective, and directs them towards the ER- associated protein degradation (ERAD) pathway. Therefore, identifying molecular targets for recovery of partially- or fully-active ATP7B mutants from the ER to appropriate functional compartment(s) like Golgi would be beneficial for a majority of WD patients.

Summary of the invention

As noted, the F508del-CFTR proteostasis machinery is well studied while the signaling networks that regulate proteostasis remain barely explored. In order to uncover the signaling networks that control proteostasis, inventors developed a novel strategy based on the analysis of the transcriptional mechanisms of action (MOAs) of drugs that regulate the proteostasis of F508del-CFTR. Given that many of the successful drugs target multiple molecular pathways (Lu et al. 2012), this approach could potentially lead to uncovering synergistically interacting molecular networks, including druggable signaling networks, that control proteostasis. In order to tease out the transcriptional changes that are correction-related from those that are due to the (correction-irrelevant) principal MOAs of the corrector drugs, inventors developed an approach based on the 'fuzzy' intersection of gene expression profiles induced by a set of proteostatic correctors, with the goal to identify genes that are commonly modified by these drugs (and should therefore relate to the correction-associated pathways targeted by the correctors), but not to those associated with their heterogeneous primary effects. Using this strategy, inventors harvested a group of few hundred genes that are regulated by most of the proteostatic correctors, and then derived a series of molecular networks from this gene pool through bioinformatic and experimental approaches. Several of these networks are signaling pathways. Silencing or targeting these pathways with chemical blockers inhibits the degradation in the ER and enhances the transport to the PM of F508del-CFTR, leading to striking levels of F508del-CFTR correction without apparent toxicity. Moreover, the large pool of ER-localized foldable F508del-CFTR that results from the inhibition of ER degradation can be acted upon by pharmacochaperones, further enhancing correction. Inventors extended the studies to other mutant proteins that are structurally similar to CFTR, for instance ATP7B the protein that is misfolded in WD patients, and found that regulatory that control CFTR proteostasis also efficiently controlled the proteostasis of other mutant proteins

Detailed description of the invention

Inventors have identified five signaling pathways that have a regulatory effect on the proteostasis of CFTR and ATP7B mutants. The best characterized two are the MLK3-JNK and CAMKK2 pathways. The inhibition of MLK3-JNK pathway (through siRNA-based depletion of its component kinases) potently activates ER retention and degradation of the misfolded CFTR and ATP7B mutants in the ER. Notably the MLK3-JNK pathway appears to be activated in cells from patients.

In addition to these, inventors have identified other signaling pathways, one of which with opposite effect on correction. The majority of the components of these pathways are kinases. Considering only the kinases composing these pathways, inventors have identified 28 kinases active on correction (Table 1). 22 of them when depleted by siRNA exert positive effects (positive or anti-correction, i.e. kinases whose inhibition induces correction), while 6 of them exert negative effects (negative or pro-correction, i.e. kinases whose inhibition suppresses correction), on correction (Table 1). Inventors have therefore inhibited the MLK3 pathway by using siRNA-based silencing of the main kinases in the pathway or by using inhibitors of these kinases [e.g. JNK inhibitors - JNKi II or SP600125 JNKi IX and JNKi XI, an inhibitor of several kinases of the MLK3-JNK pathway including VEGFR, MLK3, MKK7-(5Z)-7-Oxozeaenol (or Oxozeaenol) and Pazopanib, Dovitinib lactate and Bexarotene]. These inhibitors potently correct the defects of the mutant proteins in disease-relevant cells: immortalized lines of bronchial epithelial cells in the case of CFTR mutant and of hepatocytes in the case of ATP7B mutants. In particular, JNKi II or SP600125 and P38i SB202190, VX745, (5 Z)-7 -Oxozeaenol (or Oxozeaenol) were tested on ATP7B mutants. In the case of CFTR, they are also synergistic with the pharmacochaperone VX-809 (which is known for the treatment of cystic fibrosis) suggesting that they block the degradation of F508del-CFTR in the ER leading to the accumulation of foldable protein that can be rescued by VX-809. This effect on degradation can be easily monitored by a biochemical assay (western blotting) (Farinha et al., 2004) for F508del-CFTR to reveal both the ER localized Band B and the PM localized Band C that is of slightly higher molecular weight due to its glycosylation in the Golgi. It is therefore an object of the invention a molecule which suppresses or inhibits the expression and/or function of at least one of the following genes:

JNK2/MAPK9, CAMK1, CDC42, HPK1/MAP4K1, PRKAAl (AMPK), PRKAA2(AMPK), RAC2, TGFBR-

2, MAPK11, MAPK14, MAPK8/JNK1 , CALML5, ITPR2, RNF215, UBOX5, SART1, PDGFRB, CD2BP2, CKII/CSNK2A1, ASB8, STAG2, FBX07, PIK3CB, MLK3/MAP3K11 , CTDSP1, VEGFR2/KDR, GTSE1 ,

PRPF8, MED1, OSMR, DSN1, NFKB2, SENP6, PDGFRA, MKK7/MAP2K7, PIK3CG, MAPK15, NUP50,

CAMKK2, MIS18BP1/C14orfl06, YWHAH, VEGFR 1/FLTl, TEP1, MED13, PROKRl

for use in the treatment of a protein conformational disorder

with the proviso that said molecule is not oxozeanol, SU5402 and SU6668.

Preferably, said molecule doesn't suppress or inhibit the expression and/or function of at least one of the following genes: FGFBP1, DCLK1, DNAJC2, S100A7, MKK1/MAP2K1, ΒΓΝ2, RBM7, ERBB4, MKI67,

MKK2/MAP2K2, PIK3CD, MKK3/MAP2K3, MKK4/MAP2K4, AKAP8, CYC1.

Any combination of the above genes is comprised within the present invention.

More preferably, the molecule for use according to the invention:

a) selectively suppresses or inhibits the expression and/or function of at least one: i) of the kinases or of the kinase regulators selected from the group consisting of: JNK2/MAPK9, CAMK1, CAMKK2, CDC42, CKII/CSNK2A1, HPK1/MAP4K1, MAPK15, MKK7/MAP2K7, MLK3/MAP3K11 , PDGFRA, PDGFRB, PIK3CB, PIK3CG, PRKAA1 (AMPK), PRKAA2(AMPK), RAC2, TGFBR-2, VEGFR1/FLT1, VEGFR2/KDR, MAPK11, MAPK14, MAPK8/ JNK1, CALML5, ITPR2 or

ii) of ubiquitin ligases selected from the group consisting of: RNF215, UBX05, ASB8, FBX07

and

b) doesn't suppress or inhibit the expression and/or function of at least one of the kinases selected from the group consisting of: ERBB4, MKK1/MAP2K1, MKK2/MAP2K2, MKK3/MAP2K3, MKK4/MAP2K4, PIK3CD.

The protein conformational disorder is preferably selected from cystic fibrosis or Wilson disease.

The molecule as above defined preferably selectively suppresses or inhibits the expression and/or function of at least one of the following combinations of kinases MLK3/MAP3K11 and CAMKK2, MLK3/MAP3K11 and CKII/CSNK2A1, MLK3/MAP3K11 and RNF215, CAMKK2 and CKII/CSNK2A1.

In a preferred embodiment of the invention, the protein conformational disorder is cystic fibrosis and the molecule as above defined selectively suppresses or inhibits the expression and/or function of at least one of: JNK2/MAPK9, CAMK1, CAMKK2, CDC42, CKII/CSNK2A1, HPK1/MAP4K1, MAPK15, MKK7/MAP2K7, MLK3/MAP3K11, PDGFRA, PDGFRB, PIK3CB, PIK3CG, PRKAAl (AMPK), PRKAA2(AMPK), RAC2, TGFBR-2, VEGFR1/FLT1 and VEGFR2/KDR, or any combination thereof. In another preferred embodiment of the invention, the protein conformational disorder is Wilson disease and the molecule as above defined selectively suppresses or inhibits the expression and/or function of at least one of: MLK3/MAP3K11, MAPK8 (JNK1), MAPK11 (ρ38β) and MAPK14 (p38a), or any combination thereof. Preferably, the molecule for use according to the invention is selected from the group consisting of:

a) a polypeptide;

b) a polynucleotide coding for said polypeptide;

c) a polynucleotide able to inhibit the expression of said gene;

d) a vector comprising or expressing the polynucleotide as defined in b-c);

e) a host cell genetically engineered expressing said polypeptide or said polynucleotide; and

f) a small molecule.

More preferably, said molecule is selected from the group consisting of: JNKi IX, SP600125/JNKi II, BIRB- 796, VX-745, JNKi XI, SB202190, Pazopanib, Dovitinib lactate, Bexarotene, Flunarizine, Cannabidiol, CPI- 1189 and ENMD-2076.

In a preferred embodiment the molecule is selected from the group consisting of: JNKi IX, SP600125/XNKi II, JNKi XI, Pazopanib, Dovitinib lactate, Bexarotene and the protein conformational disorder is cystic fibrosis. In another preferred embodiment the molecule is selected from the group consisting of: VX-745, BIRB-796, JNKi II, SB202190, Bexarotene, Cannabidiol, CPI-1189 and ENMD-2076 and the protein conformational disorder is Wilson disease.

The above polynucleotide able to inhibit the expression of said gene is preferably at least one RNAi agent targeting at least one of the above disclosed gene (also defined as RNAi inhibitor). Said RNAi agent is preferably selected from the group consisting of: siRNA, miRNA, shRNA, stRNA, snRNA, and antisense nucleic acid, or a functional derivative thereof.

The molecule for use according to the invention may be in combination with a therapeutic agent. Said the therapeutic agent is preferably the pharmacochaperone VX-809 when the protein conformational disorder is cystic fibrosis.

A further object of the invention is a pharmaceutical composition comprising at least one molecule as above defined and at least one pharmaceutically acceptable carrier. Said pharmaceutical composition may be for medical use, preferably for use in the treatment of a protein conformational disorder, preferably of cystic fibrosis or WD. Another object of the invention is a method of treating and/or preventing a protein conformational disorder comprising administering to a patient in need thereof a therapeutically effective amount of at least one molecule as above defined.

Chemical structures of the above disclosed molecules are represented in table 6.

SU5402 chemical structure is:

SU6668 chemical structure is:

By the term "suppressor or inhibitor" or a "molecule which (selectively) suppresses or inhibits" it is meant a molecule that effects a change in the expression and/or function of the target. The change is relative to the normal or baseline level of expression and/or function in the absence of the "suppressor or inhibitor" or of the molecule, but otherwise under similar conditions, and it represent a decrease in the normal/baseline expression and/or function. The suppression or inhibition of the expression and/or function of the target may be assessed by any means known to the skilled in the art. The assessment of the expression level or of the presence of the target is preferably performed using classical molecular biology techniques such as (real time Polymerase Chain Reaction) qPCR, microarrays, bead arrays, RNAse protection analysis or Northern blot analysis or cloning and sequencing. The assessment of target function is preferably performed by in vitro suppression assay, whole transcriptome analysis, mass spectrometry analysis to identify proteins interacting with the target. In the context of the present invention, the target is the gene, the mRNA, the cDNA, or the encoded protein thereof. The above described molecules also include salts, solvates or prodrugs thereof. The above described molecules may be or not solvated by H2O. The polynucleotides as above described, as e.g. the siRNAs, may further comprise dTdT or UU 3 '-overhangs, and/or nucleotide and/or polynucleotide backbone modifications as described elsewhere herein. In the context of the present invention, the term "polynucleotide" includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA, siRNA, shRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The polynucleotide may be single-stranded or double-stranded. The RNAi inhibitors as above defined are preferably capable of hybridizing to all or part of specific target sequence. Therefore, RNAi inhibitors may be fully or partly complementary to all of or part of the target sequence. The RNAi inhibitors may hybridize to the specified target sequence under conditions of medium to high stringency. An RNAi inhibitors may be defined with reference to a specific sequence identity to the reverse complement of the sequence to which it is intended to target. The antisense sequences will typically have at least about 75%, preferably at least about 80%, at least about 85%, at least about 90%, at least about 95%> or at least about 99%> sequence identity with the reverse complements of their target sequences.

The term polynucleotide and polypeptide also includes derivatives and functional fragments thereof. The polynucleotide may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides).

The molecule according to the invention may be an antibody or derivatives thereof.

In the context of the present invention, the genes as above defined are preferably characterized by the sequences identified by their Gen Bank Accession numbers, as disclosed in Tables 1 and 2. The term gene herein also includes corresponding orthologous or homologous genes, isoforms, variants, allelic variants, functional derivatives, functional fragments thereof. The expression "protein" is intended to include also the corresponding protein encoded from a corresponding orthologous or homologous genes, functional mutants, functional derivatives, functional fragments or analogues, isoforms thereof.

In the context of the present invention, the term "polypeptide" or "protein" includes:

i. the whole protein, allelic variants and orthologs thereof;

ii. any synthetic, recombinant or proteolytic functional fragment;

iii. any functional equivalent, such as, for example, synthetic or recombinant functional analogues. In the present invention "functional mutants" of the protein are mutants that may be generated by mutating one or more amino acids in their sequences and that maintain their activity. Indeed, the protein of the invention, if required, can be modified in vitro and/or in vivo, for example by glycosylation, myristoylation, amidation, carboxylation or phosphorylation, and may be obtained, for example, by synthetic or recombinant techniques known in the art. The term "derivative" as used herein in relation to a protein means a chemically modified peptide or an analogue thereof, wherein at least one substituent is not present in the unmodified peptide or an analogue thereof, i.e. a peptide which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters and the like. As used herein, the term "derivatives" also refers to longer or shorter polypeptides having e.g. a percentage of identity of at least 41 % , preferably at least 41.5%, 50 %, 54.9% , 60 %, 61.2%, 64.1%, 65 %, 70 % or 75%, more preferably of at least 85%, as an example of at least 90%, and even more preferably of at least 95% with the herein disclosed genes and sequences, or with an amino acid sequence of the correspondent region encoded from orthologous or homologous gene thereof. The term "analogue" as used herein referring to a protein means a modified peptide wherein one or more amino acid residues of the peptide have been substituted by other amino acid residues and/or wherein one or more amino acid residues have been deleted from the peptide and/or wherein one or more amino acid residues have been deleted from the peptide and or wherein one or more amino acid residues have been added to the peptide. Such addition or deletion of amino acid residues can take place at the N-terminal of the peptide and/or at the C-terminal of the peptide. A "derivative" may be a nucleic acid molecule, as a DNA molecule, coding the polynucleotide as above defined, or a nucleic acid molecule comprising the polynucleotide as above defined, or a polynucleotide of complementary sequence. In the context of the present invention the term "derivatives" also refers to longer or shorter polynucleotides and/or polynucleotides having e.g. a percentage of identity of at least 41 % , 50 %, 60 %, 65 %, 70 % or 75%, more preferably of at least 85%>, as an example of at least 90%, and even more preferably of at least 95%> or 100%> with e.g. SEQ ID NO: 1-114 or with their complementary sequence or with their DNA or RNA corresponding sequence. The term "derivatives" and the term "polynucleotide" also include modified synthetic oligonucleotides. The modified synthetic oligonucleotide are preferably LNA (Locked Nucleic Acid), phosphoro-thiolated oligos or methylated oligos, morpholinos, 2'-0-methyl, 2'-0-methoxyethyl oligonucleotides and cholesterol-conjugated 2'-0-methyl modified oligonucleotides (antagomirs). The term "derivative" may also include nucleotide analogues, i.e. a naturally occurring ribonucleotide or deoxyribonucleotide substituted by a non-naturally occurring nucleotide. The term "derivatives" also includes nucleic acids or polypeptides that may be generated by mutating one or more nucleotide or amino acid in their sequences, equivalents or precursor sequences. The term "derivatives" also includes at least one functional fragment of the polynucleotide.In the context of the present invention "functional" is intended for example as "maintaining their activity". As used herein "fragments" refers to polynucleotides having preferably a length of at least 1000 nucleotides, 1100 nucleotide, 1200 nucleotides, 1300 nucleotides, 1400 nucleotides, 1500 nucleotides or to polypeptide having preferably a length of at least 50 aa, 100 aa, 150 aa, 200 aa, 250 aa, 300 aa...... The term "polynucleotide" also refers to modified polynucleotides. As used herein, the term "vector" refers to an expression vector, and may be for example in the form of a plasmid, a viral particle, a phage, etc. Such vectors may include bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, lentivirus, fowl pox virus, and pseudorabies. Large numbers of suitable vectors are known to those of skill in the art and are commercially available. The polynucleotide sequence, preferably the DNA sequence in the vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, one can mention prokaryotic or eukaryotic promoters such as CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. The expression vector may also contain a ribosome binding site for translation initiation and a transcription vector. The vector may also include appropriate sequences for amplifying expression. In addition, the vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydro folate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli. As used herein, the term "host cell genetically engineered" relates to host cells which have been transduced, transformed or transfected with the polynucleotide or with the vector described previously. As representative examples of appropriate host cells, one can cite bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, insect cells such as Sf9, animal cells such as CHO or COS, plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. Preferably, said host cell is an animal cell, and most preferably a human cell. The introduction of the polynucleotide or of the vector described previously into the host cell can be effected by method well known from one of skill in the art such as calcium phosphate transfection, DEAE-Dextran mediated transfection, electroporation, lipofection, microinjection, viral infection, thermal shock, transformation after chemical permeabilisation of the membrane or cell fusion. The polynucleotide may be a vector such as for example a viral vector. The polynucleotides as above defined can be introduced into the body of the subject to be treated as a nucleic acid within a vector which replicates into the host cells and produces the polynucleotides. Suitable administration routes of the pharmaceutical composition of the invention include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intraventricular, intraperitoneal, intranasal, intraocular, parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous), chemoembolization. Other suitable administration methods include injection, viral transfer, use of liposomes, e.g. cationic liposomes, oral intake and/or dermal application. In certain embodiments, a pharmaceutical composition of the present invention is administered in the form of a dosage unit (e.g., tablet, capsule, bolus, etc.). For pharmaceutical applications, the composition may be in the form of a solution, e.g. an injectable solution, emulsion, suspension or the like. The carrier may be any suitable pharmaceutical carrier. Preferably, a carrier is used which is capable of increasing the efficacy of the molecules to enter the target cells. Suitable examples of such carriers are liposomes. In the pharmaceutical composition according to the invention, the suppressor or inhibitor may be associated with other therapeutic agents. The pharmaceutical composition can be chosen on the basis of the treatment requirements. Such pharmaceutical compositions according to the invention can be administered in the form of tablets, capsules, oral preparations, powders, granules, pills, injectable, or infusible liquid solutions, suspensions, suppositories, preparation for inhalation. A reference for the formulations is the book by Remington ("Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins, 2000). The expert in the art will select the form of administration and effective dosages by selecting suitable diluents, adjuvants and / or excipients. Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., using a variety of well-known mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The compositions may be formulated in conjunction with one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Parenteral routes are preferred in many aspects of the invention. For injection, including, without limitation, intravenous, intramusclular and subcutaneous injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as physiological saline buffer or polar solvents including, without limitation, a pyrrolidone or dimethylsulfoxide. The compounds are preferably formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Useful compositions include, without limitation, suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain adjuncts such as suspending, stabilizing and/or dispersing agents. Pharmaceutical compositions for parenteral administration include aqueous solutions of a water soluble form, such as, without limitation, a salt of the active compound. Additionally, suspensions of the active compounds may be prepared in a lipophilic vehicle. Suitable lipophilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxym ethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers and/or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers well-known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, pastes, slurries, solutions, suspensions, concentrated solutions and suspensions for diluting in the drinking water of a patient, premixes for dilution in the feed of a patient, and the like, for oral ingestion by a patient. Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropyl- methylcellulose, sodium carboxy- methylcellulose, and/or polyvinylpyrrolidone (PVP). For administration by inhalation, the molecules of the present invention can conveniently be delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellant The moelcules may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. The compounds of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materials (for instance, in an emulsion with a pharmacologically acceptable oil), with ion exchange resins, or as a sparingly soluble derivative such as, without limitation, a sparingly soluble salt. Additionally, the compounds may be delivered using a sustained-release system, such as semi-permeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. A therapeutically effective amount refers to an amount of compound effective to prevent, alleviate or ameliorate the protein conformational disease. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the disclosure herein. Generally, the amount used in the treatment methods is that amount which effectively achieves the desired therapeutic result in mammals. In particular, the molecules administration should follow the current clinical guidelines. A suitable daily dosage will range from 0.001 to 10 mg / kg body weight, in particular 0.1 to 5 mg / kg. In the case of polynucleotides a suitable daily dosage may be in the range of 0.001 pg/kg body weight to 10 mg/kg body weight. Typically the patient doses for parenteral administration of the molecules described herein range from about 1 mg/day to about 10,000 mg/day, more typically from about 10 mg/day to about 1 ,000 mg/day, and most typically from about 50 mg/day to about 500 mg/day. The range set forth above is illustrative and those skilled in the art will determine the optimal dosing of the compound selected based on clinical experience and the treatment indication. The invention will be now illustrated by means of non-limiting examples referring to the following figures. Figure 1 : Corrector drugs modulate a set of CORE genes.

A. Schema of the FIT method. The upregulated (top 20%) and downregulated genes (bottom 20%) were fuzzy intersected to identify CORE genes. B. To obtain optimal fuzzy cut-off for the analysis, the corrector drug profiles (MANTRA dataset) as well as random profiles from MANTRA database were intersected with variable fuzzy cut-offs (represented as number of drugs out of 11). The enlargement (inset) shows that at the optimal fuzzy cut-off (0.7; 8 out of 11 drugs), the signal-to-noise ratio was close to 3 (108 probe-sets in the corrector drug intersection vs 32 in the random). C. Next, with a fuzzy cut-off of 0.7, the number of random drug profiles used was varied, and the number of probe-sets present in the intersection is shown. D. Using the optimal parameters (see B, C) the FIT analysis resulted in 402 upregulated and 219 downregulated CORE genes. E. The number of CORE genes associated with the enriched GO terms is shown. Those genes that did not associate with enriched GO terms were excluded from the chart. F. Protein-protein interactions between the CORE and the proteostasis genes (restricted to those that occur between the two groups) are shown. Figure 2 : Validation of the selected CORE genes.

A-D. CFBE cells were treated with siRNAs targeting CORE genes and changes in F508del-CFTR proteostasis monitored by western blotting. The fold change in the levels of band C obtained by downregulating negative correction (A) and positive correction (D) genes and the fold change in levels of band B (B) and band CI band B ratio (C) after downregulation of the negative correction genes are shown. The effects of negative control siRNAs (dashed line) and VX-809 (dark grey) are indicated. E. The validated CORE genes were assembled into coherent networks based on information from databases. Non-directional interactions denote protein-protein interaction, directional interactions represent phosphorylation cascades and dashed arrows indicate indirect connections through intermediaries. F. Treatment of CFBE cells with mitoxantrone (2.5 to 20 μΜ for 48h), a potential corrector identified using downregulation of anti-corrector genes as selection criteria, increased the levels of both band C and band B. G. Treatment of CFBE cells with the indicated combinations of siRNAs targeting CORE genes led to a synergistic increase in the band C levels. A representative blot is shown in the insert.

Figure 3 : Downregulation of CORE genes rescues F508del-CFTR more efficiently than the corrector drugs used originally, without altering the F508del-CFTR mRNA levels (related to Figure 2). A. CFBE cells were treated with indicated corrector drugs for 48h and then lysed and prepared for western blotting, to assay the rescue of F508del-CFTR from ERQC. The changes in the levels of band C after drug treatment are shown as mean ± SEM (n > 3).

B. CFBE cells were treated with indicated siRNAs (targeting the anti-correction genes) for 72h, and then total RNA from the cells was purified. The levels of CFTR mRNA were then quantitated by RT-PCR. The data is presented as mRNA levels relative to the negative control siRNAs. The values are expressed as mean ± SEM (n = 4). C. Representative blot used for quantitation's represented in Figure 2A-C. D. Representative blot used for quantitation's represented in Figure 2D

Figure 4: Delineation of the MLK3 pathway branch that controls F508del-CFTR proteostasis.

A. CFBE cells were treated with indicated siRNAs targeting the upstream activators of MLK3 and their effect on F508del-CFTR proteostasis monitored by western blotting. The fold change in band C levels is shown. Reduction in TGF receptor, HPK, CDC42 and RAC2 levels rescued F508del-CFTR from ERQC. The rescue obtained with TNFR2 siRNA was quite variable and so was not considered further. B. JNK isoforms were tested for their effect on F508del-CFTR proteostasis after siRNA-mediated downregulation of their levels. Downregulation of JNK2 leads to efficient rescue of F508del-CFTR that is comparable to that obtained with MLK3. C. CFBE cells were transfected with activators of the MLK3 pathway to study their effect on F508del-CFTR proteostasis. All of them reduced the levels of both band C (not shown) and band B of F508del-CFTR. The corresponding increase in the levels of phospho-c-jun indicates an increase activation of the MLK3 pathway activity. D-E. Schematic representation of the proposed MLK3 (D) and CAMKK2 (E) pathways that regulate F508del-CFTR proteostasis. The directional interactions proposed between the components of the pathways are based on published literature.

Figure 5: Delineation of the MLK3 and CAMKK2 pathway branches that regulate F508del-CFTR proteostasis (related to Figure 4).

A. HeLa cells [HeLa cells stably expressing HA-tagged F508del-CFTR] were treated with indicated siRNAs targeting MLK3 pathway components including p38 MAPK (mix of siRNAs targeting all 4 isoforms) and JNK (mix of siRNAs targeting all 3 JNKs). The effect on F508del-CFTR proteostasis monitored by western blotting. Fold Change in the levels of band C was quantitated and represented as mean ± SEM (n > 3), with a representative blot shown in the insert. The downregulation of the MLK3 pathway components (including p38 MAPK) leads to the rescue of F508del-CFTR in HeLa cells. SiRNAs targeting Rmal and Ahal used as positive controls for rescue of F508del-CFTR.

B. Screening for F508del-CFTR proteostasis regulators among the CORE genes led to the identification of CAMKK2 as an anti-correction hit. Three downstream components and 9 upstream components of the CAMKK2 signaling pathway (as derived from literature mining) were tested, by siRNA-mediated downregulation, for their role in regulation of F508del-CFTR proteostasis. CFBE cells were treated with the indicated siRNAs for 72h and their effect on F508del-CFTR proteostasis monitored by western blotting. Four of them (CALML5, ITPR2, CAMK1 and AMPK [by a mix of siRNAs targeting PRKAAl and PRKAA2]) rescued F508del-CFTR from ERQC as seen by an increase in band C levels. C. The changes in the levels of band C from (B) were quantitated and are represented as mean ±SEM (n>3). See Figure 4 for a representation of the derived CAMKK2 pathway that regulates F508del-CFTR proteostasis.

Figure 6: MLK3 pathway regulates the degradation of F508del-CFTR.

A-B. CFBE cells pretreated with siRNAs were treated with CHX (5C^g/mL) for indicated times and the levels of band B of F508del-CFTR was monitored (A). The levels were quantitated and represented in (B). Downregulation of MLK3 or JNK2 reduced the kinetics of reduction of band B of F508del-CFTR. C-D. CHX chase assay (see above) after overexpression of the activators of MLK3 pathway. The activation of MLK3 pathway increases the rate of degradation of band B (C). Quantitation of the blot is shown in (D). The results are representative of 3 independent experiments. E-F. CFBE cells were treated with indicated siRNAs followed by incubation at 26 °C for 6h followed by shift to 37 °C for the indicated time periods. The changes in band C levels were monitored as measure of PQC (C). See (F) for quantitation of band C levels. G-H. PQC assay (see above) after overexpression of CDC42 or JNK2 shows an increased rate of degradation of band C (G) upon CDC42 overexpression. JNK2 overexpression has no effect on the PQC of F508del-CFTR. The blots were quantified and presented in (H).

Figure 7: Characterization of the mode of action of the MLK3 pathway on F508del-CFTR proteostasis (related to Figure 6). A-B. CFBE cells treated with MLK3 siRNA were pulsed with radioactive [35S]- cysteine and methionine for 15 min, and then chased for the indicated times. CFTR was immunoprecipitated and processed for autoradiography (A). The signals corresponding to band B from (A) were quantitated and presented in (B). The data are representative of 2 independent experiments. Note the reduced degradation of F508del-CFTR upon downregulation of MLK3. C. Down-regulation of the MLK3-JNK pathway does not affect the activity of proteasomes. CFBE cells treated with MLK3 or JNK2 siRNA for 72h were transfected with Proteasome ZsProsensor-1 for the final 24 h, and the levels Proteasome ZsProsensor-1 monitored by fluorescence microscopy. Treatment with MG132 (20 μg/ml for 3h), a proteasomal inhibitor, was used as a positive control. While treatment with MG132 increases the fluorescence levels of ZsProsensor-1 compared to untreated cells indicating a reduced proteasome activity, downregulation of MLK3 or JNK2 did not change the levels of fluorescence, suggesting that proteasome activity is not changed under these conditions. D. CFBE cells were treated with MLK3 or JNK2 siRNA and processed for western blotting to monitor the accumulation of poly ubiquitinated proteins. There was no change in the levels of poly ubiquitinated proteins suggesting that these treatments do not affect proteasome activity. E. Down-regulation of MLK3 does not affect the folding of F508del-CFTR. CFBE cells expressing wild type CFTR or F508del-CFTR were treated with MLK3 siRNA as indicated. Untreated CFBE cells incubated at 26 °C for 24h were used as a positive control for the promotion of folding. Membrane fractions from the cells were isolated and subjected to trypsin digestion for 10 min on ice, followed by western blotting with M3A7 antibody that recognizes the NBD2 domain of F508del-CFTR, or with 3G11 antibody that recognizes NBD1. The wild-type CFTR and its NBD domains show more resistance to trypsin digestion compared to F508del-CFTR. There was no change in the stability of F508del-CFTR or its NBD domains upon down-regulation of MLK3, while the low temperature treatment enhanced the stability of F508del-CFTR and its NBD1 domain. The Western blots are representative of at least 3 different experiments.

Figure 8: Inhibitors of the MLK3 pathway rescue F508del-CFTR.

(A) CFBE cells were treated with the indicated inhibitors of the MLK3 pathway or VX-809 for 48 h, and the rescue of F508del-CFTR from was monitored by increase in band C western blotting.

(B) Fold changes in the levels of band C, normalized concentration refers to concentration [VX-809, JNKi ΓΧ and Oxozeaenol (1.25, 2.5, 5, 10 μΜ), JNKi II (6.25, 12.5, 25, 50 μΜ), JNKi XI, Pazopanib, Dovitinib lactate and Bexarotene (3.12, 6.25, 12.5, 25 μΜ)] values that were normalized to the maximum used concentrations of the respective drugs. Also refer panel A for concentrations (μΜ) [±SEM (n>3)]. C. CFBE cells were treated with inhibitors of the MLK3 pathway and/or VX-809 (5μΜ) for 48 h and changes in band C levels monitored. The concentrations of the MLK3 pathway inhibitors used were: JNKi II (12.5μΜ), JNKi IX (5μΜ), JNKi XI (25μΜ) and oxozeaenol (5μΜ). Wild type CFTR (wt-CFTR) was used as a control. D. Quantitation of band C levels from (C), normalized to the levels of band C after VX-809 treatment are shown. The results show that synergy obtained between the MLK3 pathway inhibitors and VX-809 brings the levels of band C to about 40% of the wild type levels.

Figure 9: Small-molecule inhibitors of the MLK3 pathway rescue F508del-CFTR and other structurally related mutant proteins from degradation (related to Figure 8 and Table 5).

A. CFBE cells were treated with indicated JNK inhibitors for 24h and processed for western blotting. The levels of phospho-c-jun as a measure of JNK inhibition was monitored. MLK3 pathway inhibitors reduce phospho-c-jun levels efficiently indicating a strong reduction in the activity of JNK and hence presumably of the MLK3 pathway. B. CFBE cells were treated with TAKl or MLK3 siRNA as indicated and changes in F508del-CFTR proteostasis were monitored by western blotting. TAKl does not regulate F508del-CFTR proteostasis, as evidenced by the absence of change in the levels of bands C or B. The fold change in the band C levels were quantitated and plotted as mean ±SD (n = 2).

C. CFBE cells were treated with 5 μΜ oxozeaenol for 48 h, or with MLK3 siRNA, or with both, and the correction of the F508del-CFTR folding/ trafficking defect was monitored by changes in the levels of band C. There was no additive effect observed with the combination of MLK3 downregulation and oxozeaenol treatment. The quantitated band C levels are expressed as mean ±SD (n > 3). D. CFBE cells were treated with 5 μΜ oxozeaenol for 24 h, and the activity of the JNK pathway was measured by western blotting for phospho c-jun levels and F508del-CFTR. The levels of phospho c-jun were reduced suggesting that oxozeaenol leads to a reduction in the activity of JNK. The increase in band C levels of F508del-CFTR show that the reduction in the activity of JNK is accompanied by a rescue of F508del-CFTR from ERQC. E. CFBE cells were treated with flunarizine (at concentrations 6.25-50μΜ) targeting the CAMKK2 pathway for 48h and the effect on F508del-CFTR proteostasis measured by western blotting. Treatment with flunarizine increased the levels of band C of F508del-CFTR. Other small molecules known to inhibit the CAMKK2 pathway components (verapamil and STO-609) did not show any effect on correction of F508del-CFTR. F. CFBE cells transiently transfected with the P-glycoprotein mutant (P-gp DY490), the NCC mutant (R948X), or the hERG mutant (G601S) were treated with JNKi II for 24 h, and the effect of the drug on their proteostasis monitored by western blotting. While the trafficking of P-gp DY490 out of the ER was enhanced by this treatment (seen as an increase in the Golgi-associated band C, indicated by arrows), other mutants are subjected to enhanced degradation upon drug treatment, as shown by a decrease in the levels of both bands B and C.

Figure 10: Small molecule inhibitors of MLK3 pathway rescue the channel function of F508del-CFTR. A. F508del-CFTR and Halide sensitive YFP (Galietta et al., 2001) expressing CFBE (CFBE-YFP) cells were treated with MLK3 pathway inhibitors and/or VX-809 for 48 h, and the anion transport measured as described in the Materials and methods (Halide sensitive YFP assay for CFTR activity). The rate constants of the decrease in YFP fluorescence (K), a measure of anion conductance, after inhibitor treatments are shown. The data are expressed as mean ±SEM (n > 3). B. Anion transport was measured in CFBE-YFP cells after downregulating the MLK3 pathway activity by siRNA-mediated knockdown MLK3 or JNK2. The rate constants of the decrease in YFP fluorescence (K), a measure of anion conductance, after downregulation of the indicated MLK3pathway components are shown. The data are expressed as mean ±SEM (n > 3). Treatment with VX-809 was used as a positive control for the rescue. C. CFBE41o- cells were grown under polarising conditions before addition of oxozeaenol at indicated concentrations for 48 h, followed by the measurement of short circuit currents using Ussing chamber assays. The columns show the measured values of the short circuit current after oxozeaenol treatment at the indicated concentrations. The values are mean ±SEM (n>3).

Figure 11 Silencing of several MLK3 pathways genes corrects localization and trafficking of the ATP7BH1069Q mutant.

(A) HeLa cells were incubated with siRNA, which target specific genes (indicated in graph) belonging to p38 and JNK pathways, then infected with Ad-ATP7BH1069Q-GFP (Chesi et al., 2016) and incubated for 2h with 100 μΜ CuS04. Fixed cells were then labeled for TGN46 and visualized under confocal microscope. Silencing of MAPK8, MAPK11, MAPK14 or MAP3K11 results in rescue of ATP7BH1069Q from the ER and its movement to post-Golgi vesicles (arrows) and PM. (B) Cells were treated as in panel B. The percentage of the cells (average ± SD, n=10 fields) with ATP7BH1069Q signal in the ER was calculated. RNAi of MAPK8, MAPK11, MAPK14 and MAP3K11 reduced the percentage of the cells exhibiting ATP7BH1069Q in the ER. Scale bar: 4.7 μιη.

Figure 12 Inhibitors of MLK3 pathway inhibitors correct localization and trafficking of ATP7BH1069Q mutant.

(A) HeLa cells were infected with Ad-ATP7BWT-GFP (Chesi et al., 2016) or Ad-ATP7BH1069Q-GFP, incubated overnight with 200 μΜ BCS, and incubated for an additional 2h with 100 μΜ CuS04. In response to Cu, ATP7BWT traffics from the Golgi to PM and vesicle, while ATP7BH1069Q are retained within the ER under high Cu conditions. Addition of p38 inhibitor SB202190 (5 μΜ), νΧ-745(1μΜ), JNK inhibitor SP600125 (2 μΜ) and Oxozeaenol (5 μΜ) (as indicated in corresponding panels) to the cells 24h corrects ATP7BH1069Q from the ER to PM and vesicles (arrows) (B) Cells were treated as in panel A. The percentage of the cells (average ± SD, n=50 fields) with an ATP7B signal in the ER, were calculated. The p38 inhibitors SB202190 (5 μΜ), νΧ-745(1 μΜ), JNK inhibitor SP600125 (2 μΜ) and Oxozeaenol (5 μΜ) reduced the percentage of the cells exhibiting ATP7BH1069Q in the ER and increases the number of cells in which ATP7B was corrected to PM and vesicles.

Figure 13: Small-molecule inhibitors of MLK3-JNK pathway rescue ATP7B H1069Q localization to the Golgi apparatus. Transiently ATP7B H1069Q-GFP expressing HeLa cells (A), HEPG2 cells and (C) human primary hepatocytes (E) treated with the inhibitors for 24 hours, cells are processed for immunofluorescence assay to measure the arrival of the ATP7B wt and H1069Q mutant from the ER to Golgi compartment. A, C, E) Normalized Golgi fluorescence of ATP7B is measured and plotted (n >50 cells). B, D, F) EC50 and recue effect (%) compared to the level ATP7B WT Golgi fluorescence calculated from (A), (C) and (E) respectively. Inhibitor SB202190 and VX-745 (or VX745) was used as positive control in our rescue assay and it has been shown to rescue the transport and function of ATP7B H1069Q (Chesi, Hegde et al. 2016). All the inhibitor drugs except SB202190 are in clinical trial for treatment of various other diseases.

Figure 14: The VX-745 and BIRB-796 correctors reduce Copper levels in cells expressing ATP7BH1069Q mutant. HepG2 cells overexpressing indicated ATP7B constructs were incubated with ΙμΜ each of VX-745, BIRB-796 for 24 hours, for the last 2 hours of incubation with the inhibitors 200 μΜ copper sulphate is added to the cells with or without ΙΟΟμΜ BCS (copper chelator) as indicated. Cells were incubated with HBSS for 3 hours before lysed and used for copper estimation. Fold change in copper normalized to control is plotted (n=4). Both VX-745 and BIRB-796 reduced Copper levels in ATP7BH1069Q expressing cells (BCS is used in the assay as a control for the sensitivity of the assay).

Examples

Materials and methods

Cell culture, antibodies, plasmids and transfection

CFBE cells stably expressing wild type CFTR or F508del-CFTR (Bebok et al. 2005) and stably expressing halide sensitive YFP (Pedemonte et al. 2005) and HeLa cells stably expressing HA-tagged F508del-CFTR (Okiyoneda et al. 2010) were used. CFBE cells were cultured in Minimal Essential Medium supplemented with 10% foetal bovine serum, non-essential amino acids, glutamine, penicillin/ streptomycin and 2 μg/ml puromycin. This media additionally supplemented with 50 μg/ml G418 was used for the CFBE-YFP cells. HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%> foetal bovine serum, glutamine, penicillin/ streptomycin and 1 μg/ml puromycin. The antibodies used were: anti- phospho-c-jun (Cell Signaling Technology), monoclonal anti-HA, anti-actin and anti-tubulin (Sigma), rat anti-CFTR (3G11; CFTR Folding Consortium), mouse monoclonal anti-CFTR (M3A7), HRP-conjugated anti mouse, rabbit and rat IgG (Merckmillipore) and Anti-Na/K+ATPase al (Thermoscientific). The plasmids used were: JNK2 (pCDNA3 Flag MKK7B2Jnk2a2; Addgene plasmid #19727) and MKK7 (pCDNA3 Flag MKK7M ; Addgene plasmid #14622,) from Roger Davis (University of Massachusetts Medical School, Worcester, USA), ZsProSensor-1 proteasome sensor (Clontech), VSVG tagged with GFP (Jennifer Lippincott-Schwartz, NICHD, NIH, Bethesda, USA), Cdc42 (A. Hall, Sloan-Kettering Institute, New York, NY, USA), P-glycoprotein wild type, G268V and DY490 mutants (David M. Clarke, University of Toronto, Canada) and hERG wild type and G601S mutant (Alvin Shrier, McGill University, Montreal, Canada). The reagents used include: VX-809 (Selleckchem), JNKi II (SP600125), JNKi IX and JNKi XI (Merck Millipore), oxozeaenol (Tocris Bioscience), siRNAs (Table 3), lipofectamine 2000 (Invitrogen) and ECL (Luminata crescendo from Merck Millipore), BIRB-796 (Sigma), VX-745 (Sigma), SB202190 (Sigma), pazopanib, dovitinib lactate (Sigma), bexarotene (Sigma), flunarizine (Sigma), cannabidiol (Sigma), CPI- 1189 (Sigma) and ENMD-2076 (Sigma).

Analysis of corrector-induced gene expression changes by microarray

Polarised CFBE41o-cells (cystic fibrosis bronchial epithelial cells) cultured at the air-liquid interface were treated with the corrector drugs of interest (CFBE dataset, Table 4) for 24h. Total RNA was extracted and hybridization was carried out on to Whole Human Genome 44 K arrays (Agilent Technologies, product G4112A) following the manufacturer's protocol. See (Zhang et al. 2012) for experimental details. The microarray data for ouabain and low temperature treatments have been published (Zhang et al. 2012).

FIT analysis of microarray profiles

The microarrays from the connectivity map database (https://www.broadinstitute.org/cmap/) were processed to produce prototype ranked lists (PRLs) (lorio et al. 2010). In these PRLs, cell line specific responses are diluted, thus summarising consensual transcriptional responses to drug treatment. In each PRL, microarray probe-sets are ordered from the most upregulated to most downregulated one. Inventors downloaded PRLs for the whole panel of small molecules in the connectivity map (www.connectivitymap.org) from which the MANTRA database is derived (http://mantra.tigem.it/). Inventors used these in conjunction with ranked lists of probe sets based on fold-changes (and assembled following the guidelines provided in (lorio et al., 2010)) from microarray profiles that inventors generated in house (CFBE dataset). The FIT analysis identifies microarray probe-sets that tend to respond consistently to a group of drugs (see also (lorio et al. 2010) for description of a similar method). The top and bottom 20% of the probe-sets (corresponding to the up- and downregulated probe-sets respectively) were used for the analysis. The 20% cut-off was used since the merging of individual gene expression profiles into PRLs precludes the application of other thresholds based on fold-change (or p-value) to identify significantly differentially expressed genes. To build a null model against which the significance of the final genes sets can be tested (as detailed below), a fixed number of PRLs (N) from the MANTRA dataset were randomly selected and the upregulated or downregulated probe- sets from this selection were intersected by varying the fuzzy cut-off threshold (i.e. the ratio of drugs that a given probe-set should transcriptionally respond to, in order to be considered 'consistently' regulated, hence to be included in the fuzzy intersection). After 1000 of these iterations, inventors derived an empirical null distribution of the number of probes included in the resulting fuzzy intersections and used it for p-value assignments (Figure IB). For the CFBE dataset, (generated on an Agilent platform, which is different from that used for the connectivity map and MANTRA database) inventors derived this null distribution by randomly permuting all the individual probes. Finally, inventors determined the optimal fuzzy cut-off values for the transcriptional profiles elicited by the corrector drugs (11 contained in MANTRA and 13 in the CFBE dataset). Briefly, inventors selected the value such that the number of probes present in the final fuzzy intersection was at least 3 fold higher than that expected by random chance and its p-value < 0.05 (according to the computed null models). By using this method, no significantly upregulated probes from the MANTRA dataset were identified across all of the range of tested fuzzy cut-offs. For the downregulated probe-sets a fuzzy cut-off of 8 (out of 11 corrector drugs) or above produced significant fuzzy intersection of probe-sets. For the CFBE dataset, a significant cut-off of 6 drugs (out of 13) and above was identified. To further optimise the selection of these cut-offs, inventors chose the maximal cut-off yielding a fuzzy intersection of probe-sets enriched in one or more Gene Ontology terms. With this criterion, inventors obtained a final cutoff value of 8 for the MANTRA down-regulated probe set and cut-off of 9 for the CFBE dataset. Intersecting the corrector-induced gene expression profiles using this optimal fuzzy cut-off resulted in 541 upregulated probe-sets (mapping 402 unique genes) and 191 downregulated probe-sets (mapping to 117 unique genes) for the CFBE dataset, and 108 downregulated probe-sets (mapping 102 genes) for the MANTRA dataset. Note, that most of the CORE genes (about 500 out of the 600) are derived from the CFBE dataset. This inventors suppose is due to the use of PRLs in the case of cMAP dataset and use of data derived from a single cell line in the case of CFBE dataset. The use of single cell line derived data can potentially lead to high number of false positives since perturbation-independent response of cell lines to treatments is usually stronger than the perturbation-dependent response (Iorio et al. 2010). Inventors finally validated the optimal number of drugs that need to be considered for a fuzzy cut-off of 70% (corresponding to 8 out 11 drugs cut-off from the MANTRA dataset), providing a minimum number of false positives in the intersection (i.e. genes expected to be contained in the resulting intersections by random chance). This was performed by a permutation test where, in a series of iterations, the fuzzy cut-off is kept constant and the number of randomly selected drugs varied within a given range (specifically from 1 to 20). At each of these iterations inventors computed the cardinality 1 of the resulting fuzzy intersections, observing that this value reached a plateau at 10 drugs (Figure 1C), which suggests that the number of drugs that was used in the analysis (i.e. 11 drugs in the cMAP dataset) was fairly close to the optimal level.

Protein-protein interaction

The protein-protein interactions were downloaded from the STRING database (http://string-db.org/) (Franceschini et al. 2013), and those with a confidence level of >0.7 were used for the analysis. To build the proteostasis gene (PG) dataset, inventors included known proteostatic regulators of CFTR i.e., proteins where their expression/ activity level changes have been shown to affect CFTR proteostasis. Inventors also included the interactors of CFTR and CF pathology related genes/ proteins present in GeneGO Metaminer Cystic Fibrosis database. The number of interactions observed among the CORE gene dataset and the proteostasis gene dataset as well as among the CORE gene dataset were more than expected on a random basis and were statistically significant. For details on the statistical test used see (Franceschini et al. 2013).

Ingenuity pathway analysis (IP A) The gene sets were analyzed using the CORE analysis application of the Ingenuity pathway analysis, a web- based software application. The default settings of the analysis were used. Each network had an assigned significance score based on the p-value (calculated using Fischer's exact test) for the probability of finding the focus genes in a set of genes randomly selected from the global molecular network. The upregulated and downregulated genes of the CFBE dataset and the downregulated genes of the cMAP dataset were analyzed separately and also together, to infer common pathways or networks embedded among them.

Cell lysis, western blotting and analysis

Cells were washed three times in ice-cold Dulbecco's phosphate-buffered saline, and lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 20 niM Tris-HCl, pH 7.4), supplemented with protease inhibitor cocktail and phosphatase inhibitors. The lysates were clarified by centrifugation at 15000 x g for 15 min, and the supematants were resolved by SDS-PAGE. BCA Protein Assay kit (Pierce) was used to quantitate protein levels before loading. The western blots were decorated with appropriate antibodies and developed using ECL. The blots were then exposed to x-ray films and exposure time was varied to obtain optimal signal. The x-ray films were then scanned and the bands were quantitated using ImageJ gel-analysis tool. The protein concentration and the exposures used for quantitation of the blots were optimized to be in a linear range of detection.

Biochemical Screening assay:

Each gene was targeted by 3 siRNAs and as control non-targeting siRNAs provided by the manufacturer were used (Table 3). A gene was considered as active if: (1) at least two different siRNAs targeting a gene gave concordant changes in the levels of band C that was >2 SD from the mean value of the control siRNAs and (2) the change in band C levels was ±20%> of the level of band C obtained with the control siRNAs. Those genes that increased band C levels significantly upon their downregulation were termed anti-correction genes and those that decreased band C levels were termed pro-correction genes.

Immunoprecipitation

HeLa cells cultured in 10-cm plates (80%> confluence) were treated with appropriate corrector drugs for 24h. The cells then were washed three times in ice-cold Dulbecco's phosphate-buffered saline, and lysed in immunoprecipitation buffer (150 mM NaCl, 1% Triton X-100, 20 mM Tris-HCl, pH 7.4) on ice for 30min. The lysates were clarified by centrifugation at 15000 x g for 15 min, and the protein content of the supematants BCA quantitated by BCA Protein Assay kit (Pierce). Equal amounts of proteins from control and treated cell lysates were incubated with Protein-G sepharose beads conjugated with anti-HA antibody (Sigma) overnight at 4 °C. The beads were then washed in the immunoprecipitation buffer 5 times and the bound proteins eluted with HA-peptide (Sigma) at a concentration of 100 μg/ml. The eluted proteins were then resolved by SDS-PAGE and then immunoblotted.

Partial trypsin digestion of CFTR The trypsin digestion assay was similar to that described previously (Zhang, Kartner, and Lukacs 1998). Cells were grown in a 10-cm plate and post-treatment they were washed three times with 10 mL phosphate- buffered saline (PBS). They were then scraped in 5 ml PBS, and pelleted at 500 x g for 5 min in 4 °C. The cell pellet was resuspended in 1 mL of hypertonic buffer (250 niM sucrose, 10 niM Hepes, pH 7.2) and the cells were then homogenized using a ball bearing homogenizer. The nuclei and unbroken cells were removed by centrifugation at 600x g for 15 min. The membranes were then pelleted by centrifugation at 100,000x g for 30 min, and then resuspended in digestion buffer (40 niM Tris pH 7.4, 2 niM MgC12, 0.1 niM EDTA). Then membranes corresponding to 50 μg of protein were incubated with different concentrations of trypsin (1 to 50 μg/ml) on ice for 15 min. The reactions were stopped with the addition of soya bean trypsin inhibitor (Sigma) to a final concentration of 1 niM, and the samples were immediately denatured in sample buffer (62.5 niM Tris-1 HCL, pH 6.8, 2% SDS, 10% glycerol, 0.001% bromophenol, 125 mM dithiothreitol) at 37 °C for 30 min. The samples were resolved on 4%> to 16% gradient SDS-PAGE (Tris-glycine) and transferred onto nitrocellulose membranes. These membranes were developed with the 3G11 anti-CFTR antibodies (that recognize nucleotide binding domain 1 - NBD1) or the M3A7 clone (that recognizes nucleotide binding domain 2 -NBD2).

Plasma membrane quality control assay

The PQC assay was essentially as described previously (Okiyoneda et al. 2010). CFBE cells were untreated or treated with siRNAs for 72 h and for the final 31 h they were kept at low temperature (26 °C) and for an additional 5 h at 26 °C with CHX (100 μg/ml). Then the cells were shifted to 37 °C for 1.5 h with 100 μg/ml CHX before the turnover measurements started at 37 °C. The cells were lysed at 0, 1, 3 and 5 h and the kinetics of degradation of band C was examined by immunoblotting.

Halide sensitive YFP assay for CFTR activity

Twenty-four hours after plating, the CFBE cells that stably expressed halide sensitive YFP were incubated with the test compounds at 37 °C for 48 h. At the time of the assay, the cells were washed with PBS (containing 137 mM NaCl, 2.7 mM KC1, 8.1 mM Na2HP04, 1.5 mM KH2P04, 1 mM CaC12, 0.5 mM MgC12) and stimulated for 30 min with 20 μιη forskolin and 50 μιη genistein. The cells were then transferred to a Zeiss LSM700 confocal microscope, where the images were acquired with a 20x objective (0.50 NA) and with an open pinhole (459 μιη) at a rate of 330 ms/frame (each frame corresponding to 159.42 μιη x 159.42 μιη), at ambient temperature. The excitation laser line 488nm was used at 2%> efficiency coupled to a dual beam splitter (621nm) for detection. The images (8-bit) were acquired in a 512x512 format with no averaging to maximize the speed of acquisition. Each assay consisted of a continuous 300-s fluorescence reading with 30 s before and the rest after injection of an iodide-containing solution (PBS with CI- replaced by I-; final I- concentration in the well, 100 mM). To determine the fluorescence-quenching rate associated with I- influx, the final 200 s of the data for each well were fitted with a mono-exponential decay, and the decay constant K was calculated using GraphPad Prism software. Ussing chamber assay for short circuit current recordings

Short-circuit current (Isc) was measured across monolayers in modified Ussing chambers. CFBE41o- cells (1x106) were seeded onto 12-mm fibronectin-coated Snapwell inserts (Corning Incorporated) and the apical medium was removed after 24h to establish an air-liquid interface. Transepithelial resistance was monitored using an EVOM epithelial volt-ohmmeter and cells were used when the transepithelial resistance was 300- 400 Ω.αη2. CFBE41o- monolayers were treated on both sides with optiMEM medium containing 2% (v/v) FBS and one of the following compound: 0.1% DMSO (negative control), or compounds at the stated dosage for 48h before being mounted in EasyMount chambers and voltage clamped using a VCCMC6 multichannel current-voltage clamp (Physiologic Instruments). The apical membrane conductance was functionally isolated by permeabilising the basolateral membrane with 200μg/ml nystatin and imposing an apical-to- basolateral CI- gradient. The basolateral bathing solution contained 1.2 mM NaCl, 115 mM Na-gluconate, 25 mM NaHC03, 1.2 mM MgC12, 4 mM CaC12, 2.4 mM KH2P04, 1.24 mM K2HP04 and 10 mM glucose (pH 7.4). The CaC12 concentration was increased to 4mM to compensate for the chelation of calcium by gluconate. The apical bathing solution contained 115 mM NaCl, 25 mM NaHC03, 1.2 mM MgC12, 1.2 mM CaC12, 2.4 mM KH2P04, 1.24 mM K2HP04 and 10 mM mannitol (pH 7.4). The apical solution contained mannitol instead of glucose to eliminate currents mediated by Na+-glucose co-transport. Successful permeabilization of the basolateral membrane was obvious from the reversal of Isc under these conditions. Solutions were continuously gassed and stirred with 95% 02-5% C02 and maintained at 37°C. Ag/AgCl reference electrodes were used to measure transepithelial voltage and pass current. Pulses (lmV amplitude, I s duration) were delivered every 90s to monitor resistance. The voltage clamps were connected to a PowerLab/8SP interface for data collection. CFTR was activated by adding 10μΜ forskolin to the apical bathing solution.]).

Immunofluorescence assay for correction of ATP7B

Cells were fixed with 4% paraformaldehyde in 0.2 M HEPES for 10 mins, permeabilized, labeled with primary and secondary antibodies, and examined with a ZEISS LSM 700 confocal microscope equipped with a 63 x 1.4 numerical aperture oil objective. The cells were scored based on the disappearance of ATP7B from the ER.

Morphological assay to estimate the exit ot ATP7B exit from ER to Golgi:

Cells were transfected with ATP7B-WT-GFP or ATP7B-H1069Q-GFP, incubated overnight with 200 μΜ BCS and/or drugs. Fixed cells were further labeled for TGN46 to mark and visualize the Golgi area under a confocal microscope. Under low copper conditions ATP7B-WT traffics to the Golgi from the ER, while ATP7B-H1069Q is retained within the ER. If the drug treatments induce the rescue of trafficking from the ER to the Golgi, the ATP7B-H1069Q-GFP fluorescence in the Golgi area increases. This is measured by quantifying (in 10 different microscopy fields in 100 cells) the increased in fluorescence of ATP7BWT-GFP or ATP7BH1069Q-GFP in the Golgi area (marked by TGN46) and normalizing this value to total cell fluorescence

Copper detection by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)

To determine intracellular Cu concentrations, cell pellets were lysed. The protein concentration in each sample was evaluated using Bradford Protein Assay (BioRad, Segrate, Italy). Cu concentration in the cell lysates was analyzed by ICP-MS. An aliquot of each sample was diluted 1 :10 v/v with 5% HN03 and analyzed with an Agilent 7700 ICP-MS (Agilent Technologies, Santa Clara, CA, USA) all values of Cu concentration were normalized for protein content in corresponding cell lysates.

Copper detection Coppersensor 3 (CS3):

Coppersensor 3 (CS3), which becomes fluorescent in the presence of bioavailable Cu (Dodani, Domaille et al. 2011). For fluorescent Cu detection, cells were incubated with 5 μΜ CS3 solution for 15 min at 37°C. CS3 was excited with 561 nm laser of LSM710, and its emission was collected from 565 to 650 nm. The signals were measured using ZEISS ZEN 2008 software and reported in arbitrary units.

Copper estimation:

Cells are lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 20 mM Tris-HCl, pH 7.4). 500 μg of total protein lysate in 100 μΐ is taken for copper estimation using Copper assay kit (MAK127 sigma-aldrich) according to the manufacturer's protocol.

RESULTS

Proteostasis correctors have a shared transcriptional signature

As noted, the proteostasis regulators share the ability to correct (albeit weakly) the F508del-CFTR folding- trafficking defect but have principal pharmacological effects not related to F508del-CFTR correction. Since the correction-related MOAs of these drugs are transcription-dependent, the gene signatures of the correctors should comprise genes related to F508del-CFTR correction in addition to those related to the principal actions of these drugs. If the correctors act through common mechanisms, the former genes, but not the latter, should be shared by all or most of the corrector gene signatures. To uncover this potential correction-related (CORE) gene pool, inventors developed a method based on the fuzzy intersection of transcriptional profiles (FIT) (Figure 1A), by which corrector gene signatures are 'intersected' to identify their commonalities (Figure 1A). The intersections among the majority of the signatures should include the CORE genes but exclude genes related to the heterogeneous principal effects of the drugs. The main parameters of the FIT analysis (number of correctors; number of genes to be analyzed in each signature, and cut-off threshold for inclusion in the correction relevant gene pool (Figure 1B-C)) were selected to identify a sufficiently large CORE gene pool for pathway analysis, and also to minimize the number of 'false' CORE genes. Inventors included in our analysis two sets of correctors (24 drugs/conditions altogether) with different chemical structures and pharmacological activities (Table 4) while excluding known pharmacochaperones. The gene signatures of 13 correctors were obtained in our laboratories using immortalised CF bronchial epithelial (CFBE41o-) cells (Kunzelmann et al. 1993) and an Agilent microarray platform (CFBE dataset; and GEO accession number GSE67698 for the expression profiles). Another 11 signatures were extracted from the MANTRA (Mode of action by network analysis; www.mantra.tigem.it; MANTRA dataset) (Iorio et al. 2010). The transcriptional profiles of glafenine and ouabain obtained from CFBE dataset were similar to those present in the MANTRA database (Zhang et al. 2012) suggesting that profiles obtained from CFBE and those downloaded from MANTRA database are similar enough to be treated together.

The FIT analysis of the gene signatures resulted in 219 downregulated and 402 upregulated CORE genes (Figure 1D,E). Each of these CORE genes was shared by 70% of the corrector signatures. The number of CORE genes was 3-fold higher than that expected on a random basis. This indicates that common transcriptional programmes are indeed embedded in the signatures of proteostasis correctors.

Identification of CORE genes/ pathways involved in F508del-CFTR correction

To understand the relation of CORE genes to CFTR proteostasis, inventors built a dataset of known F508del- CFTR proteostasis-relevant genes by assembling literature data and mapped their interactions with the CORE pool using STRING (Franceschini et al. 2013). Inventors found extensive and statistically significant protein-protein interactions among the nodes of the union of these two datasets (Figure IF), indicating that (at least a fraction of) the CORE genes are related to CFTR proteostasis. Significant interactions were also found between the CORE genes from CFBE and the MANTRA datasets, suggesting they were related and thus can be analyzed together. Inventors next applied standard bioinformatic tools to the CORE gene pool to identify functionally coherent pathways/ networks/ groups. Gene Ontology (GO)-based searches for proteostasis components among CORE genes retrieved 48 folding/ degradation and 24 transport-machinery components, some of which are known to be involved in F508del-CFTR proteostasis. A search for signaling molecules yielded 24 kinases and 6 phosphatases. STRING and the Ingenuity pathway analysis (IP A) tool identified several statistically significant networks. The IPA networks comprised also (predicted) interactors of CORE genes, some of which were network hubs. Such hubs were often constituents of signaling pathways such as growth-factor-mediated pathways (e.g., receptors for vascular endothelial growth factor [VEGF] and platelet- derived growth factor [PDGF], phosphatidylinositol 3 -kinase [PI3K], and mitogen-activated protein kinases [MAPKs]), inflammation-associated pathways (NF-κΒ subunits, Toll-like receptor 4 [TLR4]), stress activated protein kinase (SAPK) pathways (MKK3/6, MKK4/7), and casein-kinase pathway (CSNK2A1/ CKII). These hubs might control the CORE genes. Of note, many of the hubs were frequently present in the gene signatures of the individual correctors, although below the fuzzy cut-off threshold of 0.7 required for inclusion in the CORE gene pool.

Analysis of the promoters of CORE genes aimed at the identification of upstream transcription factors did not generate interpretable results.

Inventors then turned to experimental validation of the role of the CORE genes in the regulation of F508del- CFTR proteostasis. Experiments were carried out using a characterised biochemical assay that detects both the amount of core-glycosylated CFTR trapped in the ER (band B with Western blotting) and the amount of CFTR fully glycosylated in the Golgi (most of which presumably resides at the PM; band C with Western blotting). As a model system, inventors used non-polarised CFBE41o- cells stably expressing F508del-CFTR (Bebok et al. 2005) (hereafter referred to as CFBE); but many experiments were carried out also in HeLa, BHK and polarized CFBE cells, with results that were in good qualitative agreement with the CFBE data (unless specified otherwise).

While this assay is not suitable for large-scale screening, it provides quantitative information on the main proteostasis parameters including CFTR accumulation in the ER, ER-associated CFTR degradation, and transport and processing in the Golgi complex. Moreover, this assay is specific for proteostasis as it separates the effects on the F508del-CFTR protein from the effects on conductance as revealed by faster chloride- permeability assays (Pedemonte et al. 2005). Experimental validation was restricted to a limited set of genes: downregulated CORE genes (to exploit the availability of siRNA based downregulation and of small- molecule inhibitors) that showed functional coherence, i.e., were found in protein-protein interaction networks or in enriched GO groups; or were network hubs from Ingenuity analysis, or ubiquitin ligases and signaling molecules. In total, this resulted in a group of 108 genes. Notably, these genes had no previously reported role in the regulation of F508del-CFTR proteostasis.

CFBE cells were treated with siRNAs against these genes and the effects on both bands B and C were monitored. As a reference for correction, inventors used the investigational drug VX-809 (Van Goor et al. 2006), a robust corrector that acts as a pharmacochaperone. VX-809 treatment increased band C levels by 4- 5 -fold over control in most experiments. In all, 47 (Table 2) out of the 108 genes tested were found to be active in regulating F508del-CFTR proteostasis (Figure 2A-D). Of these, 32 genes (when depleted) enhanced the levels of bands B and C by 1.5 -fold to more than 10-fold over controls (importantly, they also increased the ratio between bands C and B; see below), while 15 decreased bands B and C by 20 to 80% of the control levels. Inventors refer to these as negative correction and positive correction genes, respectively. Among these genes 30 were CORE genes and 17 were hubs in IP A networks. Notably, the correction that was induced by depletion of many negative correction genes was greater than that achieved by VX-809 (Figure 2A), or by the corrector drugs originally included in the study (Figure 3A). This was in particular the case for a group of four poorly characterised ubiquitin ligases (RNF215, UBX05, ASB8, FBX07) that were not known to regulate F508del-CFTR proteostasis. RNF215 depletion increased the levels of bands C to over 10- fold the control levels (Figure 2A). Given these strong effects, RNF215 is a worthy candidate for further studies as a potential ERAD machinery component. Also notably, the depletion of many negative correction genes not only enhanced the bands B and C but also markedly increased (to different extents) the band C/band B ratio (Figure 2C), suggesting that these genes affect the efficiency of export of F508del-CFTR protein from the ER and/or the stability of this protein after export. It is to be noted that the downregulation of negative correction genes did not change the levels of CFTR mRNA (Figure 3B) and thus the observed effect is not due to increased synthesis. It might appear surprising to find both positive correction and negative correction genes within the downregulated CORE gene pool. However, these genes are, presumably, components of complex transcriptional modules whose role is to control cellular functions in a balanced manner. To this end, the concomitant operation of regulatory systems of opposite signs is probably necessary (Hart and Alon 2013). These observations are therefore a reflection of the organization of the transcriptional programs that regulate proteostasis. Based on these results, inventors sought to identify putative pathways/ networks/ groups (collectively, networks) within the 47 active CORE gene pool, using literature data and pathway building tools (Figure 2E). This resulted in several small potential networks (each comprising 2 to 6 connected elements), 4 of which were composed of signaling molecules and will be referred to by the name of their 'central' components: MAP3K11 (MLK3), CAMKK2 ΡΙ3Κ-β and γ, and CKII (with predominantly negative correction activity) and ERBB4 (with positive correction activity). A recent kinome-wide screening identified several kinases that regulate the rescue of F508del-CFTR (Trzcinska-Daneluti et al. 2015), with no overlap with the hits identified by the present inventors (possibly due to the different functional assays and cell types used in this versus our study). Other 3 of the networks shown in Figure 2 comprised spliceosome, centromere and mediator complex components; and 2 were groups of ubiquitin-ligases and kinases. Of note, to minimise the possibility of false positives due to off-target effects of siRNA, in addition to the 3 siRNAs used in the screening, inventors also additionally tested up to 5 siRNAs for selected genes (MLK3, CAMKK2, RNF215, NUP50 and CD2BP2; Table 3) and found concordant effects on correction. Moreover, the presence of networks and pathways among the active CORE genes provides further evidence that the observed effect on correction was not due to off-target or other non-specific effects of the siRNAs used. Proteostasis corrector drugs act in part by modulating the expression of CORE genes

Inventors next sought to verify whether the effects of correctors on the CORE genes might explain the action of these drugs. Inventors first analyzed the frequency of the active CORE genes among the genes downregulated by the corrector drugs. The CORE genes were about ~3-fold more enriched in the signatures of correctors compared to those of other ~ 200 drugs taken at random from the MANTRA database. Inventors next searched for MANTRA drugs that significantly downregulate the CORE genes (anti-correctors) using Gene set enrichment analysis (GSEA; specifically two-tailed symmetric GSEA as implemented in MANTRA; www.mantra.tigem.it). The top 25 hits included 3 of the correctors that inventors had used for the FIT analysis. From the remaining 22 inventors selected 8 drugs (based on availability) for testing in the correction assay. Among these, mitoxantrone was found to potently increase both band C and band B. (Figure 2F); in addition, among the top 5 hits was Vorinostat, an HDAC inhibitor that was shown to act as a corrector (Hutt et al. 2010). Thus, at least 20% of the short-listed drugs were correctors, while among a large number (>20) of randomly selected drugs none showed correction activity. These data suggest that the downregulation of CORE genes is a useful criterion to identify correctors. Inventors then extended our analysis of the top hits by comparing the 5 active drugs with those that failed to correct and also by examining upregulated by their gene expression profiles. The correctors showed a high frequency (2 to 3 fold more than non-corrector drugs) of up-regulation of the potent pro-corrector genes MKK1, MKK3 and FGFBP1 (Figure 2D) while the non-corrector drugs up-regulated more frequently (2 to 3 fold more than corrector drugs) the anti corrector genes NF-KB2 and MKK7. These results thus suggest that considering also the upregulation of CORE genes will help in further defining the search space for new correctors. Altogether, the above data indicate that the drug-induced modulation of CORE genes is a significant component of the MOAs of corrector drugs. Thousands of gene signatures of drugs and perturbagens are being deposited in specialized databases (http://www.lincscloud.org/). These and a more extensive search for CORE genes will provide useful tools for a more refined bioinformatic identification of new correctors.

Epistatic interactions between CORE pathways

As described earlier, the advantage of using this approach (deconvolution of drug MOA) to identify regulatory pathways is the possibility of discovering synergistic pathways. So in order, to explore the possible epistatic interactions between the CORE networks/ pathways, siRNAs against selected targets were combined and tested on F508del-CFTR rescue. These candidates were chosen for their potential druggability and/or strong effects on correction. Strong synergistic interactions were observed between various combinations of siRNAs against CKII, CAMKK2, MLK3 and NUP50 (a spliceosomal network component) (Figure 2G), thus validating our choice of the method. As a note of caution here, the efficacy of the combined siRNA treatments was more variable than that observed with single siRNAs. In our experience, this is because siRNAs in combinations are less effective than the individual siRNAs in depleting their target proteins, and a depletion threshold must be reached to achieve synergy. Inventors conclude that, using the FIT technique and a series of bioinformatic and experimental filters, inventors have identified a set of synergistic molecular networks that show strong control over F508del-CFTR proteostasis.

Delineation of the MLK3 and CAMKK2 signaling pathways regulating F508del-CFTR proteostasis

Next, inventors sought to define the composition and the role in correction of two representative CORE- networks, namely, the MLK3 and the CAMKK2 pathways. MLK3 (or MAP3K11) is part of a group of 14 MAP3 kinases that act through cascades of MAP2K and MAPK enzymes. MLK3 can be activated by various PM receptors, which include the TNF-a, TGF-β, VEGF and PDGF receptors, through at least two MAP4Ks (haematopoietic progenitor kinase [HPK] 1 and germinal centre kinase [GCK]) and glycogen synthase kinase (GSK)3P, or via the CDC42/Rac family [summarised in (Karen Schachter 2006)]. MLK3 can also be activated by stress, e.g., oxidative stress (Lee et al. 2014) (i.e., it is a Stress Activated Protein Kinase, or SAPK). It can, in turn, trigger three main kinases: p38 MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal regulated kinase (ERK), depending on cell type and conditions, through the intermediate kinases MAP2K3/6, MAP2K4/7 and MAP2K1/2, respectively (Karen Schachter 2006). MLK3 is also known to be an upstream activator of NF-kB (Hehner et al. 2000). Inventors thus sought to determine which components of the MLK3 pathway have roles in F508del- CFTR correction. The VEGF and PDGF receptors, MAP2K7 (MKK7), and NF-KB2, like MLK3, appear to be components of the correction-relevant branch of the MLK3 pathway, as indicated by the screening data in Figure 2A. Among the components upstream of MLK3, inventors found TGF receptors, CDC42, Rac2, and HPK1 to be active in correction (i.e. their depletion induced correction) (Figure 4A). Within the cascade downstream of MLK3, MKK7 (Figure 2A) and further downstream, JNK2 (Figure 4B) were active components (JNK2, is highly expressed in bronchial epithelial cells (http://biogps.org). The p38 MAPK, also downstream of MLK3 (through MAP2K3 and 6) was inactive in CFBE but moderately active in HeLa cells indicating some cell-type-dependent specificity in the effects of these kinases (Figure 5 A). Thus, altogether, suppression of the MKK7-JNK branch of the MLK3 pathway induced F508del-CFTR correction. Conversely, when the activity of the MLK3 pathway was enhanced by transfection of the MLK3 activator CDC42 or MKK7 or JNK2 into the CFBE cells, the levels of both bands B and C dropped markedly (Figure 4C), confirming that the MLK3 pathway has a tonic negative effect on the proteostasis of F508del-CFTR. In sum, as shown in Figure 4D, a signal regulating F508del- CFTR proteostasis flows from the ligands and receptors upstream of MLK3, through HPK1 and CDC42/Rac2, to impinge on MLK3 and is then passed on through the XNK2 arm. NF-KB2 is also a probable downstream component of this proteostasis regulatory pathway. Inventors also tested (again by siRNA silencing) seven other MAP3Ks (including TAK1/MAP3K7, see below) that can activate JNK or p38, for their effect on F508del- CFTR proteostasis. They had no effect. This highlights the remarkable specificity of MLK3 in the regulation of proteostasis, possibly due to spatial/temporal compartmentalization of the MAPK networks (Engstrom, Ward, and Moorwood 2010). A similar series of experiments was performed to characterise the CAMKK2 pathway in F508del-CFTR correction. The results are reported in detail (Figure 5 B-C, Figure 4E), and indicate that the CAMKK2 pathway has negative effects on F508del-CFTR proteostasis similar to those found for the MLK3 pathway.

The MLK3 pathway exerts complex regulatory effects on F508del-CFTR proteostasis.

The increase in band B induced by inhibition of the MLK3 pathway might be due to increased synthesis or to decreased degradation of F508del-CFTR. Downregulation of MLK3 did not increase the CFTR mRNA levels (Figure 3 B), speaking against the former possibility. Inventors then examined the degradation of band B using both a cycloheximide (CHX) chase and a radioactive pulse-chase assays. Downregulation of the MLK3 pathway markedly slowed the degradation of band B when measured by CHX chase (Figure 6A,B) and similar effects were obtained with the radioactive pulse-chase method (Figure 7A,B). Inventors also examined the effects of enhancing the activity of MLK3 pathway by overexpressing CDC42 or MKK7 or JNK2: under these conditions the rate of degradation of band B increased 2- fold [Figure 6C,D; see also (Ferru-Clement et al. 2015)]. The ubiquitin-proteasome system itself was not detectably affected by modulation of the MLK3 pathway activity, as judged by the lack of effects on both the proteasome sensor- ZsProsensor-1 (Figure 7C) and the accumulation of poly-ubiquitinated proteins (Figure 7D). Thus, the MLK3 pathway appears to regulate the ERQC/ERAD of F508del-CFTR at a step prior to proteasomal digestion. In addition, silencing of the MLK3 pathway (and of several CORE genes) increased also the band CI band B ratio (see Figure 2C). This is not explained by reduced ERAD alone and suggested that the MLK3 pathway might have additional effects on the folding/ export of F508del-CFTR, or on the stability of band C at the PM (or both). The trypsin susceptibility assay to assess the folding status of F508del-CFTR and an assay for protein transport out of the ER using vesicular stomatitis virus G protein (VSVG), a classical probe to study secretory trafficking, ruled out large effects of the MLK3 pathway on F508del-CFTR folding or on the general ER-export machinery (Figure 7E,F). Inventors next tested the effect of MLK3 on the stability of F508del- CFTR at the PM. Inventors depleted MLK3 and exposed the cells to low temperature (26 °C), to accumulate F508del-CFTR at the cell surface, and then shifted the cells back to 37 °C, a temperature at which the F508del-CFTR at the PM is subjected to accelerated ubiquitination and degradation (Okiyoneda et al. 2010). Under these conditions, the depletion of MLK3 slowed the degradation rate of band C, increasing the tl/2 from ~ 2 to ~ 4 h (Figure 6E,F), whereas overexpression of CDC42 to activate MLK3 enhanced band C degradation rate (Figure 6G, H). These data suggest that also the peripheral QC of F508del-CFTR is regulated by MLK3. In contrast, the knockdown of XNK2 (or its overexpression) did not change the degradation kinetics of band C, although it increased the band C/band B ratio, suggesting that JNK2 may have additional effects on the folding and/or export of F508del-CFTR. Similar effects on F508del-CFTR folding seem likely to be induced also by the CORE genes whose depletion greatly increases the band C/band B ratio, in some cases up to 4-fold over control levels (Figure 2C). In conclusion here, the receptor and stress- activated MLK3 signaling pathway markedly activates both the ER-associated and peripheral degradation processes of F508del-CFTR while possibly at the same time reducing the efficiency of F508del-CFTR folding/ER export. As a consequence, inhibition of the MLK3 pathway results in large increases in the levels of the Golgi-processed mature form of F508del-CFTR. 22 ROS and the CF modifiers TNF-a, TGF-β enhance F508del-CFTR degradation in an MLK3-dependent fashion.

Inventors next examined the effects on F508del-CFTR proteostasis of agents known to activate MLK3 such as TNF-a, TGF-β (Karen Schachter 2006) and reactive oxygen species (ROS) (Lee et al. 2014). TNF-a and TGF-β have been proposed to be genetic modifiers of CF (Cutting 2010) and ROS have been reported to 1 be enhanced in CF cells (Luciani et al. 2010) and to be massively produced by neutrophils during the inflammatory reactions that are common in CF patients (Witko-Sarsat et al. 1995). Inventors treated CFBE cells with TNF-a, TGF-β or H202 (to increase ROS), and monitored the effects on F508del-CFTR. The effects of H202 at non-toxic concentrations were dramatic, with a marked drop of the F508del-CFTR levels within a few minutes. Also TNF-a and TGF-β induced rapid, though less complete (50%) decreases in levels of F508del-CFTR. Under these conditions, the reduction in F508del-CFTR levels was completely abolished by MLK3 downregulation, confirming the crucial role of MLK3 pathway in F508del-CFTR QC/degradation. These results, and in particular the effects of H202, provide evidence for extremely rapid and potent mechanisms of protein degradation that involve the MLK3 pathway and act on F508del-CFTR (and presumably on other misfolded mutant proteins). These regulatory mechanisms might have pathological relevance, as discussed below.

Chemical inhibitors of the MLK3 pathway act as CFTR correctors and potently synergize with the pharmacochaperone VX-809 Inventors next tested the effect of selected kinase inhibitors on F508del-CFTR proteostasis in CFBE cells. A well-known characteristic of the kinase inhibitors is their promiscuity. In our experience, inhibitors that nominally target the same kinases can cause divergent effects on correction (see below), most likely because they target other kinases with different or competing effects. Inventors sought to overcome this difficulty by selecting kinase inhibitors with different structures and modes of action, and by using information from the KTNOMEscan library (http://lincs.hms.harvard.edu/data/kinomescan/). For JNK, inventors tested a set of 10 reported JNK inhibitors (JNKi), three of which led to robust increases in the levels of band B and band C (Figure 8A,B; JNKi II, JNKi IX, JNKi XI) at concentrations that were required for JNK inhibition in CFBE cells (Figure 9A). These JNK inhibitors have different chemical structures; moreover, while JNKi II and JNKi IX are ATP-competitive inhibitors of JNK, JNKi XI is an inhibitor of substrate/ scaffold binding to JNK. These JNK inhibitors therefore appear to be reliable tools to correct F508del-CFTR by targeting the MLK3-JNK pathway. For MLK3, a previously proposed MLK3 inhibitor (K252a) had no clear effects on correction, perhaps because of its weak effect on MLK3 itself and diverging effects on other kinases (see http://www.kinase-screen.mrc.ac.uk/screening-compounds/34589 2). Inventors thus searched the KTNOMEscan library for a molecule that had a suitable inhibitory pattern on the MLK3 pathway. (5Z)-7-oxozeaenol (herein referred as oxozeaenol) (Ninomiya-Tsuji et al. 2003) potently inhibits the MLK3 pathway members VEGF and PDGF receptor kinases and (less potently) MLK3 itself and MKK7, as well as, more weakly, a few kinases with antagonistic effects on correction (http://lincs.hms.harvard.edu/db/datasets/20211/). Oxozeaenol treatment markedly increased the bands B and C of F508del-CFTR (Figure 8A). This drug had earlier been identified as a corrector in a screening study, and had been proposed to have F508del-CFTR corrector properties as an inhibitor of TAK1 (MAP3K7) (Trzcinska- Daneluti et al. 2012). However, the downregulation of TAK1 itself had no effect on correction (Figure 9B). The data thus indicate that oxozeaenol acts by inhibiting the kinases of the MLK3 pathway. In line with this notion, the corrective effects of oxozeaenol were not additive with MLK3 knockdown (Figure 9 C) and were accompanied by a reduction in phospho c-jun levels (c-jun phosphorylation is diagnostic of JNK activation) (Figure 9D). Further inventors also tested 25 drugs that are either FDA-approved or under clinical trial kinase inhibitors that target MLK3-JNK pathway (Figure 4D) anti-correction kinases but not pro- correction kinases. Among them Pazopanib, Dovitinib lactate and Bexarotene led to robust increases in the levels of band B and band C (Figure 8A,B).

Thus, selected chemical blockers of the MLK3 (and CAMKK2; Figure 9E) pathway potently increase band C (Figure 8A,B). The level of correction obtained with these inhibitors is higher than the effects of the corrector compounds from which the pathways were deduced (Figure 3 A), and similar to, or higher than, the effects of VX-809. Inventors also noted that, while inhibitors of MLK3 pathway lead to large increases in the ER- localised band B, the pharmacochaperone VX-809 increases F508del-CFTR exit from the ER with a limited increase in band B (Figure 8A), presumably because it primarily enhances F508del-CFTR folding. Inventors reasoned that if the band B protein accumulating in the ER following the inhibition of MLK3 pathway is in a foldable state, VX-809 might enhance its folding, and greatly increase the generation of the band C mature protein. Indeed when inventors added both MLK3 pathway inhibitors and VX-809, there was potent synergy between them (Figure 8C,D; Figure 10) with increases in levels of band C that were over 20-fold the basal band C level and 4-fold over those obtained with VX-809. Though VX-809 alone had only limited benefits in clinical trials (Clancy et al. 2012), recent laboratory-based research and additional clinical trials have shown promising results with combination therapies of VX-809 and other pharmacochaperones or potentiators (Jones and Barry 2015, Okiyoneda et al. 2013, Phuan et al. 2014). Given these observations, the observed additive/ synergistic effects with MLK3 pathway inhibitors provide a potential therapeutic opportunity. The MLK3 pathway exerts selective effects on the proteostasis of F508del-CFTR and of structurally related mutant proteins. Inventors next examined the effects of the MLK3 pathway inhibition on the proteostasis of other conformational disease mutants. Inventors transfected CFBE (and HeLa) cells with different conformational mutants (i.e., Sodium-chloride symporter [NCC, R948X mutant]; P-glycoprotein, [P-gp, G268V and DY490 mutants]; human Ether-a-go-go-Related Gene [hERG, G601 S mutant]; Wilson's disease associated protein [ATP7B, H1069Q and R778L mutants]) and then treated cells with JNKi II and monitored these proteins by assessing changes in their glycosylation patterns (NCC, P-gp, hERG mutants) or in their intracellular movement from the ER to the Golgi complex (ATP7B mutants). JNKi II rescued some of these mutants (P-gp DY490 and ATP7B mutants), while it had no effects or had 'negative' effects, on others (Figure 9 F and Table 5). The effects on ATP7B were large and led to almost complete correction.

Both P-glycoprotein and ATP7B, like CFTR, have two groups of transmembrane domains with an interconnecting nucleotide-binding domain. Moreover, the mutations (DY490 and H1069Q) are located in the nucleotide binding domains of these proteins, and result from either a loss or substitution of aromatic amino acids, as for F508del-CFTR. These similarities suggest that common proteostatic machinery might be involved in the detection of these defects and might be targeted by the MLK3 pathway in a selective fashion. Prompted by the effects of the MLK3 kinase cascade on the CFTR-D-508 mutant, inventors examined the effects of the MLK3 pathway inhibition on the Wilson's disease (WD) associated protein mutants (ATP7B, H1069Q and R778L mutants, the main mutations found in Wilson patients). This is because CFTR and ATP7B are structurally similar, and the above mutations (DY490 and H1069Q) are located in the nucleotide- binding domains of the protein, and result from either a loss or substitution of aromatic amino acids, as for F508del-CFTR. These similarities suggest that the same proteostatic machinery acting on CFTR-D-508 might be involved in the detection of these defects and might be targeted by the MLK3 pathway in a selective fashion. This led us to test the relevance of the MLK3 pathway components and inhibitors on Wilson's disease ATP7B, H1069Q and R778L mutants.

MLK3, p38 MAPK and JNK as new targets for correction of Wilson disease-causing ATP7B mutants.

Inventors silenced MAP3K11, the upstream activator of both p38 and JNK, and isoforms of p38 (MAPK11 - MAPK14) and JNK (MAPK8-MAPK10), in HeLa cells expressing the ATP7BH1069Q (Fig. 11 A, B) to evaluate whether this treatment improves ATP7BH1069Q recovery from the ER. Both a reduction in ER retention and a recovery of Golgi and vesicle targeting of the mutant was detected after depletion of MAP3K11, MAPK8 (JNKl), MAPKl l (ρ38β) and MAPK14 (p38a) (Fig. 11). Thus, siRNA depletion of these kinases strongly corrected the trafficking defect of mutant ATP7B.

Inventors then tested the chemical inhibitors of p38 and JNK VX-745, SB202190 (SB90), Oxozeaenol and SP600125 (SP125) respectively (Fig 12). Both p38 and JNK inhibitors (added for 24h) did not affect ATP7BWT but strongly corrected the ATP7BH1069Q defect.

Altogether, the finding in this study is that MAP3K11, MAPK8 (JNKl), MAPKl l (ρ38β) and MAPK 14 (p38a) p38 and JNK kinases play an important role in WD by promoting retention and degradation of the ATP7BH1069Q mutant in the ER. Thus, suppression of these kinases allows ATP7BH1069Q to reach the post-Golgi vesicles and the apical surface in hepatocytes, from where it can contribute to the removal of excess Cu from the cell. As a consequence, treatments with the appropriate kinase inhibitors restore normal trafficking dynamics of the ATP7B mutants and reduce Cu accumulation in cells expressing them. Thus, MAP3K11, MAPK8 (JNKl), MAPKl l (ρ38β) and MAPK14 (p38a) represent attractive targets for correction of the ATP7B mutant localization and function and could be considered for development of new therapeutic strategies.

Screening assays

About 70 repositionable clinical phase drugs were acquired and tested in screening assays in cells expressing ATP7B H1069Q-GFP.

1) Traffic-based screening in Hela cells

This screening was based on a morphological assays that reveals the ability of the H1069Q to exit the ER and reach the Golgi complex.

Inventors found that 5 inhibitors (BIRB-796, Bexarotene, Cannabidiol, CPI-1189, ENMD-2076) potently rescue the mutant protein localization. A large fraction of the cellular of the mutant protein exits the ER and reaches the Golgi compartment upon inhibitor treatments (Fig.13 A,B).

2) Traffic-based screening in hepatocytes

Liver hepatocytes are the main cells that express ATP7B and the Wilson disease affects primarily liver cells. HEPG2 cells (hepatocytes from human liver carcinoma) and human primary hepatocytes are therefore a disease-relevant models to study the efficacy of the rescue by drugs. Inventors have therefore used the assay developed in HeLa cells to test drugs that rescue the ATP7B-H1069Q also in HEPG2 cells and human primary hepatocytes expressing ATP7B H1069Q-GFP. Inventors found that BIRB-796 and VX-745 rescue H1069Q potently in these cells (Fig.13 C-F).

3) Test of copper excretion in hepatocytes

ATP7B protein functions in the excretion of copper out of cells and tissue. As ATP7B H1069Q trafficking to the plasma membrane is impaired, the cells cannot excrete the copper, which leads to higher level of intracellular copper. If the corrector drugs promote the correct localization of the mutant, then the copper should be excreted, leading to lower intracellular levels. Inventors have tested the two best correctors of the localization defect of the ATP7B H1069Q mutant by estimating their intracellular copper levels upon treatment with BIRB-796 and VX-745 Inventors found that cells treated with VX-745 and BIRB-796 show low intracellular copper levels, indicating that the copper excretion function is recovered up on drugs treatments (Fig. 14).

Discussion

In this study, inventors have developed a bioinformatic method based on the fuzzy intersection of drug transcriptomes (FIT) that reveals the transcriptional components of the MOAs of proteostasis correctors. Using this method, inventors have uncovered a set of correction relevant genes (CORE genes), some of which belong to signaling networks that potently and selectively regulate the proteostasis of F508del-CFTR and of structurally related protein mutants. These are the first example of signaling cascades that specifically control the proteostasis machinery acting on AF508-CFTR. Physio-pathological significance of the CORE signaling networks. Based on literature data, interaction databases and our own experimental findings, the correction-relevant components inventors identified can be organised into five signaling cascades, which, for brevity, inventors refer to here by the names of their 'central' components: namely, MLK3, CAMKK2, PI3K, CKII, and ERBB4. Other networks are made up of constituents of the spliceosome, centromere and mediator (transcriptional) complexes, or are groups of ubiquitin ligases.

The physiological role of the CORE signaling systems might be to regulate the stringency of the QC and degradation processes according to cellular needs. Most of the CORE pathways enhance the efficiency of QC and degradation. This is the case of the MLK3 pathway, which is activated by selected cytokines and by cellular stresses. The ERBB4 pathway, in contrast, is activated under growth conditions, and appears to have the effect of suppressing the QC and degradation processes. It may be speculated that cells under stress need to reduce the toxic burden of unfolded proteins to survive, while growing cells might need to 'tolerate' higher levels of folding/ unfolded proteins to proliferate, and that the CORE pathways regulate the proteostasis machinery according to needs. In addition, the CORE pathways might function as part of an internal control system (Cancino et al. 2014, Luini et al. 2014) that senses, and reacts to the presence of misfolded proteins. Interestingly in this regard, MLK3 interacts directly with (and might be activated by) HSP90 (Zhang et al. 2004), a component of the F508del-CFTR folding and QC machinery. More in general, the function of the CORE networks, considering that they exert selective effects on the degradation of different protein classes (Figure 9F), might be to 'sculpt' the proteome according to functional requirements. With respect to cystic fibrosis and similar diseases, the MLK3 and other CORE networks can be deleterious in that they enhance the degradation of mutants that retain the potential to function, such as F508del-CFTR. Also important, they can be hyper activated under pathological conditions, leading to vicious circles. For example, large amounts of ROS are produced by neutrophils in the inflamed lungs of CF patients (Witko-Sarsat et al. 1995); and elevated serum VEGF are detected in some CF patients (McColley et al. 2000). Both of these molecules act via the MLK3 pathway to enhance the degradation of F508del-CFTR, and in particular the ROS do so with striking efficacy and speed (Figure 5A,B). These effects most probably results in lowering F508del-CFTR below the levels determined by the primary folding defect, which might be harmful because even low residual levels of F508del-CFTR may help to improve the CF phenotype in the long term (Amaral 2005). Blocking the MLK3 pathway is thus probably necessary to stop maladaptive processes that can adversely affect therapeutic efforts. Similar considerations apply to the CAMKK2 and other CORE -derived pathways. Mechanism of action of the MLK3 signaling network

The ER quality control relies on chaperones such as HSP90 and HSC70 that are also involved in folding and can switch between folding and quality control /degradation roles depending on their dwell-time on the folding client proteins (Zhang, Bonifacino, and Hegde 2013). The simplest interpretation of the data is therefore that inhibition of the MLK3 pathway regulates this folding/degradation switch by impairing the entry of F508del-CFTR into the degradation pathway and giving the mutant more time to fold and exit the ER. It cannot be excluded, however, although MLK3 does not measurably affect the folding of F508del- CFTR as detected by trypsin assay, that MLK3 (and other CORE genes) might exert subtle direct actions on the folding/ ER export mechanisms. This is supported by the strong effects of some of the CORE pathways on the band C/band B ratio, and by the observation that the inhibition of MLK3 stimulates a mutant of ATP7B (similar in structure to CFTR) to leave the ER in a functional form (see Table 5).

At the molecular level, the mechanisms underlying these rescue effects remain unclear. Some initial insight might come from our observation that the phosphoprotein HOP co-precipitates much less efficiently with F508del-CFTR in cells treated with TNK inhibitors that in control cells. HOP serves as a link between HSC70 and HSP90, and its depletion induces rescue of F508del-CFTR (Marozkina et al. 2010), possibly by acting on the folding/ ERQC switch discussed above. It is thus possible that a reduced interaction of HOP with the F508del-CFTR-associated QC/folding complex might be one of the modes of action of MLK3 on F508del- CFTR rescue. However, a complete analysis of the effects of the MLK3 pathway on the interactions and posttranslational modifications of the ERQC/ERAD machinery components remains a task for future work. Relevance of the CORE signaling networks for the pharmacological correction of F508del-CFTR.

Signaling cascades are eminently druggable (the majority of the known drug targets are signaling components (miming, Sinning, and Meyer 2006)), and an enormous repertoire of drugs directed at kinases and other related molecules has been developed by the pharmaceutical industry for the therapy of major diseases. For instance, over 120 inhibitors against the correction-related kinases identified in this study are currently in clinical trial. Moreover, as shown for the case of oxozeaenol (Figure 8A-B), suitable kinase inhibitors can be selected in a rational fashion by matching the list of CORE kinases with the kinase inhibitory patterns of the many available drugs of this class, according to polypharmacology principles (Aggarwal et al. 2007). It is thus quite possible that some of these drugs may be repositioned for CF therapy. In addition, the group of CORE ubiquitin ligases, particularly RNF215, are also attractive targets in view of their potent effect on F508del-CFTR correction (Figure 2A). Although the technology for developing ubiquitin ligase inhibitors is still in its early stages, robust progress is being made in this direction (Goldenberg et al. 2010).

A further consideration is that the inhibitors of the CORE pathways show corrective effects that are (partially) selective for F508del-CFTR (and structurally related mutants) (see Figure 9 F); and that these effects are complementary and synergic with those of the pharmacochaperone VX-809. Since these synergies lead to levels of correction that are several-fold higher than those achieved by VX-809 alone, it is possible that they result in combination therapies of clinical interest. Also of note is that the MOA-based approach used here can be exploited further in the future to identify more CORE pathways as well as more effective and specific correctors. In addition to the above, a key requirement for translating our findings towards clinical treatments is the conservation of the CORE pathways in epithelial bronchial cells in situ. Inventors have observed fundamentally similar role of the CORE networks across several human and mammalian cell lines, both under polarized and non-polarized conditions, suggesting that these networks are well conserved. Moreover, JNK has been reported to be hyperactive in the lungs of a mice model of CF (Grassme et al. 2014), as is p38 MAPK (also activated by MLK3) in the lungs of CF patients (Berube et al. 2010) indicating that a SAPK pathway is stimulated under these conditions. Also notably, the MLK3 pathway inhibitor oxozeaenol has been shown to be effective in correcting the F508del-CFTR proteostasis defect in the primary human bronchial epithelial cells (Trzcinska-Daneluti et al. 2012). These observations, together with the fact that the CF genetic modifiers TNF-a and TGF-β potently affect F508del-CFTR proteostasis, support the notion that a regulatory network similar to that uncovered in CFBE cells operates on the proteostasis machinery in bronchial cells in CF patients. In sum, this study builds on previous screening studies and on the accumulated knowledge on the F508del-CFTR proteostasis machinery (Balch, Roth, and Hutt 2011,Farinha, Matos, and Amaral 2013, Lukacs and Verkman 2012, Turnbull, Rosser, and Cyr 2007) to identify the first signaling pathways acting on F508del-CFTR proteostasis, and thereby opens new exciting possibilities to pharmacologically correct the folding and trafficking defect of this mutant protein. To establish the efficacy of these interventions in human bronchial epithelia and relevant animal models (Y an et al. 2015) will be the next stage towards the rational development of effective F508del-CFTR proteostasis regulators for CF patients.

Tables

Table 1 - Kinases active on correction:

The anti-corrector kinases when depleted by siRNA rescue F508del-CFTR from degradation and increase band C levels which can function at PM. The pro-corrector kinases when depleted by siRNA increase degradation of F508del-CFTR and band C levels reduce. Table 2 - CORE genes regulating the F508del-CFTR.

The anti-corrector when depleted by siRNA rescue F508del-CFTR from degradation and increase band C levels which can function at PM. The pro-corrector when depleted by siRNA increase degradation of F508del-CFTR and band C levels reduce.

Table 3 - The siRNAs used in the study.

Gene Symbol siRNA ID/ Sense siRNA Sequence (5'-3')/ catalogue no. siRNA 1 siRNA 2 siRNA 3 siRNA 4 siRNA 5

AKT1 s659 s660 s661

AKT2 S1215 S1216 S1217

CALM1 S2340 S2341 S2342

CALM2 S2343 s2344 S2345

CALM3 S2346 S2347 S2348

CALML3 S2349 S2350 S2351

CEN PA S2906 S2907 S2908

CEN PE S2917 S2915 S2916

CSN K2A2 S3639 S3640 s3641

CSN K2B S3642 S3643 s3644

CYC1 S3790 S3791 S3792

ELAVL1 S4608 S4609 S4610

ERBB4 S4781 S4782 S4783 FARSA S5027 S5028 S5029

FLN B S5278 S5279 S5280

H N F4A S6696 S6697 S6698

ON ECUTl S6702 S6703 s6704

ITPR1 S7631 S7632 S7633

ITPR2 S7634 S7635 S7636

ITPR3 s265 s266 s267

IVL S7640 S7641 S7642

KDR S7822 S7823 S7824

KRT34 s8011 S8012 S8013

LMN B1 S8224 S8225 S8226

MAL s8472 s8473 s8474

MITF S8790 s8791 S8792

MKI67 S8796 s8797 S8798

MAP3K11 s8814 s8815 s8814

N FKB1 s9504 s9505 S9506

PDE3A S10183 S10184 S10185

PDGFRA S10234 S10235 S10236

PDG FRB S10242 S10240 S10241

PIK3CA S10520 S10521 S10522

PIK3CB S10524 S10525 S10526

PIK3CD S10529 S10530 S10531

PIK3CG S10532 S10533 S10534

MED1 S10889 S10890 S10891

MAPK1 S11137 S11138 S11139

MAPK3 S11141 S230179 S230180

MAPK6 S11146 S11147 S11148

MAPK7 S11149 S11150 S11151

MAP2K1 S11167 S11168 S11169

MAP2K2 S11170 S11171 S11172

MAP2K3 S11173 S11175 S11176

MAP2K5 S11176 S11177 S11178

MAP2K6 S11180 S11181 S11182

MAP2K7 S11182 S11183 S11184

PXN S11627 S11628 S11629

RELB S11917 S11918 S11919

S100A7 S12419 S12420 S12421

MAP2K4 S12703 S12701 S12702

SPRR1A S13381 S13382 S13383

SPRR1B S13383 S13384 S13385

SPRR3 S13397 S13398 S13399

TEP1 S13985 S13986 S13987

TLR4 S14194 S14195 S14196

TOP3A S14310 S14311 S14312

TP53 s605 s606 s607

VH L S14789 S14790 S14791

YWHAH S14967 S14968 S14961

ZAP70 S14973 S14974 S14975 AKAP1 S15665 S15666 S15667

PPAP2B S16384 S16385 S16386

PRPF4B S17018 S17017 S17018

NOL3 s300 s301 s302

SART1 S17343 S17344 S17345

OSMR S17542 S17543 S17544

DCLK1 S17584 S17585 S17586

WTAP S18431 S18432 S18433

DHX38 S18906 S18907 S18908

MED13 S19365 S19366 S19367

FGFBP1 S19392 S19393 S19394

SC02 S19424 S19425 S19426

AKT3 S19427 S19428 S19429

TROAP S229661 S229662 S229663

KIF20A S19676 S19677 S19678

RBM7 S19835 S19836 S19837

AKAP8 S20070 S20068 S20069

RGS19 S20107 S20108 S20109

CD2BP2 S20381 S20382 S20383

PRPF8 S20796 S20797 S20798

CAMKK2 S20925 S20926 S20927

STAG 2 S21089 S21090 S21091

NUP50 S21138 S21139 S21140

E HD1 S21513 S21514 S21515

WDR6 S22068 S22069 S22070

UBOX5 S22595 S22596 S22597

ZC3H3 S23133 S23134 S23135

DICERl S23754 S23755 S23756

PATZ1 S24176 S24177 S24178

FBX07 S24491 S24492 S24493

SEN P6 S25023 S25024 S25025

DNAJC2 S25685 S25686 S25687

GEM IN4 S27064 S27065 S27066

PDE11A S27187 S27188 S27189

BIN2 S28102 S28103 S28104

GTS El S28240 S28241 S28242

CALML5 S28669 S195236 S195237

SHC3 S28721 S28722 S28723

CYCS S28896 S28897 S28898

DGCR8 S29061 S29062 S29063

EXOSC4 S29112 S29113 S29114

C14orfl06 S30720 S30721 S30722

CTDSP1 S33804 S33805 S33806

DSN1 S36760 S36761 S36762

ALPK1 S37074 S37072 S37073

ASB8 S44282 S44283 S44284

CALML6 S46468 S46469 S46470

CSN K2A1 S3636 S3637 S3638

The Supplier for the siRNAs corresponding to from "AKT1" to "non targeting-CHGG_05426" is Life Technologies. The Supplier for the siRNAs corresponding to from "MAPK8" to "NUP50" and to "siControl- 2" is Sigma- Aldrich (USA). The Supplier for the siRNAs corresponding to "siControl- 1 (AUStars Negative Control siRNA) is Qiagen (Germany).

Table 4: The list of corrector drugs used in the study with their corresponding known primary MOAs.

ABT888 (Anjos et al., A poly(ADP-ribose) polymerase (PARP) -1 and -2 inhibitor with chemosensitizing 2012) and antitumor activities. ABT-888 inhibits PARPs, thereby inhibiting DNA repair and potentiating the cytotoxicity of DNA-damaging agents.

Glafenine (Robert et al., An anthranilic acid derivative with analgesic properties used for the relief of all 2010) types of pain (1)

GSK339 Androgen receptor ligand (Norris et al., 2009).

Ibuprofen (Carlile et al., Ibuprofen is a nonsteroidal anti-inflammatory drug. It is a non-selective inhibitor 2015) of cyclooxygenase.

JFD03094 PARP inhibitor

KM1 1060 (Robert et PDE5 inhibitor (an analog of sildenafil).

al., 2008)

Latonduine (Carlile et PARP3 inhibitor

al., 2012)

Minocycline H (D Y A tetracycline analog that inhibits protein synthesis in bacteria. Also known to Thomas lab inhibit 5-lipooxygenase in the brain (2).

unpublished)

Ouabagenin (Zhang et A cardiaoactive glycoside obtained from the seeds of Strophanthus gratus. Acts by al., 2012) inhibiting Na+/K+ ATPase, resulting in an increase in intracellular sodium and calcium concentrations (2).

Ouabain (Zhang et al., A cardiaoactive glycoside obtained from the seeds of Strophanthus gratus. Acts by 2012) inhibiting Na+/K+ ATPase, resulting in an increase in intracellular sodium and calcium concentrations (2).

PJ34 (Anjos et al., PARP1 inhibitor

2012)

Low temperature

(Denning et al., 1992)

Drugs of the Primary Use/ Class

MANTRA dataset

(Reference for

correction activity)

Chloramphenicol Inhibitor bacterial protein synthesis by binding to 23 S rRNA and preventing (Carlile et al., 2007) peptidyl transferase activity (2).

Chlorzoxazone (Carlile Muscle relaxant. Acts by inhibiting degranulation of mast cells and preventing the et al., 2007) release of histamine and slow-reacting substance of anaphylaxis. It acts at the level of the spinal cord and subcortical areas of the brain where it inhibits multi- synaptic reflex arcs involved in producing and maintaining skeletal muscle spasm (2).

Dexamethasone Is a synthetic glucocorticoid agonist. Its anti-inflammatory properties are thought (Caohuy et al., 2009) to involve phospholipase A2 inhibitory proteins, lipocortins (2).

Doxorubicin (Maitra et DNA intercalator that inhibits topoisomerase II activity by stabilizing the DNA- al., 2001) topoisomerase II complex (2).

Glafenine (Robert et al., An anthranilic acid derivative with analgesic properties used for the relief of all 2010) types of pain (1).

Liothyronine (Carlile et L-triiodothyronine (T3, liothyronine) thyroid hormone is normally synthesized and al., 2007) secreted by the thyroid gland. Most T3 is derived from peripheral monodeiodination of T4 (L-tetraiodothyronine, levothyroxine, L-thyroxine). The hormone finally delivered and used by the tissues is mainly T3. Liothyronine acts on the body to increase the basal metabolic rate, affect protein synthesis and increase the body's sensitivity to catecholamines (such as adrenaline). It is used to treat hypothyroidism (2).

MS-275 (Hurt et al., Also known as Entinostat. An inhibitor of Class Ihistone deacetylases 2010) (preferentially HDAC 1, also HDAC 3) (Hu et al., 2003).

Scriptaid (Hurt et al., An inhibitor of Class I histone deacetylases (HDAC1, HDAC3 and HDAC 8) (Hu 2010) et al., 2003).

Strophanthidin (Carlile

et al., 2007) A cardioactive glycoside that inhibits Na+/K+ ATPase. Also known to inhibit the interaction of MDM2 and MDMX (1).

Thapsigargin (Egan et A sesquiterpene lactone found in roots of Thapsia garganica. A non-competitive al., 2002) inhibitor of sarco/endoplasmic Ca 2+ ATPase (SERCA) (1).

Trichostatin-A (Hurt et An inhibitor of histone deacetylases (HDAC1, HDAC3, HDAC 8 and HDAC7) al., 2010) (Hu et al., 2003).

(1) http://pubchem.ncbi.n rn.nih.gov/ (2) www.drugbank.ca

Table 5: MLK3 pathway regulates the proteostasis of mutant proteins that are structurally related to CFTR.

ATP7B H1069Q 32 80

ATP7B R778L 12 40

Note: * in case of ATP7B mutants denotes fraction of protein in Golgi as calculated by fluorescence microscopy, and in other cases the protein that was processed by the Golgi are calculated by a biochemical assay similar to the one used for CFTR.

CFBE or HeLa cells (in case of ATP7B) were transfected with constructs encoding the indicated mutant proteins and treated with JNKi II for 48h. The effect of JNKill on proteostasis of these mutants was monitored by western blotting (to measure the change in Golgi processed band C or ER localized band B; or in the case of ATP7B using fluorescence microscopy to monitor the efficiency of translocation of the ER- localized mutant proteins to the Golgi. Treatment with JNKi II corrects the folding-trafficking defects of mutant proteins that have similar structure to F508del-CFTR (P-gp and ATP7B) while it does not have any effect or has an opposite effect on other multi-transmembrane proteins. ATP7B mutants displayed efficient correction after downregulation of the MLK3 pathway, where the localization of the mutant proteins to the Golgi reached almost the WT levels. Table 6. Chemical structures of tested molecules

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