PL221889B1 - Functional modulators of the mutated CFTR protein and the use of the modulators for the treatment of diseases associated with abnormal functioning of the CFTR protein - Google Patents

Description

Description of the invention

The invention relates to the use of a modulator of the function of a mutant CFTR protein or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for the treatment of cystic fibrosis. It is an object of the present invention to provide compositions, pharmaceutical preparations and methods for correcting the cell processing of a mutant CFTR protein caused by an AF508-CFTR mutation or other class II mutations.

Cystic fibrosis is one of the most common genetic diseases in humans. It inherits as an autosomal recessive trait and affects an average of one child in 2,500 live births (1). The cause of cystic fibrosis are mutations in the cftr gene, which lead to a malfunction of the CFTR protein (Cystic Fibrosis Transmembrane Conductance Regulator), which acts as an ion channel of epithelial cells (2, 3). As a result of impaired function of this protein, severe disease symptoms appear related to failure of the respiratory, digestive and male reproductive systems (4).

So far, over 1,600 mutations in the cftr gene have been identified and described (5). Based on the molecular mechanisms leading to disturbances in the functioning of the CFTR protein, a classification of mutations has been made, based on which we can distinguish five classes (6, 7). Class I mutations contribute to the production of a protein of incomplete length and are usually associated with a complete loss of its activity (e.g., G542X). Class II mutations lead to abnormal protein maturation in the endoplasmic reticulum and golgi apparatus. The effect of these mutations is premature degradation, as a result of which CFTR does not reach the cell membrane where it should perform its function (e.g. AF508, ΔΙ507, S549R) (8). The protein product of a gene with a class III mutation is correctly synthesized, transported, and incorporated into the cell membrane, but has decreased activity due to mismanagement. These mutations are usually located within one of the nucleotide binding domains (e.g. G551D / S). Class IV mutations mainly cause disturbances in the endothelial structure of the protein, thus reducing the flow of chloride ions (e.g. R117H, R334W). Mutations affecting mRNA stability are class V mutations in the cftr gene (3849 + 10kbC-> T, 5T).

The most common mutation present in at least one allele in about 90% of patients (9) is a mutation that deletes phenylalanine at position 508 of the CFTR protein XF508 CFTR). This is a classic example of a type 2 mutation that causes premature protein degradation (8, 10). The lack of a protein responsible for the regulation of water and electrolyte balance (including the transport of chloride ions outside the cell (11), regulation of Na ⁺ ion absorption into the cell (12)) results in the appearance of severe phenotypic symptoms. The most serious are the thickening and viscosity increase of mucus in the upper and lower respiratory tract, causing lung damage and providing a favorable environment for the development of bacterial infections caused by, for example, Pseudomonas aeruginosa. Moreover, the secretory tubules of the pancreas are blocked and the digestive system is seriously disturbed (13).

CFTR is a glycoprotein consisting of 1480 amino acids and is characterized by a structure typical of proteins from the family of "ABC" (ATP Binding Cassette) transporters. Five domains can be distinguished in the protein structure - two nucleotide binding domains NBD1 and NBD2 (Nucleotide Binding Domain), regulatory domain RD (Regulatory Domain) and two transmembrane domains TMD1 and TMD2. The activity of the protein is regulated by the multiple phosphorylation of the regulatory domain RD by cAMP-dependent PKA kinase and the binding of two ATP molecules by the domains of NBD1 and NBD2 (14).

The patent applications WO2005120497 (published 2005-12-22) and US20080319008 (published 2008-12-25) describe compounds increasing the activity (ion transport) of the mutant CFTR protein, as well as their use. The invention provides compositions, pharmaceutical preparations and methods for increasing the activity (ion transport) of a mutant CFTR protein, namely ∆F508 CFTR, G551D-CFTR, G1349D-CFTR, or D1152H-CFTR, which are useful in the treatment of cystic fibrosis (CF). Compositions and pharmaceutical preparations according to the invention may include one or more phenylglycine-containing compounds or sulfonamide-containing compounds or an analog or derivatives thereof.

The patent application WO2009051910 (published 2009-04-23) describes compounds showing an activity increasing the ion transport of the mutant CFTR protein, as well as their use. The invention provides compositions, pharmaceutical formulations, and methods of increasing the activity, namely, ion transport of a mutant CFTR protein. The pharmaceutical preparations and formulations of the invention have utility in the research and treatment of diseases associated with the mutant CFTR protein, such as cystic fibrosis. Compositions of the invention may include one or more phenylglycine-containing compounds or an analog or derivatives thereof.

Patent application US5948814 (published 1999-09-07) describes the use of genistein for the treatment of cystic fibrosis. The invention describes a method of treating cystic fibrosis by generating CFTR function in cells containing mutant CFTR and a therapeutic composition. The method of treatment comprises administering an effective dose of genistein, or an analog or derivative thereof, to a person suffering from cystic fibrosis.

The patent application US20040006127 (published 2004-01-08) describes a method of activating the CFTR chloride channel. Fluorescein and its derivatives are used in the treatment of animals, including humans, whose disease is related to the activity of CFTR chloride channels, e.g. cystic fibrosis, disseminated bronchiectasis, pulmonary infections, chronic pancreatitis, male infertility and long QT syndrome.

The patent applications WO2006101740 (published 2006-09-28) and US20080318984 (published 2008-12-25) describe compounds that correct the cell processing of the mutant CFTR protein and their use. The invention provides compositions, pharmaceutical preparations and methods for correcting cell processing of a mutant CFTR protein (namely AF508 CFTR) that are useful in the treatment of cystic fibrosis. The pharmaceutical compositions and preparations may include one or more compounds containing an aminobenzothiazole, aminoarylthiazole, quinazolinylaminopyrimidinone, bisaminomethylbithiazole or phenylaminoquinolines or analogs or derivatives thereof.

The patent application WO2009051909 (published 2009-04-23) describes compounds that improve the processing of the mutant CFTR protein in the cell and their use. The invention provides compositions, pharmaceutical preparations and methods for increasing the activity of a mutant CFTR protein. The pharmaceutical compositions and methods have utility in the research and treatment of diseases associated with CFTR mutations such as cystic fibrosis. Compositions of the invention may include one or more bitiazole-containing compounds or an analog or derivatives thereof.

Despite the prior art and the knowledge that phenylalanine 508 in the CFTR protein is present on the surface of the NBD1-CFTR domain, the structural and biophysical studies to date do not show significant differences between the wild protein domain and the domain of the AF508 mutant that could affect the folding kinetics or thermodynamic stability. The solved crystal structures of both domains show only slight differences in the rearrangement of the amino acids close to the site that F508 should occupy (10).

It is an object of the present invention to provide compositions, pharmaceutical preparations and methods for correcting the cell processing of mutant CFTR protein. The effect of the F508 deletion on the structure of the NBD1 domain observed in the X-ray results does not explain the dramatic difference in the behavior of the mutant and the native form of CFTR protein in the cell. For the purposes of the present invention, the structural data of both protein forms were subjected to computer simulation to determine the dynamic properties of NBD1. The solution according to the invention uses molecular dynamics, which is a method based on iterative calculations of interactions between atoms forming a simulated system and solving the equations of their motion (15). The result of the above simulations are the sets of structures obtained (for both tested forms of NBD1) that could be adopted by the tested protein in accordance with the assumed physical conditions - the so-called trajectories.

Based on the analysis of the trajectories of the molecular dynamics of both domains, it was possible to identify the conformation of the mutant protein, which differs significantly from the conformational states assumed by the wild protein. One of the hallmarks of this conformation are two significant depressions - "pockets" on the protein surface located on either side of the ATP binding site. The structure of this conformation was used to develop substances correcting the action of the AF508-CFTR mutant.

The present invention has achieved the realization of such a defined goal and solving the problems described in the prior art related to the use of compounds used in the treatment of cystic fibrosis while obtaining the highest possible efficiency of the process.

The object of the invention is the use of a modulator of the function of a mutant CFTR protein or a pharmaceutically acceptable derivative thereof, wherein the modulator is described by the formula

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for the manufacture of a medicament for the treatment of cystic fibrosis.

Preferably, the modulator influences CFTR-dependent ion flux across the cell membrane and / or increases the amount of mutant CFTR protein that reaches the cell membrane.

Preferably, the modulator affects the stabilization of the structure of the mutant CFTR protein and / or blocks interactions with cellular proteins responsible for the premature degradation of the CFTR mutant.

Preferably, the mutation of the CFTR protein is an AF508-CFTR mutation, or another class II mutation.

The attached figures allow a better explanation of the essence of the invention.

Figure 1 shows the effect of the tested molecules on the efflux of iodide ions, where:

Figure 1A shows the curves depicting iodide ion flux from HeLa cells stably transferred with AF508-CFTR and treated with test compounds for 24 hours at a concentration of 10 pM. The CFTR channel response was induced by 10 pM forskolin (Fsk) and 30 pM genistein (Gsk). An example is the iodide efflux curve in cells treated with miglustatat (100 pM / 2 h).

Figure 1B. Bar graph showing the Fsk / Gsk amplitudes of the dependent iodide efflux in cells treated with the test compounds for 24 h. at a concentration of 10 µM. The chart shows the mean values of three independent trials. * p <0.05, ** p <0.01. HeLa-F508del-CFTR cells. Compounds 1a, 1b significantly restored the function of the delF508 mutant (after 10 pM / 24 h incubation).

Figure 1C shows the curves showing the flow of iodide ions in the presence of the test molecule (incubation 1 µM / 24 h). An example is the iodide efflux curve in cells treated with miglustatat (100 pM / 2 h).

Figure 1D is a bar graph showing the index of CFTR channel activity: Fsk / Gsk amplitude of dependent iodide efflux from cells incubated with test compounds for 24 h. at a concentration of 1 µM. HeLa-F508del-CFTR cells.

Positive control = cells incubated with miglustat (100 µM / 2 h).

Figure 2a shows exemplary immunoblots of wild-type (WT CFTR) and mutant (F508del) proteins expressed in HeLa cells. F508del cells were additionally incubated with test compounds at a concentration of 10 µM for 24 hours.

C-band CFTR - corresponds to a protein with a mass of 170 kDa

CFTR band B - corresponds to the protein of 145 kDa

Figure 2b shows exemplary immunoblots of wild-type (WT CFTR) and mutant (F508del) proteins expressed in HeLa cells. F508del cells were additionally incubated with test compounds at a concentration of 1 µM for 24 hours.

C-band CFTR - corresponds to a protein with a mass of 170 kDa

CFTR band B - corresponds to the protein of 145 kDa

Figure 3 shows the list of molecules tested.

Figure 4 shows the study of the cytotoxicity of the test molecules.

For a better understanding, exemplary embodiments of the above-defined invention are presented below.

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Examples

1. Materials and methods

Antibodies

The following antibodies were used in the experiments: MAB25031 (clone 24-1, R&D systems, USA) and MM13-4 (Upstate), monoclonal antibodies (mAb) for CFTR channel detection and murine mAb for cytokeratin 18 (Santa Cruz) staining, secondary fluorescent antibodies

Alexa 594 and 488 provided by Molecular Probes (Cergy Pontoise, France).

Cell culture

Stably transferred HeLa cells (with the control plasmid alone (pTracer) and expressing WT-CFTR (spTCFWT), F508del-CFTR s (pTCF-F508del) were guided by Pascal Fanen (Inserm U.468, Creteil, France) according to the procedure described in literature [16]. Cell culture was performed on DMEM (Dulbecco's Modified Eagles Medium) supplemented with heat-inactivated 10% calf serum (FCS), penicillin (100 μg / ml), streptomycin (100 μg / ml) and Zeocin (250 μg / ml) Cells were cultured at 37 ° C in an appropriate humidity incubator with 5% CO ₂ concentration The expression of the wild wt-CFTR protein and the AF508-CFTR mutant in the cells was verified by immunoprecipitation and immunocytobiochemistry throughout the experiment. were treated with test compounds at concentrations of 1 and 10 μΜ after reaching 75% confluence.

Immunoprecipitation ₂

Cell cultures grown in flasks (75 cm ² ) were washed twice with chilled PBS buffer, detached by resuspending in 2 ml PBS and centrifuged (600 g / 5 min). The pellets were suspended in 300 µl of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, pH 7.5) at 4 ° C for 30 minutes with shaking. After centrifugation (15,000 g 30 min), the supernatant was immunoprecipitated as described in the literature [17] with minor modifications. Briefly, 5% of the lysate was used for protein concentration analysis by DC (Bio-Rad, Hercules, CA) and for input samples.

The remaining material (500 µg) was incubated with 2 µg of Mab-24-1 at 4 ° C for 45 minutes with shaking. Then 20 µl of Protein G Bio-Adembaeds (Ademtech) suspended in the lysis buffer was added. The pellets were washed three times with lysis buffer. The total material after immunopercipitation was mixed with 20 µl of Leameli buffer and heated (37 ° C / 15 minutes). The samples were then separated on 8% SDS-PAGE and transferred to PVDF membranes. The analysis was then performed according to the manufacturer's instructions for the Odyssey infrared scanner (LI-COR Biosciences, NE, USA). Membranes were blocked with Odyssey buffer (ScienceTec, Paris, France) for 1 hour and hybridized with anti-CFTR MM13-4 monoclonal antibodies (1/1000). Proteins were labeled by incubation with IRdye-labeled secondary antibodies (1/1000). The images were obtained with the Odyssey infrared imaging system. The quantitative analysis of the bands was performed using the Odyssey software.

The influence of iodide ions

The chloride channel activity was tested by measuring the efflux of iodide ( ¹²⁵ I ^- ) ions from transferred CHO cells as previously described [18]. Cells were grown for 4 days in 96-well plates, washed twice with 2 ml Erle's modified salt (137 mM NaCl,

5.36 mM KCl, 0.4 mM Na2HPO4, 0.8 mM MgCl2, 1.8 mM CaCl2, 5.5 mM glucose and 10 mM HEPES,

125 pH 7.4) and incubated in the same medium with the addition of 1 mM KI (1 mCi Na ¹²⁵ I / ml, NEN Life Science Products) for 30 minutes at 37 ° C. The cells were then washed and re-incubated with 1 ml of fresh aliquot of Erle's modified salt. After one minute, the medium was removed for radioactivity counting and immediately replaced with another fresh aliquot. The procedure was repeated eight times every minute. The first three fractions were used to obtain a uniform background (line podsta125 wowej characterized passive output ¹²⁵ ion I ^-) in the buffer discharge. In subsequent cycles, medium enriched with forskolin (10 µM) and genistein (30 µM) were added to activate the CFTR channel. At the end of the incubation, the medium was harvested and the cells were dissolved in 1N NaOH for determination of residual radioactivity inside the cells. Radioactivity was measured

125 counts per minute (cpm) using gamma counter (LKB). The total amount of ¹²⁵ I ^- (cpm) at the start time t0 was calculated as the sum of the cpm counted in each sample taken every minute

125 and cpm in the NaOH fraction. The amount of ¹²⁵ I (cpm) in each fraction collected in the following was counted

125 time slots. The discharge of ¹²⁵ I iodide ions as a function of time was calculated from the relationship described by Becq et al. [19]:

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In ( ¹²⁵ It1 / ¹²⁵ It2) / (t1-t2) where:

125 125

It - intracellular concentration of I at time tt ₁ and _{t 2} - consecutive time points

The results are presented on a graph in the form of a curve showing the dependence of the flow rate of iodide ions on time. Results are reported as mean values +/- S.E from n independent experiments. Student's t-statistical test was used to compare the effect of the tested molecules with the control samples. The results were considered statistically significant for p <0.05.

Cell Survival Test:

HeLa cells were seeded in 96-well cell culture plates, then incubated with test compounds, and assayed with MTT after 24 hours.

Metabolically active cells metabolize the soluble MTT derivative (yellow) into purple formazan (purple) insoluble in aqueous medium, which is then dissolved in DMS and in Sorson buffer (pH 10.5). Product quantification was performed by spectrophotometry on a plate reader at a wavelength of 570 nm.

Identification of molecules

The procedure leading to the identification of the molecules was based on the use of Virtual Screening methods of the bases of low-molecular compound structures with the use of docking programs. In the initial phase, the conformational space of small molecules inside two potential binding sites on the protein surface was scanned, and then gradually minimized in the force field, allowing the ligands themselves and then the entire complexes to reach an energy minimum. At individual stages, a number of functions validating potential complexes were used, and finally, on their basis, compounds for biological research were selected.

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73100 Pok2e

2- (9H-fluoren-2ylcarbamoyljterephthalic acid

9608 / Poklf

-naphthalen-2-yl-9,10dioxoanthracene-2-carboxyhc acid

123526 / Pok2d

2-amino-3- [9- (4chlorophenyl) fluoren-9 yljsulfanylpropanoic acid

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11237 / Pok2a

[4 - [(4-diniethylaminopheayl) - {2 'hydroxy> 3, ó-disulfonaphthalen-1 yl) methyl idene] cyclobex .a-2,

-dien-1 -y lidene J-dimethylazanium

105687 / PokZb

N- {2- (3,4-dimethylbenzoy i) phenyi] -4methylbenzenesułfonamide

Description of the results

Effect of tested compounds on the efflux of iodide ions

In order to confirm the hypothesis about the influence of the compounds obtained with the in silico method on the activity and / or localization of the mutant CFTR protein, the effect of the molecules on the CFTR channel function in cell lines was first checked by an automated method of studying the outflow of radioactive iodide ions from cells. HeLa cells expressing AF508-CFTR not treated with our compounds showed little response to stimulation with forskolin and genistein (Fig. 1A). After 24-hour incubation of the cells with the tested molecules at a concentration of 10 μΜ, for molecules 37173 and 130813, a cAMP dependent increase in the efflux of iodide ions from the cells was observed. For comparison, Figure 1A also shows the activity of miglustat at a concentration of 100 µM - the known corrector for the AF508-CFTR protein. Compounds 130813 and 73100 also showed activity at a concentration of 1 µΜ (Fig. 1C and 1D).

Effect of test compounds on AF508-CFTR maturation

In further studies of the molecules in the context of their activity of correcting the AF508-CFTR protein, the degree of glycosylation of the CFTR mutant was checked in the presence of the designed compounds. Figure 2A shows the immunoblot of the immunoprecipitated wild-type CFTR protein (WT-CFTR). The first fraction (band C) corresponds to the mature 170 kDa protein which is fully glycosylated. It is a mature protein that has been processed by the Golgi apparatus. The second fraction (band B) is a 145 kDa protein, which is an incompletely mature, partially glycosylated protein present only in the endoplasmic reticulum (ER). Only the second (band B) fraction can be detected in AF508-CFTR cells expressing the mutant protein.

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The presence of molecules that influence the maturation of AF508-CFTR protein results in the appearance of a fully glycosylated protein fraction, which is verified by immunoblotting after incubating the cells with the test compounds at a concentration of 10 pM for 24 hours. As shown in Fig. 2A, strip C was detected in the presence of molecules 37173 and that could simultaneously restore the ion channel function of the AF508-CFTR protein. Figure 2A also shows the weaker effect of the molecule

125 9608, which did not increase I ^- ion efflux. The mature form of the AF508-CFTR protein was also detected when the cells were incubated with three compounds designed to bind in the second "pocket" (123526, 11237 and 105687 not published) - which had no effect on the function of AF508-CFTR protein. Moreover, the molecules 130813 and 73100 at the concentration of 1 pM showed an effect on the glycosylation of the AF508-CFTR protein (Fig. 2B).

Cytotoxicity

All molecules except 73100 did not reduce the survival of treated cells, as demonstrated by the MTT assay (Fig. 4).

Literature:

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2. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science, 1989 Sep 8; 245 (4922): 1066-73.

3. Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science, 1989 Sep 8; 245 (4922): 1059-65.

4. Castellani C, Cuppens H, Macek M, Jr., Cassiman JJ, Kerem E, Durie P, et al. Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice. J Cyst Fibros, 2008 May; 7 (3): 179-96.

5. Cystic Fibrosis Mutation Database http://www.genet.sickkids.on.ca/cftr/app

6. Welsh MJ, Smith AE. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell, 1993 Jul 2; 73 (7): 1251-4.

7. Wilschanski M, Zielenski J, Markiewicz D, Tsui LC, Corey M, Levison H, et al. Correlation of sweat chloride concentration with clases of the cystic fibrosis transmembrane conductance regulator gene mutations. J Pediatr, 1995 Nov; 127 (5): 705-10.

8. Ward CI., Omura S, Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell, 1995 Oct 6; 83 (1): 121-7.

9. Bobadilla JL, Macek M, Jr., Fine JP, Farrell PM. Cystic fibrosis: a worldwide analysis of CFTR mutations-correlation with incidence data and application to screening. Hum Mutat, 2002 Jun; 19 (6): 575-606.

10. Lewis HA, Zhao X, Wang C, Sauder JM, Rooney I, Noland BW, et al. Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. J Biol chem, 2005 Jan 14; 280 (2): 1346-53.

11. Schwiebert EM, Benos DJ, Egan ME, Stutts MJ, Guggino WB. CFTR is a conductance regulator as well as a chloride channel. Physiol Rev, 1999 Jan; 79 (1 Suppl): S145-66.

12. Reddy MM, Light MJ, Quinton PM. Activation of the epithelial Na + channel (ENaC) requires CFTR CI-channel function. Nature, 1999 Nov 18; 402 (6759): 301-4.

13. Ahmed N, Corey M, Forstner G, Zielenski J, Tsui LC, Ellis L, et al. Molecular consequences of cystic fibrosis transmembrane regulator (CFTR) gene mutations in the exocrine pancreas. Gut, 2003 Aug; 52 (8): 1159-64.

14. Riordan JR. Assembly of functional CFTR chloride channels. Annu Rev Physiol, 2005; 67: 701-18.

15. Allen MP, Tildesley DJ. Computer simulation of liquids: Oxford, Clarendon Press; 1987.

16. Jungas, T., et al., Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells ex pressing wild-type and mutant cystis fibrosis transmembrane conductance regulator.

J Biol Chem, 2002. 277 (31): p. 27912-8.

17. Clain, J., et al., Two mild cystis fibrosis-associated mutations result in severe cystic fibrosis when combined in cis and reveal a residue import ant for cystic fibrosis transmembrane conductance regulator processing and function. J Biol Chem, 2001. 276 (12): p. 9045-9.

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18. Marivingt-Mounir, C., et al., Synthesis, SAR, crystal structure, and biological evaluation of benzoquinoliziniums as activators of wild-type and mutant cystic fibrosis transmembrane conductance regulator channel. J Med. Chem, 2004. 47 (4): p. 962-72.

19. Becq, F., et al., Development of substituted Benzo [c] quinolizinium compounds as novel activators of the cystic fibrosis chloride channel. J Biol Chem, 1999, 274 (39): p. 27415-25.

Claims (4)

Patent claims 1. The use of a modulator of the function of a mutant CFTR protein or a pharmaceutically acceptable derivative thereof, wherein the modulator is described by the formula ο for the manufacture of a medicament for the treatment of cystic fibrosis. 2. Use of a modulator according to claim 1 The method of claim 1, wherein the modulator influences CFTR-dependent ion flux across a cell membrane and / or increases the amount of mutant CFTR protein that reaches the cell membrane. 3. Use of a modulator according to claim 1 The method of claim 1, wherein the modulator affects the stabilization of the structure of the mutant CFTR protein and / or blocks interactions with cellular proteins responsible for premature degradation of the CFTR mutant. 4. Use of a modulator according to claim 1 The method of claim 1, wherein the mutation of the CFTR protein is a ΔF508-CFTR mutation, or another class II mutation.