PL223680B1 - 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 present invention relates to the use of a modulator of the function of a mutant CFTR protein in the treatment of diseases associated with disorders in the proper functioning of the CFTR protein. It is an object of the present invention to provide pharmaceutical preparations and methods for correcting the cell processing of a mutant CFTR protein caused by the 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 mutation classification has been made, on the basis of 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, AI507, S549R) (8). The protein product of a gene with a class III mutation is properly 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. Rl17H, 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 approx. 90% of patients (9) is a mutation that deletes phenylalanine at position 508 of the CFTR protein (AF508 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 AF508 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 mutated 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 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 forms of protein 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 isolate 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 mutant AF508-CFTR protein.

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 protein or its physiologically acceptable salt CFTR, wherein the modulator is described by the formula (I)

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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 contributes to the stabilization of the structure of the mutant CFTR protein and / or blocks interactions with cellular proteins responsible for premature degradation of the CFTR m utant.

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 µM. The CFTR channel response was induced by 10 µM forskolin (Fsk) and 30 µM genistein (Gsk). * Lack of activity is due to the use of DMSO as a solvent in which the molecule is insoluble.

The last plot also shows the iodide flux curve in cells treated with 100 µM miglustatat for 2 hours - Figure 1B. Bar graph showing the index of CFTR channel activity: Fsk / Gsk amplitude of iodide-dependent efflux from cells incubated with test compounds for 24 h. at a concentration of 10 μM. Hela-F508del-CFTR cells.

The chart shows the mean values of three independent trials. * p <0.05, ** p <0.01.

Compounds 1c and 1e significantly affect the restoration of the delF508 mutant function (after incubation with 10 μΝ ^^.

Figure 1C shows the curves illustrating the discharge of iodide ions in the presence of the tested molecules (incubation at 1 µM / 24 h). As an example, the iodide efflux curve in cells treated with miglustatat (100 μM / 2 h) is presented.

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 and 2b show the effect of test compounds on the processing of CFTR protein in cells.

Characteristic immunoblots for WT-CFTR wild protein and AF508-CFTR mutant derived from HeLa cell lines treated with test compounds for 24 hours at 10 µM (2A) and 1 µM (2B) concentrations (proteins were labeled with Mab 24-1). Mature and immature protein fractions are marked on the immunoblots.

Figure 3 shows the effect of test compounds at 1 µM on the localization of CFTR in cells. The confocal microscopy image shows the presence of the wild-type WT-CFTR protein in the membrane (panel a) and the presence of the AF508-CFTR protein inside the cell (panel b). The effect of compounds on the localization of AF508-CFTR is illustrated in panels e-f. The arrows indicate labeled CFTR and membrane localized AF508-CFTR.

Figure 4 shows the list of molecules tested.

Figure 5 shows the cytotoxicity study of the test molecules.

For better understanding, an exemplary embodiment of the above-defined invention is 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 In the literature [16], the cells were grown on DMEM (Dulbecco's Modified Eagles Medium) enriched with heat-inactivated 10% calf serum (FCS), penicillin (100 gg / ml), streptomycin (100 gg / ml) and Zeocin (250 gg / ml) Cells were cultured at 37 ° C in an appropriate humidity incubator with a 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 gM after reaching 75% confluence.

Immunoprecipitation

Cell cultures grown in flasks (75 cm) 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 g) was incubated with 2 g of Mab-24-1 at 4 ° C for 45 minutes with shaking. Then 20 g of Protein G Bio-Adembaeds (Ademtech) suspended in 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 (1/10000). The images were obtained with the Odyssey infrared imaging system. The quantitative analysis of the bands was performed using the Odyssey software.

Immunofluorescent staining

Hela cells were grown on coverslips and treated as above. They were then fixed with 4% formaldehyde and permeabilized with 0.1% Triton in PBS. Blocked with 1% bovine serum albumin in PBS / Triton and incubated at 4 ° C overnight with primary antibodies, 24-1 (1: 300) or anti-cytokeratins 18 (1:00). After washing and blocking with 5% normal goat serum, cells were incubated with secondary antibodies and then coverslip slides were sealed using Vectashield (Vectashield Mounting Medium Vector laboratories). The preparations prepared in this way were scanned with a confocal laser microscope (Zeiss, LSM 510).

Iodide outflow

125

The activity of the chloride channel was investigated by measuring the efflux of iodide ions (I. from transfected CHO cells as previously described [18]. Cells were grown for 4 days in 96-well plates, washed twice with 2 ml of Erie's modified salt (137 mM NaCl, 5.36 mM KCl, 0.4 mM Na₂HPO4, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 5.5mM glucose and 10mM HE125

PES, 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 (baseline characterizing the passive I ^- output) in the outflow buffer alone. In the next

During cycles, medium enriched with forskolin (10 µM) and genistein (30 µM) was 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 in counts per minute (cpm) using a gamma counter (LKB). The total amount of I ^- (cpm) at the start time t ₀ was calculated as the sum of the cpm counted in each sample taken every minute

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

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

ln ( 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 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 was searched, and then they were subjected to gradual minimization in the force field, allowing the ligands themselves, and then the entire complexes, to reach the 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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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 the concentration of 10 μΜ (molecules: 118208, 11668), a cAMP-dependent increase in the efflux of iodide from the cells was observed. For comparison, Figure 1A also shows miglust activity at a concentration of 100 µM - the known corrector for the AF508-CFTR protein. Compounds 118208 and 407882 also showed activity at a concentration of 1 µΜ (Figures 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.

The presence of molecules that influence the maturation of the AF508-CFTR protein results in the appearance of a fully glycosylated protein fraction, which is verified by immunoblot after incubating the cells with the test compounds at a concentration of 10 μM for 24 hours. As shown in Fig. 2A, strip C was detected in the presence of the molecule 118208, which could simultaneously restore the function of the AF508-CFTR protein ion channel. Moreover, molecules 118208 and 407882 also at a concentration of 1 μM showed an effect on the glycosylation of the AF508-CFTR protein (Fig. 2B).

Effect of studied molecules on CFTR immunolocation

Figure 3A shows typical staining for CFTR protein on the membrane in HeLa cells expressing WT-CFTR. The AF508-CFTR mutant in cells is located throughout the cytoplasm (Fig. 3B). Treatment of cells expressing AF508-CFTR with compounds 11668, 118208 and 407882 at a concentration of 10 µM for 24 hours resulted in the appearance of a stained mutant in the cytoplasmic membrane, indicating the ability of these molecules to inhibit the degradation of AF508-CFTR protein.

Cytotoxicity

All tested molecules did not reduce the survival of the treated cells, as demonstrated by the MTT test (Fig. 5).

L i t e r a t u r a:

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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.

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8. Ward CL, 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.

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12. Reddy MM, Light MJ, Quinton PM, Activation of the epithelial Na ⁺ channel (ENaC) requires CFTR Cl- 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.

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Claims (4)

1. The use of a modulator of the function of a mutant CFTR protein or a physiologically acceptable salt thereof, wherein the modulator is described by the formula (I) 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 in the CFTR protein is an AF508-CFTR mutation or another class II mutation.