Patent Application
20200022957
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.
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 (Camino 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 (Hutt 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.
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
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, (5Z)-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, PRKAA1(AMPK), PRKAA2(AMPK), RAC2, TGFBR-2, MAPK11, MAPK14, MAPK8/JNK1, CALMLS, ITPR2, RNF215, UBOXS, SART1, PDGFRB, CD2BP2, CKII/CSNK2A1, ASB8, STAG2, FBXO7, PIK3CB, MLK3/MAP3K11, CTDSP1, VEGFR2/KDR, GTSE1, PRPF8, MED1, OSMR, DSN1, NFKB2, SENP6, PDGFRA, MKK7/MAP2K7, PIK3CG, MAPK15, NUP50, CAMKK2, MIS18BP1/C14orf106, YWHAH, VEGFR1/FLT1, TEP1, MED13, PROKR1 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, BIN2, 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, UBXO5, ASB8, FBXO7 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, PRKAA1(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 (p38β) and MAPK14 (p38α), 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/JNKi 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 H20. 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′-O-methyl, 2′-O-methoxyethyl oligonucleotides and cholesterol-conjugated 2′-O-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.
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.
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 C/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 μM for 48 h), 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.
B. CFBE cells were treated with indicated siRNAs (targeting the anti-correction genes) for 72 h, 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
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.
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 Rma1 and Aha1 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 72 h 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 PRKAA1 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
A-B. CFBE cells pretreated with siRNAs were treated with CHX (50 μg/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 6 h 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).
(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 IX and Oxozeaenol (1.25, 2.5, 5, 10 μM), JNKi II (6.25, 12.5, 25, 50 μM), JNKi XI, Pazopanib, Dovitinib lactate and Bexarotene (3.12, 6.25, 12.5, 25 μM)] values that were normalized to the maximum used concentrations of the respective drugs. Also refer panel A for concentrations (μM) [±SEM (n>3)]. C. CFBE cells were treated with inhibitors of the MLK3 pathway and/or VX-809 (5 μM) for 48 h and changes in band C levels monitored. The concentrations of the MLK3 pathway inhibitors used were: JNKi II (12.5 μM), JNKi IX (50 μM), JNKi XI (25 μM) and oxozeaenol (5 μM). 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.
A. CFBE cells were treated with indicated JNK inhibitors for 24 h 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 TAK1 or MLK3 siRNA as indicated and changes in F508del-CFTR proteostasis were monitored by western blotting. TAK1 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 μM 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 μM 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 6.25-50 μM) targeting the CAMKK2 pathway for 48 h 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.
(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 2 h with 100 μM CuSO4. 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 μm.
(A) HeLa cells were infected with Ad-ATP7BWT-GFP (Chesi et al., 2016) or Ad-ATP7BH1069Q-GFP, incubated overnight with 200 μM BCS, and incubated for an additional 2 h with 100 μM CuSO4. 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 μM), VX-745(1 μM), JNK inhibitor SP600125 (2 μM) and Oxozeaenol (5 μM) (as indicated in corresponding panels) to the cells 24 h 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 μM), VX-745(1 μM), JNK inhibitor SP600125 (2 μM) and Oxozeaenol (5 μM) 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.
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.
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 α1 (Thermoscientific). The plasmids used were: JNK2 (pCDNA3 Flag MKK7B2Jnk2a2; Addgene plasmid #19727) and MKK7 (pCDNA3 Flag MKK7b1; 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, N.Y., 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 CFBE410-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 24 h. 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) (Iorio 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 (Iorio 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 (Iorio 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 (
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 (IPA)
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 mM Tris-HCl, pH 7.4), supplemented with protease inhibitor cocktail and phosphatase inhibitors. The lysates were clarified by centrifugation at 15000×g for 15 min, and the supernatants 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 24 h. 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 30 min. The lysates were clarified by centrifugation at 15000×g for 15 min, and the protein content of the supernatants 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×g for 5 min in 4° C. The cell pellet was resuspended in 1 mL of hypertonic buffer (250 mM sucrose, 10 mM 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 600×g for 15 min. The membranes were then pelleted by centrifugation at 100,000×g for 30 min, and then resuspended in digestion buffer (40 mM Tris pH 7.4, 2 mM MgCl2, 0.1 mM 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 mM, and the samples were immediately denatured in sample buffer (62.5 mM 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 KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2) and stimulated for 30 min with 20 μm forskolin and 50 μm genistein. The cells were then transferred to a Zeiss LSM700 confocal microscope, where the images were acquired with a 20× objective (0.50 NA) and with an open pinhole (459 μm) at a rate of 330 ms/frame (each frame corresponding to 159.42 μm×159.42 μm), at ambient temperature. The excitation laser line 488nm was used at 2% efficiency coupled to a dual beam splitter (621 nm) for detection. The images (8-bit) were acquired in a 512×512 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 Cl— 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 (1×106) were seeded onto 12-mm fibronectin-coated Snapwell inserts (Corning Incorporated) and the apical medium was removed after 24 h 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·cm2. 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 48 h 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 200 μg/ml nystatin and imposing an apical-to-basolateral Cl− gradient. The basolateral bathing solution contained 1.2 mM NaCl, 115 mM Na-gluconate, 25 mM NaHCO3, 1.2 mM MgCl2, 4 mM CaCl2, 2.4 mM KH2PO4, 1.24 mM K2HPO4 and 10 mM glucose (pH 7.4). The CaCl2 concentration was increased to 4mM to compensate for the chelation of calcium by gluconate. The apical bathing solution contained 115 mM NaCl, 25 mM NaHCO3, 1.2 mM MgCl2, 1.2 mM CaCl2, 2.4 mM KH2PO4, 1.24 mM K2HPO4 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% O2-5% CO2 and maintained at 37° C. Ag/AgCl reference electrodes were used to measure transepithelial voltage and pass current. Pulses (1 mV amplitude, is duration) were delivered every 90 s to monitor resistance. The voltage clamps were connected to a PowerLab/8SP interface for data collection. CFTR was activated by adding 10 μM 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 63×1.4 numerical aperture oil objective. The cells were scored based on the disappearance of ATP7B from the ER.
Morphological Assay to Estimate the Exit of ATP7B Exit from ER to Golgi:
Cells were transfected with ATP7B-WT-GFP or ATP7B-H1069Q-GFP, incubated overnight with 200 μM 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% HNO3 and analyzed with an Agilent 7700 ICP-MS (Agilent Technologies, Santa Clara, Calif., 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 μM 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 μl 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) (
The FIT analysis of the gene signatures resulted in 219 downregulated and 402 upregulated CORE genes (
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 (
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 (
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. (
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) (
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-α, 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)3β, 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-κB2, like MLK3, appear to be components of the correction-relevant branch of the MLK3 pathway, as indicated by the screening data in
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 (
In addition, silencing of the MLK3 pathway (and of several CORE genes) increased also the band C/band B ratio (see
Inventors next examined the effects on F508del-CFTR proteostasis of agents known to activate MLK3 such as TNF-α, TGF-β (Karen Schachter 2006) and reactive oxygen species (ROS) (Lee et al. 2014). TNF-α 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-α, TGF-β or H2O2 (to increase ROS), and monitored the effects on F508del-CFTR. The effects of H2O2 at non-toxic concentrations were dramatic, with a marked drop of the F508del-CFTR levels within a few minutes. Also TNF-α 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 H2O2, 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 KINOMEscan library (http://lines.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 (
Thus, selected chemical blockers of the MLK3 (and CAMKK2;
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 (
Inventors then tested the chemical inhibitors of p38 and JNK VX-745, SB202190 (SB90), Oxozeaenol and SP600125 (SP125) respectively (
Altogether, the finding in this study is that MAP3K11, MAPK8 (JNK1), MAPK11 (p3813) and MAPK14 (p38α) 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 (JNK1), MAPK11 (p3813) and MAPK14 (p38α) 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 (
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 (
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 (
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 (
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 JNK 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 (Imming, 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 (
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
Tables
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.
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.
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 (AllStars Negative Control siRNA) is Qiagen (Germany).
Thapsia garganica. A non-competitive
CFBE or HeLa cells (in case of ATP7B) were transfected with constructs encoding the indicated mutant proteins and treated with JNKi II for 48 h. The effect of JNKiII 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.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 102015000084815 | Dec 2015 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/081578 | 12/16/2016 | WO | 00 |
