WO2020242225A1 - COMPOSITION FOR PREVENTING OR TREATING PROTEIN CONFORMATIONAL DISORDERS, COMPRISING IRE1α KINASE ACTIVATOR AS ACTIVE INGREDIENT - Google Patents

Abstract

The present invention relates to a composition for preventing or treating protein conformational disorders, a screening method therefor and a composition for inhibiting an unconventional protein secretion (UPS) pathway. The present invention may be usefully employed as a therapeutic agent composition effective against various protein conformational disorders caused by a folding transport defect of a mutant protein, and also provides a highly-reliable novel target for developing a therapeutic agent.

Description

Composition for preventing or treating diseases of abnormal protein form comprising IRE1α kinase activator as an active ingredient

The present invention relates to a composition for the prevention or treatment of abnormal protein form diseases comprising an IRE1α kinase activator, for example, CSTMP as an active ingredient.

Deficiency in protein folding and migration is a common pathogenesis mechanism that inhibits the homeostasis of living organisms and contributes to a variety of human diseases (1-3). In eukaryotes, secreted proteins, including transmembrane proteins, are synthesized in ribosomes and then migrated to the endoplasmic reticulum (ER). ER contains a variety of chaperones and folding catalysts to ensure proper folding of proteins (4). The secreted proteins that enable the ER quality control mechanism come out of the endoplasmic reticulum (ER), move to the Golgi, and eventually to the plasma membrane or extracellular medium. In contrast, incompletely folded proteins remain in the ER and are selectively targeted with ER-associated degradation (ERAD) in order to prevent the risk of abnormal proteins that occur when they exit the ER (5). Many disease-causing mutations in cystic fibrosis membrane conduction regulators (CFTR, ABCC7 ) and pendrin ( SLC26A4 ) cause protein folding defects. In conclusion, the misfolded protein cannot enter the normal Golgi-mediated protein secretion pathway, ultimately causing cystic fibrosis (CF) and congenital hearing loss, respectively.

CFTR, which has anion channel activity, is a cyclic AMP-regulated transport protein and conducts Cl ^- and HCO ₃ ^- on the apical surface (tip surface) of epithelial cells in secretory organs including airways, pancreas, small intestines, and exocrine glands ( 6). CFTR is one of the qualitative management systems of ER and has two N-linked glycosylation sites that are initially core glycosylated in the ER (band B) and mediate the interaction with the ER lectin chaperone (7). After migration to the Golgi, these glycosylation sites are complex-glycosylated (band C), and the complex-glycosylated CFTR is directed to the adhesiv surface of the epithelial tissue (8). Of the 2,000 identified mutations, the mutation in which the 508th phenylalanine residue is deleted (ΔF508) is the most common disease-causing CFTR mutation (9), causing protein misfolding, ER retention, and degradation by ERAD (10). In conclusion, the ΔF508-CFTR protein remains in a core-glycosylated form in the ER, and only a small amount is expressed on the plasma membrane surface (11).

Is a transmembrane protein of transporting anions such as (12) ^-, I ^{- -} or HCO ₃ gave pen is Cl in the inner ear (inner ear), and thyroid follicles. The loss of function of fendrin due to genetic mutations can be the cause of deafness with an enlarged vestibular aqueduct (DFNB4) and Pendrid syndrome (PDS). The most common disease-causing mutation of pendrin, p.H723R (His723Arg), induces protein misfolding, ER retention, and degradation by ERAD, like the ΔF508-CFTR protein (13).

Most secreted proteins use a typical Golgi-mediated secretory pathway when moving from the ER to the plasma membrane. In addition to these traditional secretion pathways, a new mechanism called “atypical protein secretion (UPS)” mediates the transport of secreted proteins into cells, including transmembrane proteins (14). Previous studies have shown that the ER-Golgi transport pathway is blocked or under stress conditions of the ER, the immature core-glycosylated CFTR and pendrin can reach the plasma membrane via the Golgi-independent UPS pathway ( 15, 16). Once at the cell surface, core-glycosylated CFTR and pendrin have anionic hydrogen capacity, albeit somewhat reduced (15, 16). Therefore, if UPS can be selectively activated without causing significant cellular stress, it can be used as a promising therapeutic means for diseases that occur due to defects in the folding of membrane proteins and migration to the cell surface. However, conventional methods of activating the UPS of CFTR and pendrin (e.g., overexpression of GRASP55 in ΔF508-CFTR and overexpression of MVB12B in p.H723R-fendrin) (15, 16) are CF, DFNB4, or It is clinically unsuitable to treat human patients with Pendrid syndrome.

Although recent studies have identified several key molecules involved in the UPS of membrane proteins, the overall picture of the UPS regulation mechanism is not yet known. In general, most UPSs are stress-induced rather than structural (19). Blocking of ER-Golgi transport, for example, induces a stress related signal with respect to the UPS of membrane proteins. Blocking typical protein secretion from the ER to the Golgi causes unfolded proteins to accumulate in the ER lumen, inducing ER stress and an adaptive cellular response called UPR (unfolded protein response) (20). There are three main types of UPR sensors in eukaryotic cells: inositol-desiring enzymes 1α (IRE1α) and IRE1β; RNA-like ER kinase (PERK), which is a protein kinase; And activating transcription factors 6α (ATF6α) and ATF6β that exist in the ER membrane and transmit ER stress signals. Interestingly, IRE1α, the evolutionarily most conserved form of the UPR signal, appears to play an important role in the ER stress-induced UPS of membrane proteins. Deficiency of IRE1α does not induce CFTR and Fendrin UPS following ER-Golgi pathway blockade (16, 21). Nevertheless, the clear mechanism of UPS regulation by IRE1α is not known.

IRE1 is a type I ER transmembrane protein consisting of an ER-luminal domain that serves as a sensor for a protein that is not folded during ER stress, and a cytoplasmic domain including an endoribonuclease domain and a Ser/Thr protein kinase domain. The activated mammalian IRE1α protein transmits the ER stress signal by cleaving the mRNA of the transcription factor XBP1 (X-box-binding protein 1), and thus activated XBP1 (spliced XBP1) regulates protein folding and protein quality. And UPR-related genes involved in ERAD (22). Using this mechanism, the burden by the unfolded protein in the ER is alleviated and the ER regains homeostasis. The IRE1-dependent decay (RIDD) process is also recognized as a complementary mechanism to reduce ER loading by degrading the mRNA of various proteins (23). In addition, activation of IRE1 protein kinase triggers the'alarm stress pathway' using an adapter protein such as TRAF2 (TNF receptor-associated factor 2) to trigger ASK1 (apoptosis signal-regulating kinase 1) and its downstream effector, JNK. (JUN N-terminal kinase) is activated (24).

The present inventors investigated the role of IRE1α in UPS by analyzing each signal arm. The present inventors' findings showed that the UPS of CFTR and pendrin was activated in vivo and in vitro through the IRE1α kinase-mediated signaling cascade, not by the XBP1- and RIDD-dependent pathways requiring IRE1α RNase activity. The IRE1α kinase pathway can provide a novel target for the development of therapeutic agents for diseases caused by protein folding and migration defects.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made intensive research efforts to discover new highly reliable therapeutic targets for various protein morphological disorders caused by the abnormal three-dimensional structure of the protein, and to develop an efficient new therapeutic composition based on this. As a result, when the compound of Formula 1 is administered, the'Unconventional Protein Secretion (UPS)' is activated without causing apoptosis, and the expression of the cell surface of the secreted protein having folding and mobility defects due to mutation, etc. By finding a dramatic recovery, the present invention was completed.

Accordingly, an object of the present invention is to provide a composition for preventing or treating protein conformational disorders.

Another object of the present invention is to provide a method for screening a composition for preventing or treating diseases of abnormal protein form.

Another object of the present invention is to provide a composition for inhibiting atypical protein secretion (UPS).

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a composition for preventing or treating protein conformational disorders comprising a compound represented by the following general formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient do:

General Formula 1

In the above general formula, R ₁ to R ₃ are each independently C ₁ -C ₃ alkyl, and X is halogen.

The present inventors have made intensive research efforts to discover new highly reliable therapeutic targets for various protein morphological disorders caused by the abnormal three-dimensional structure of the protein, and to develop an efficient new therapeutic composition based on this. As a result, when the compound of Formula 1 is administered,'Unconventional Protein Secretion (UPS)' is activated without causing apoptosis, and expression of the cell surface of the secreted protein having folding and mobility defects due to mutation, etc. By finding a dramatic recovery, the present invention was completed.

According to a specific embodiment of the present invention, the protein morphological abnormality disease prevented or treated with the composition of the present invention is a disease caused by unfolding or misfolding of the protein due to amino acid mutation.

In the present specification, the term "protein" refers to a series of macromolecules formed by bonding of amino acid residues to each other by peptide bonds. Proteins are linear molecules consisting of consecutive bonds of amino acid units, but their three-dimensional shape and state change tendency are affected by the overall size, charge and hydrophobicity of all or each constituent residue, and whether or not covalent or non-covalent bonds are formed. And if the tendency is abnormal, it may cause various PCD (protein conformational disease) diseases.

Specifically, the protein is a secretory protein. Secreted proteins, including transmembrane proteins, move from the endoplasmic reticulum (ER) to the Golgi and are ultimately sent to the plasma membrane or extracellularly. Proteins with folding defects do not enter the normal Golgi-mediated protein secretion pathway, and vesicle-related degradation mechanisms (ERAD, ER-associated degradation) causes various diseases because cell surface expression is not achieved.

In the present specification, the term "folding defect" means that a polypeptide cannot be folded normally so that a protein acquires a three-dimensional structure having its own function and activity. Thus, the term “folding defect” is meant to include “misfolding” and “unfolding”.

In the present specification, the term “treatment” refers to (a) inhibition of the development of a disease, disease or condition; (b) alleviation of a disease, disease or condition; Or (c) to eliminate the disease, disease or condition. The therapeutic composition discovered through the method of the present invention inhibits cell surface expression of secreted proteins by activating atypical protein secretion in individuals suffering from PCD disease, more specifically due to folding and migration defects caused by mutations in secreted proteins. It serves to inhibit, eliminate, or alleviate the development of symptoms caused by. Accordingly, the composition of the present invention may itself be a therapeutic composition for PCD, or may be administered together with other pharmacological components and applied as a therapeutic adjuvant for the disease. Accordingly, the term “treatment” or “therapeutic agent” in the present specification includes the meaning of “treatment aid” or “treatment aid”.

In the present specification, the term “prevention” refers to suppressing the occurrence of a disease or disease in a subject that has not been diagnosed as having a disease or disease, but is likely to have such disease or disease.

In the present specification, the term “administration” or “administer” refers to the formation of the same amount in the body of the subject by directly administering a therapeutically effective amount of the composition of the present invention to the subject. The "therapeutically effective amount" of the composition means an amount of extract sufficient to provide a therapeutic or prophylactic effect to a subject to which the composition is to be administered, and includes a "prophylactically effective amount". As used herein, the term “subject” includes, without limitation, human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.

In the present specification, the term “alkyl” refers to a linear or branched saturated hydrocarbon group, and C ₁ -C ₃ alkyl refers to an alkyl group having 1 to 3 carbon atoms, and when C ₁ -C ₃ alkyl is substituted, a substituent The carbon number of is not included.

According to a specific embodiment of the present invention, R ₁ to R ₃ in the general formula 1 of the present invention are C ₁ alkyl.

According to a specific embodiment of the present invention, X in the general formula 1 of the present invention is Cl.

The compound of Formula 1 wherein R ₁ to R ₃ are C ₁ alkyl (methyl) and X is Cl is (E)-2-(2-chlorostyryl)-3,5,6-trimethyl-pyrazine (CSTMP), CSTMP Activates the IRE1α kinase. As described in detail in the following examples, the present inventors first confirmed through various experiments that IRE1α kinase must be activated in order to restore the surface expression of a secreted protein having a folding/migration defect by activating UPS. This reverses common sense in the art that suggested that the induction of splicing (activation) of XBP1 through IRE1α endonuclease activation is a key target for UPS promotion, suggesting the most efficient and direct target for treating PCD disease. will be.

According to a specific embodiment of the present invention, the concentration of the compound of Formula 1 of the present invention is 5 μM-50 μM, more specifically 5 μM-30 μM, and most specifically 10 μM-20.

According to a specific embodiment of the present invention, the protein is selected from the group consisting of a cystic fibrosis membrane conduction regulator (CFTR), pendrin, and combinations thereof.

More specifically, the protein form abnormal disease prevented or treated with the composition of the present invention is cystic fibrosis or congenital hearing impairment.

CFTR is a protein constituting the qualitative management system of the endoplasmic reticulum, and is transferred to the Golgi, complex-glycosylated, and then sent to the attachment surface of the epithelial tissue. Cystic fibrosis is caused by being degraded by ERAD without being expressed on the plasma membrane surface.

Pendrin is a transmembrane protein that transports negative ions in the inner ear and thyroid follicles, and a series of processes leading to protein misfolding, ER retention, and ERAD caused by mutations cause congenital hearing impairment. Specifically, the hearing impairment is deafness with an enlarged vestibular aqueduct (DFNB4) or Pendrid syndrome (PDS).

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It does not become. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and specifically, it is administered parenterally.

The appropriate dosage of the pharmaceutical composition of the present invention is prescribed in various ways depending on factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. A preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001-100 mg/kg on an adult basis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs. Or it can be made by incorporating it into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating a protein conformational disorder comprising the following steps:

(1) contacting a test substance with a biological sample containing IRE1α kinase; And

(2) measuring the activity or expression of IRE1α kinase in the sample,

When the activity or expression of the IRE1α kinase is increased, the candidate substance is determined as a composition for preventing or treating abnormal protein morphology.

Since the protein morphology abnormal disease indicated in the present invention has already been described above, description thereof will be omitted to avoid excessive duplication.

In the present invention, the term "biological sample" refers to any sample including cells expressing IRE1α obtained from mammals including humans, and includes, but is not limited to, tissues, organs, cells, or cell culture.

The term “test substance” used while referring to the screening method of the present invention is added to a sample containing IRE1α-expressing cells, and is used in screening to test whether it affects the activity or expression level of IRE1α kinase. Means substance. The test substances include, but are not limited to, compounds, nucleotides, peptides, and natural extracts. The step of measuring the expression level or activity of IRE1α kinase in a biological sample treated with the test substance may be performed by various methods known in the art for measuring expression levels and activities. As a result of the measurement, when the expression level or activity of IRE1α kinase is increased, the test substance may be determined as a composition for preventing or treating diseases of abnormal protein form.

In the present specification, the term “increased expression” means that the expression level of IRE1α kinase increases to the extent that the symptoms of protein morphological disorders are alleviated or improved or the risk is reduced by promoting UPS of a mutant protein having a folding/migration defect. do. Specifically, it may mean a state in which the activity or expression is increased by 20% or more, more specifically, by 40% or more, and more specifically, by 60% or more compared to the control group.

In the present specification, the term "increase in activity" refers to a significant increase in the intrinsic function of the protein in vivo compared to the control group. Specifically, the UPS of a mutant protein having a folding/migration defect is promoted to cause abnormal protein morphology. It refers to an increase in the activity of IRE1α kinase to the extent that symptoms of a disease are alleviated or improved, or the risk thereof is reduced. Increasing activity includes not only an increase in function, but also an increase in ultimate activity due to an increase in stability.

According to another aspect of the present invention, the present invention provides a composition for inhibiting atypical protein secretion (UPS) comprising an inhibitor of IRE1α kinase as an active ingredient.

In the present specification, the term “inhibitor” refers to a substance that causes a decrease in the activity or expression of IRE1α kinase, whereby the activity or expression of the IRE1α kinase becomes undetectable or exists at an insignificant level, as well as in the IRE1α kinase. It refers to a substance that decreases the activity or expression of IRE1α kinase to the extent that the UPS pathway induced by it can be significantly reduced.

Inhibitors of IRE1α kinase include, for example, shRNA, siRNA, miRNA, ribozyme, peptide nucleic acids (PNA) or Antisense oligonucleotides and antibodies or aptamers that inhibit at the protein level, as well as compounds, peptides, and natural products that inhibit the activity of IRE1α kinase, but are not limited thereto.

As used herein, the term “atypical protein secretion (UPS) inhibition” significantly inhibits the UPS pathway induced by IRE1α, and ultimately inhibits the movement of abnormal proteins with folding defects to the plasma membrane or secretion outside the cell. it means. Accordingly, the term “atypical protein secretion (UPS) inhibition” has the same meaning as “prevention or treatment of diseases caused by excessive secretion of proteins with folding defects”.

According to a specific embodiment of the present invention, the inhibitor of IRE1α kinase of the present invention is APY29.

According to another aspect of the present invention, the present invention provides a protein conformational disorder comprising administering a composition comprising a compound represented by the following general formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. ) To provide a method of preventing or treating:

General Formula 1

Since the general formula 1 compound used in the present invention and protein morphological disorders that can be prevented or treated through the same have been described above, description thereof will be omitted to avoid excessive redundancy.

According to another aspect of the present invention, the present invention provides a method of inhibiting atypical protein secretion (UPS) in a subject comprising administering an inhibitor of IRE1α kinase.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for preventing or treating a protein conformational disorder, and a screening method thereof.

(b) The present invention also provides a composition for inhibiting the atypical protein secretion (UPS) pathway.

(c) The present invention can be usefully used as an efficient therapeutic composition for various protein morphological disorders caused by defects in folding and migration of mutant proteins, and provides a novel target with high reliability for the development of therapeutic agents.

1 is a diagram showing that XBP1 splicing and IRE1α endonuclease activity are independent of the UPS of ΔF508-CFTR. ASK1 phosphorylation is activated due to Arf1-Q71L-induced ER-to-Golgi blockade, but XBP1 splicing is not. HEK293 cells were transformed with Arf1-Q71L plasmid for 48 hours or treated with 5 μM of tapsigargin for 12 hours to induce ER stress. Representative immunoblot results of IRE1α, phosphoric acid-IRE1α, phosphoric acid-ASK1, and spliced XBP1 are shown in FIG. 1A, and the quantification results for the results of multiple experiments are summarized in FIG. 1B (n=3). IRE1α, not XBP1, is required for the UPS in Arf1-Q71L-derived ΔF508-CFTR. Figure 1c is a picture showing the results of the surface biotinylation analysis of HEK293 cells transformed with plasmid and/or siRNA as an immunoblot, and the results of multiple experiments are summarized in Figure 1d (n=4). The cell surface-specific labeling of the protein was confirmed by the absence of the cytoplasmic protein aldolase A in the biotinylated fragment. In the above-described method, instead of XBP1 siRNA, STF-083010 (60 μM, 12 h) was treated to perform surface biotinylation analysis. Representative results are shown in Fig. 1e, and the results for multiple experiments are summarized in Fig. 1f (n=4). Histogram data are expressed as mean ± standard error. ** P <0.01, ns no significance. Data was analyzed for one-way variance and then Tukey multiple comparison test was performed.

Figure 2 is a diagram showing that IRE1α kinase activity is required for UPS induction of ΔF508-CFTR. After inducing ΔF508-CFTR UPS with Arf1-Q71L in HEK293 cells, APY29 (100 μM) or protein synthesis inhibitor cycloheximide (0.1 mg/mL) was treated, and surface biotinylation assay was performed. The effect of ΔF508-CFTR on UPS was investigated. Representative immunoblot results are shown in FIG. 2A, and results for multiple experiments are summarized in FIG. 2B (n=5). APY29 selectively reduces the surface ΔF508-CFTR without affecting the overall protein level. HeLa cells were transformed with ΔF508-CFTR expression plasmids in the presence and absence of Arf1-Q71L, respectively, and APY29 (100 μM) was added to the medium for a period of time. CFTR was immunostained with anti-M3A7 CFTR antibody (green), and IRE1α was labeled with anti-IRE1α antibody (red). Representative immunofluorescence images are shown in Fig. 2c, and the quantification results for surface CFTR intensity are summarized in Fig. 2d (n=5-14). Arrows indicate ΔF508-CFTR expressed on the cell surface. Scale bar: 10 μm. Graph data are expressed as mean ± standard error. ** P <0.01. Data was analyzed for one-way variance and then Tukey multiple comparison test was performed.

3 is a diagram showing that ASK1 is required for a UPS of a core-glycosylated CFTR. The ASK1 inhibitor, MSC2032964A (ASK1-Inh), inhibits the UPS of Arf1-Q71L-induced CFTR. Surface biotinylation assays were performed in HEK293 cells transformed with plasmids expressing WT-CFTR, ΔF508-CFTR and/or Arf1-Q71L. Several cells were treated with MSC2032964A (10 μM) for a period of time. Representative surface biotinylation results for WT-CFTR and ΔF508-CFTR are shown in Figs. 3A and 3B, respectively. The results for the multiple experiments are summarized in the graph of FIG. 3C (n=6). The inhibitory effect of MSC2032964A on ASK1 activity could be confirmed through reduced phosphorylation. ** P <0.01, compared to Arf1-Q71L, 0h. Surface biotinylation analysis was performed in the manner described above by co-transformation of ASK1-specific siRNA (100 nM). Representative surface biotinylation results of WT-CFTR and ΔF508-CFTR are shown in FIGS. 3D and 3F, respectively. The results for the multiple experiments are summarized in FIGS. 3E and 3G (all n=3). b , core-glycosylated CFTR. c , complex-glycosylated CFTR. Graph data are expressed as mean ± standard error. ** P <0.01. Data were analyzed for one-way variance, followed by Tukey multiple comparison test or two-sided Student's t-test.

4 is a diagram showing that activation of IRE1α kinase by CSTMP induces UPS of ΔF508-CFTR. HEK293 cells were treated with 3-100 μM CSTMP for 48 hours. The activation of the apoptosis signal was analyzed by cleaving PARP and caspase 3 (arrow). Representative immunoblot results are shown in FIG. 4A, and results for multiple experiments are summarized in FIG. 4B (n=5). CSTMP at a concentration of 10 μM activated ASK1, but did not activate the apoptosis signal (red arrow). Surface biotinylation assays were performed in HEK293 cells expressing ΔF508-CFTR. Several cells were treated with CSTMP (10 μM) for a period of time. Representative results are shown in Fig. 4c, and a summary of the results of multiple experiments is shown in Fig. 4d (n=6). * P <0.05, ** P <0.01, compared to untreated group (first lane). ## P <0.01, compared to the group treated with GRASP55-Myc alone (lane 4). HeLa cells were transformed with a plasmid encoding extracellular HA-tagged ΔF508-CFTR. Several cells were incubated with CSTMP (10 μM) for 12 or 24 hours before fixation. CFTR on the cell surface before membrane permeation was immunostained with an anti-HA antibody (green), and after permeation, the entire CFTR was stained with an anti-R4 CFTR antibody (red). Arrows indicate surface CFTR. Morphological quantification of surface CFTR strength is shown in Fig. 4f (n=5). Scale bar: 10 μm. ** P <0.01, compared to untreated group (first lane). Data were analyzed for one-way variance and then Tukey multiple comparison test was performed.

5 is a diagram showing that CSTMP (10 μM) treatment does not induce cytotoxicity. 5A and 5B show the effect of ectopic Arf1-Q71L on UPS, activation of ASK1 and caspase 3 cleavage of ΔF508-CFTR over time. Representative immunoblot results are shown in FIG. 5A, and results for multiple experiments are summarized in FIG. 5B (n=6). Caspase 3 was cleaved after 72 hours of transformation with the plasmid encoding Arf1-Q71L (arrow). Figures 5c and 5d show the effect of CSTMP (10 μM) on UPS, activation of ASK1 and caspase 3 cleavage of ΔF508-CFTR over time. Representative immunoblot results are shown in Fig. 5c, and the results for multiple experiments are summarized in Fig. 5d (n=6). CSTMP (10 μM) did not cause cleavage of caspase 3 until 72 hours. Camptothecin (2 μM, 12h) was used as a positive control to induce cleavage of caspase 3 (arrow). Graph data are expressed as mean ± standard error.

6 is a diagram showing that the Cl ^- channel function of ΔF508-CFTR is restored by CSTMP. Total cell currents were recorded from HEK293 cells transformed with the specified plasmid. Current was induced by applying ramp pulses from -100 mV to +100 mV (0.8 mV/ms, fixed voltage 0 mV) at intervals of 10 seconds. CFTR Cl ^- current was activated by cAMP (Forskolin 5 μM + IBMX 100 μM) and inhibited by CFTR _inh -172 (5 μM). 6A is a diagram showing the current density measured at -80 mV. The number of copies (n) was indicated for each lane. 6B-6F show representative total cell currents for 48 hours in cells transformed with mock, WT-CFTR or ΔF508-CFTR plasmid. Several cells were treated with CSTMP (10 μM) for 24 hours, thereby inducing a clear CFTR current in cells expressing ΔF508-CFTR. Histogram data are expressed as mean ± standard error. ** P <0.01. Data were analyzed by unpaired Student's t -test.

7 is a diagram showing that CSTMP recovers the surface expression of misfolded fendrin. PANC-1 cells stably expressing p.H723R-fendrin were transfected with a plasmid encoding Arf1-Q71L and incubated with CSTMP (10 μM or 30 μM) for 48 hours. A representative surface biotinylation analysis result is shown in FIG. 7A, and the results for multiple experiments are summarized in FIG. 7B (n=5). * P <0.05, ** P <0.01, compared to untreated group (first lane). ## P <0.01, compared to the group that introduced only Arf1-Q71L (second lane). By recording the pH _i , Cl ^- _o /HCO ₃ ^- _i exchange activity was measured, and representative anion exchange measurement results are shown in Fig. 7c, and quantification of multiple experimental values is shown in Fig. 7d (n=7-8). CSTMP (30 μM) is expressed in the Cl gave p.H723R- pen ^cells increased the activity significantly exchange ^- / HCO _3. Histogram data are expressed as mean ± standard error. ** P <0.01, compared with the group treated with p.H723R-fendrin only. Data were analyzed for one-way variance and then Tukey multiple comparison test was performed.

8A shows the result of performing a surface biotinylation analysis using epithelial cells collected from the colonic mucosa. A protein sample from HEK293 cells was used as a control. Wild-type (Cftr ^WT ) or ΔF508-CFTR (Cftr ^F508del ) 6-week-old mice were administered vehicle or CSTMP (2.59 mg/kg, per os, once a day) for 5 days, respectively. Representative surface biotinylation analysis results are shown in Fig. 8A, and the results of multiple experiments are summarized in Fig. 8B (n=4). Figure 8c shows the immunohistochemistry of CFTR. The longitudinal/transverse sections of mouse colon tissue were immunostained with anti-CFTR R4 rabbit polyclonal antibody. Arrows indicate CFTR expression in the colon apical membrane. CSTMP treatment induced cell surface expression of CFTR in Cftr ^F508del mouse colon. Scale bar: 10 μm. 8D and 8E It shows the result of measuring short circuit current (I _sc ) in the mouse colon. The epithelial Na ⁺ channel was blocked by treatment with amiloride (100 μM) on the tip of the mouse colon. When forskolin (10 μM), an adenylyl cyclase activator, was administered to the tip, a lumen-negative I _sc was induced, which was delivered to the base of Na ⁺ -K ⁺ -2Cl ^- bumetanide (100 μM). In Cftr ^F508del mouse colon, CSTMP treatment induced CFTR-dependent I _sc . Representative I _sc measurement results are summarized in FIG. 8D, and results for multiple experiments are summarized in FIG. 8E (n=6-9). b , core-glycosylated CFTR. c , complex-glycosylated CFTR. Bar graph data are expressed as mean±standard error. ns no significance. ** P <0.01. Data were analyzed for one-way variance and then Tukey multiple comparison test was performed.

9 is a diagram showing the control experiment results of FIG. 1. HEK293 cells were transformed with a plasmid encoding Arf1-Q71L for 48 hours or treated with tapcigagin (5 μM) for 12 hours to induce ER stress, and incubated with cross-linking reagent EGS (500 μM) for 30 minutes ( Fig. 9a). Arrow heads represent cross-linked IRE1α polymer (top band) and monomer (bottom band) forms, respectively. Representative immunoblot results are shown in the left panel, and the quantification results for multiple experiments are summarized in the right panel (n=3). 9B is a diagram showing the result of quantifying the mRNA of XBP1 by qPCR. RNA samples were prepared 48 hours after transforming HEK293 cells with XBP1-specific siRNA. Cells were transformed with a plasmid encoding Arf1-Q71L for 48 hours or treated with tapcigagin (5 μM) for 12 hours, and the quantification results for the multiple experiments were summarized (n=3). Only tapcigagin, not Arf1-Q71L, increased the level of XBP1 mRNA silenced by siRNA against XBP1 . 9C is a diagram showing the effect of IRE1α- and XBP1-specific siRNAs on XBP1 splicing. HEK293 cells were treated with tapcigagin (5 μM) for 12 hours or transformed with a designated siRNA (48 hours). Tapsigagin increased the protein levels of spliced XBP1 (XBP1s) reduced by siRNA against IRE1α or XBP1. Representative immunoblot results are shown in the left panel, and the quantification results of multiple experiments are summarized in the right panel (n=3). 9D is a diagram showing that STF-083010 reduces the RNase activity of IRE1α. HEK293 cells were treated with tapcigagin (5 μM, 12 hours) and/or STF-083010 (60 μM, 12 h), and then immunoblot analysis for XBP1 protein was performed. Representative immunoblot results are shown in the left panel, and the quantification results for multiple experiments are summarized in the right panel (n=3). Bar graph data are expressed as mean±standard error. ** P <0.01. The data were analyzed through the Tukey multiple comparison test after one-way variance analysis.

10 is a diagram showing the control experiment results of FIG. 2. 10A and 10B Figure shows that APY29 dose-dependently inhibits phosphorylation of IRE1α and ASK1. Phosphorylation of IRE1α and ASK1 was induced by transforming with Arf1-Q71L plasmid for 48 hours. A representative immunoblot is shown in Fig. 10A, and the results of multiple experiments are summarized in Fig. 10B (n=3). The IC ₅₀ value of APY29 for ASK1 phosphorylation is 6.4 μM. 10C to 10F show the results of analyzing the protein stability of WT- and ΔF508-CFTR in HEK293 cells treated with cycloheximide, a protein synthesis inhibitor. Cells were transformed for 48 hours with a plasmid encoding WT- or ΔF508-CFTR. Several cells were co-transformed with the Arf1-Q71L plasmid to induce UPS. After incubation with cycloheximide (0.1 mg/mL) for a predetermined time, surface biotinylation analysis was performed. Representative results of WT-CFTR and ΔF508-CFTR are shown in Figs. 10C and 10D, respectively. CFTR levels and total cell lysates at the control and Arf1-Q71L-transformed cell-surface are summarized in Figures 10E and 10F, respectively (n=3). The protein stability of ΔF508-CFTR was lower in all conditions than WT-CFTR. Data are expressed as mean±standard error. ** P <0.01, compared to WT-CFTR. The data were analyzed through the Tukey multiple comparison test after one-way variance analysis.

11 is a diagram showing that overexpression of IRE1α activates the UPS of ΔF508-CFTR. Only IRE1α overexpression activates the UPS of ΔF508-CFTR and reinforces the effect of tarpsigagin. HEK293 cells were co-transformed with plasmids encoding ΔF508-CFTR and/or IRE1α (0.5 or 1 μg/mL, 48 hours). Several cells were treated with tapcigagin (5 μM, 12 hours). Representative surface biotinylation analysis results are shown in Fig. 11A, and a summary of the results of multiple experiments (n=3) is shown in Fig. 11B. HEK293 cells were transformed with the defined plasmid for 48 hours. Representative surface biotinylation analysis results are shown in Fig. 11c, and a summary of the results of multiple experiments (n=5) is shown in Fig. 11d. Bar graph data are expressed as mean±standard error. * P <0.05, ** P <0.01, compared to untreated group (1st lane). # P <0.05, ## P <0.01, compared to the group treated with only IRE1α (lane 3 or 4). The data were analyzed through the Tukey multiple comparison test after one-way variance analysis.

12 is a diagram showing that CSTMP further increases the UPS of Arf1-Q71L-induced ΔF508-CFTR. Surface biotinylation analysis was performed on HEK293 transformed with the specified plasmid for 48 hours. Some cells were treated with CSTMP (10 μM) for 24 hours and 48 hours. Representative results are shown in Fig. 12A, and a summary of the results of multiple experiments (n=3) is shown in Fig. 12B. ** P <0.01, compared to untreated control (first lane). # P <0.05, ## P <0.01, compared to the group treated with only Arf1-Q71L (second lane). The data were analyzed through the Tukey multiple comparison test after one-way variance analysis.

13 is a diagram showing that apoptosis is induced when Arf1-Q71L overexpression is prolonged. Transform only ΔF508-CFTR plasmid into HeLa cells (Fig. 13A) or Arf1-Q71L plasmid together for 24 hours (Fig. 13AB), 48 hours (Fig. 13BC) or 72 hours (Fig. 13BD) During co-transformation. Before fixation, the apoptosis marker Annexin V was labeled on living cells. After fixation and permeabilization, cells were stained with anti-CFTR (M3A7) antibody. Transformation with Arf1-Q71L plasmid for 72 hours induced apoptosis. Similar results were found in three independent experiments. Scale bar: 10 μm.

14 is a diagram showing that CSTMP (10 μM) activates the UPS of CFTR without causing apoptosis. HeLa cells were transformed with ΔF508-CFTR and with 10 μM CSTMP for 0 hours (FIG. 14A), 24 hours (FIG. 14AB), 48 hours (FIG. 14BC) or 72 hours (FIG. 14BD) Cultured. Before fixation, apoptosis marker Annexin V was labeled on living cells. After fixation and permeabilization, cells were stained with anti-CFTR (M3A7) antibody. UPS of ΔF508-CFTR was initiated at 24 hours due to CSTMP treatment, but apoptosis was not induced until 72 hours. Arrow heads indicate CFTR expressed on the cell-surface. Similar results were found in three independent experiments. Scale bar: 10 μm.

15 is a diagram showing the LD ₅₀ value of CSTMP orally administered to a mouse. Vehicle (saline solution) or CSTMP in 4 groups consisting of 4 mice (12 weeks old), 2.59 mg/kg (corresponding to 10 μM, assuming that CSTMP is evenly distributed throughout the body), 25.9 mg/kg (corresponding to 100 μM) , Or 259 mg/kg (equivalent to 1,000 μM) was administered orally once a day for 5 days. The LD ₅₀ value was 25.9 mg/kg/day. No mice died on the 2.59 mg/kg/day dosing schedule.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experiment method

Cell culture, plasmids, siRNA and mice.

HEK293, HeLa and PANC-1 cells were treated with 10% Fetal Bovine Serum (FBS) and 1% 100X antibiotic/antifungal (100 units/mL penicillin, 100 units/mL streptomycin and 250 ng/mL amphotericin B) (Gibco # 15240062) supplemented with DMEM (Dulbecco's modified Eagle's medium-high glucose, Gibco #11995-065, Carlsbad, CA) medium. Cells were cultured in a 37° C. 5% CO ² incubator. A mammalian expression plasmid encoding human IRE1α-pcDNA3.EGFP was purchased from Addgene (gene ID: 2081). Plasmids encoding human pCMV-ΔF508-CFTR, pCMV-WT-CFTR (pCMVNot6.2), pCMV-GRASP55-Myc, and extracellular tagged HA-ΔF508-CFTR and pcDNA3-HA-Arf1-Q71L have been described in conventional literature. It is described above (15, 29). ON-TARGETplus human ERN1 siRNA (IRE1α, gene ID: 2081) and human XBP1 siRNA (gene ID: 7494) were purchased from SMARTpool (Dharmacon, Lafayette, CO, USA). Transfection of plasmid DNA and siRNA into HEK293 or HeLa cells was performed using the TransIT-X2 Dynamic Delivery System (Mirus Bio LLC, Madison, WI) according to the manufacturer's instructions. The CftrF508del mouse is a Dr. Obtained from KR Thomas (University of Utah, Salt Lake City, UT) (33) by a previously reported method (15). Mice were bred and managed according to the animal research regulations of Yonsei Medical Center.

Compounds and antibodies.

Tapsigagin (Sigma Aldrich, T9033), STF-083010 (TOCRIS, 4509), APY29 (TOCRIS, 4865), MSC 2032964A (TOCRIS, 5641) and cyclohexide (Sigma Aldrich, C4859) were purchased commercially. CSTMP was synthesized by Cayman chemical (Michigan, USA) (CAS registration number 1000672-89-8). The following antibodies were commercially purchased: anti-CFTR M3A7 (Millipore, Billerica, MA), anti-IRE1α (Cell Signaling Technology, 3294), anti-phosphate S724 IRE1α (Abcam, ab48187), anti-XBP1 (Abcam, ab198999 ), anti-ASK1 (Cell Signaling Technology, 3762), anti-phosphate Thr845 ASK1 (Cell Signaling Technology, 3765), anti-BiP (Cell Signaling Technology, 3177), anti-CHOP (Cell Signaling Technology, 2895), anti- pro/p17-caspase 3, anti-cleaved PARP1 (Abcam, ab136812), anti-HA (Cell Signaling Technology, 2367), anti-Myc (Cell Signaling Technology, 2276), anti-aldolase A (Abcam, ab78339), anti-β-actin (Santa Cruz, sc47778) and anti-fendrin (Santa Cruz, sc23779). Anti-R4 polyclonal antibody was obtained using a peptide corresponding to amino acids 1458-1471 of human CFTR as an antigen as previously reported (15).

Quantitative PCR analysis (qPCR).

Total RNA was extracted from HEK293 cells according to the manufacturer's instructions using Tri-RNA reagent (Favorgen Biotech Corp, Taiwan). In order to reverse transcription of RNA to cDNA, the purified RNA sample was combined with cDNA EcoDry Premix (Takara Bio Inc., Shiga, Japan), and the mixture was incubated at 42°C for 1 hour, followed by incubation at 70°C for 10 minutes.

qPCR was performed using the StepOne system (Applied Biosystems, Foster City, CA, USA). Real-time PCR reaction was measured by detecting the binding of double-stranded DNA and fluorescent SYBR Greendye. For PCR amplification, mix with 100 ng cDNA, 2 μL primer set, 10 μL 2x SYBR premix Ex Taq and 0.4 μL 50× ROX standard dye (Takara, RR420L), and then use RNase-free water to reduce the total reaction volume to 20 μL. Adjusted. Amplification was carried out under the following cycle conditions: 95°C for 15 minutes, followed by 40 cycles at 95°C for 15 seconds, and 60°C for 40 seconds. Analysis was performed 3 times for each cDNA. Relative mRNA expression levels were calculated by normalizing gene expression to the housekeeping gene GAPDH using a comparative threshold cycle (Ct) method, and ΔCt values were calculated as follows: ΔCt = Ct(GAPDH)-Ct (Target gene). The average ΔCt was subtracted from each experimental condition to obtain a ΔΔCt value. The fold change in gene expression was normalized to GAPDH and the relative value for the control sample was calculated as 2 ^-ΔΔCt . The primer sequences used for the qPCR analysis are as follows: hIRE1α, positive "nun* primer 5'-CGG GAG AAC ATC ACT GTC CC-3', reverse"nun* primer 5'-CCC GGT AGT GGT GCT TCT TA- 3'; hXBP1, positive "nun* primer 5'-TTG TCA CCC CTC CAG AAC ATC-3', reverse"nun* primer 5'-TCC AGA ATG CCC AAC AGG AT-3'; hXBP1 (spliced), positive&quot;nu* primer 5'-TGC TGA GTC CGC AGC AGG TG-3', reverse&quot;nu* primer 5'-GCT GGC AGG CTC TGG GGA AG-3'; GAPDH, positive "Nun* primer 5'-AAT CCC ATC ACC ATC TTC CA-3', reverse "Nun* primer 5'-TGG ACT CCA CGA CGT ACT CA-3'.

Surface biotinylation, cross-linking analysis and immunoblot analysis.

For biotinylation, HEK293 cells grown in ⁶ -well plates (1×10 ⁶ ) were incubated at 4° C. for 5 minutes and washed 3 times with refrigerated PBS (phosphate buffered saline). For biotinylation in the mucous membrane of the mouse colon, the colon tissue was cut lengthwise and the connective tissue and muscle were stripped. Cultured cells or transmembrane proteins in the plasma membrane of the colon mucosa were mixed with 1 mL biotin solution (0.3 mg/mL Sulfo-NHS-SS-biotin in refrigerated PBS, Thermo Pierce, 21331) at 4°C for 30 minutes in dark conditions. It was biotinylated while gently stirring and cultivation. Cells or colon tissues were incubated in PBS with a quenching buffer containing 0.5% bovine serum albumin (BSA) at 4° C. for 10 minutes to remove excess biotin, followed by washing three times with PBS. Next, the surface-biotinylated cells were prepared with 25 mM Tris (pH 7.4), 1% (v/v) NP40, 150 mM NaCl, 10% glycerol and 1 mM EDTA-Na ² and a protease inhibitor cocktail (Roche, Germany). ) Was collected in lysis buffer supplemented. The cell lysate was homogenized with ultrasound for 20 seconds (1 s pulse) and then centrifuged at 4° C. for 20 minutes at 16,000 g . The supernatant containing 400 μg of total protein was incubated with 200 μL 10% streptavidin agarose (Thermo Pierce, 20347). Streptavidin-bound, biotinylated protein was centrifuged and washed 5 times with lysis buffer. The biotinylated protein was eluted in a 2×SDS sample buffer supplemented with DTT (0.02 g/mL) and separated by SDS-polyacrylamide gel electrophoresis. The separated protein was transferred to a nitrocellulose membrane and blotted with appropriate primary and secondary antibodies in 5% skim milk. Protein bands were detected by chemiluminescence, and the density of each protein band was quantified using imaging software (Multi Gauge ver. 3.0; Fujifilm, Tokyo, Japan).

Cross-linking analysis was performed by the previously reported method (29). HEK293 cells were washed three times with PBS and incubated at 37°C for 30 minutes in PBS (pH 8.2) with 500 μM ethylene glycol succinimidyl succinate (EGS; Thermo Scientific, 21565). After cross-linking, the cells were sequentially incubated in 20 mM Tris (pH 7.4) for 10 minutes to quench EGS to terminate the reaction. After the cells were lysed and homogenized, immunoblotting was performed using an anti-IRE1α antibody made into a sample in a 2×SDS sample buffer.

Immunocytochemistry, immunohistochemistry, and morphometric analysis.

Immunofluorescence staining was performed by slightly modifying the previously reported methods (15, 18). For immunohistochemistry in the colonic mucosa, mouse tissues were embedded in an optimal incision temperature (OCT) compound (Miles, Elkhart, IN, USA), cooled in liquid nitrogen, and cut into 4-μm sections. Then, the mouse colon sections were fixed and permeabilized by incubating for 5 minutes at -20°C with cold methanol. For immunocytochemical analysis, HeLa cells were cultured on 18-mm round coverslips and fixed with 3.7% formaldehyde for 6 minutes at room temperature. Thereafter, the cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature. Cells grown on tissue sections or coverslips on glass slides were washed 3 times with PBS and cultured at room temperature for 1 hour in PBS containing 1% BSA and 5% serum of the appropriate species (horse/donkey/goat serum) for non-specific binding. The site was blocked. After blocking, tissue sections or cells were stained by incubating with an appropriate primary antibody, and stained with a fluorophore-conjugated secondary antibody. For the surface-specific labeling of CFTR, cells that were not permeabilized after fixation were cultured with a blocking solution and stained with an anti-HA antibody to detect the extracellular HA-epitope of CFTR. The cells on the coverslip were mounted on a slide glass with a fluorescent mounting medium (Dako, S3025, US). Fluorescence images were captured with a laser scanning confocal microscope (LSM 780; Carl Zeiss, Berlin, Germany) with a 63 x 1.4 numerical aperture oil objective.

Morphological analysis of the captured confocal images was performed using MetaMorph microscope analysis software (version 7.1; Molecular Devices, Sunnyvale, USA) according to the previously reported method (18). The concentration profile of each single cell was expressed as the standard deviation of the mean concentration over the entire area.

Cl ^- Measurement of channel activation and short circuit current (Isc).

Total cell recordings were performed on CFTR-transformed HEK293 cells according to a previously reported method (15). The cells were transferred to a bath mounted on a stage of a conduction microscope (Ti2, Nikon) and the membrane was teared after gigaohm (Ω) sealing to obtain a whole-cell patch. The bath solution was perfused at 5 mL/min. Voltage and current recording was performed at room temperature (22-25°C). A patch pipette of 2-4 MΩ resistance was connected to the head stage of a patch clamp amplifier (Axopatch-200B, Molecular Devices, Sunnyvale, CA, USA). The bath solution contained 140 mM N-methyl-D-glucamine chloride (NMDG-Cl), 1 mM CaCl ₂ , 1 mM MgCl ₂ , 10 mM glucose and 10 mM HEPES (pH 7.4). The pipette solution contained 140 mM N-methyl-D-glucamine chloride, 5 mM EGTA, 1 mM MgCl ₂ , 3 mM MgATP and 10 mM HEPES, pH 7.2. In order to investigate the current-voltage (I/V) relationship, a voltage clamp mode and I/V curve were obtained by applying a ramp pulse (0.8 mV/ms, fixed voltage 0 mV) of -100 to 100 mV. CFTR currents were activated by cAMP (5 μM forskolin and 100 μM 3-isobutyl-1-methylxanthine [IBMX]). The current generated by CFTR was confirmed by adding CFTR _inh -172 (10 μM), a CFTR inhibitor. PClamp 10.2 and Digidata 1550B (Molecular Devices) were used to obtain data and apply command pulses. Voltage and current flow were analyzed by storage in pClamp 10.2 and Origin 8.0 (OriginLab Corp., Northampton, MA, USA). The current was filtered at 5 kHz and sampled at 1 kHz. All data were normalized to total-cell capacitance (pF).

The previously reported method (15) was slightly modified to measure the mouse colon I _sc (15). After anesthesia with CO ₂ , a specimen of the colon from the cecum to the rectum of the sacrificed mouse was recovered and opened along the mesenteric boundary. The contents of the tube were washed with HCO ₃ -buffered solution and the specimen was mounted in a Ussing chamber (World Precision Instruments, Stevenage, UK) so that the exposed surface area was 12.6 mm ² . Tissues with 10 mL of HCO ₃ -buffered solution containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 1 mM CaCl ₂ , 10 mM D-glucose, 5 mM HEPES and 25 mM NaHCO ₃ (pH 7.4). Together, each slide was batched at 37° C., 95% O ₂ -5% CO ₂ . Tissues were voltage-fixed using an EVC-4000 voltage clamp (World Precision Instruments) and I _sc was continuously recorded using a PowerLab data acquisition system (AD Instruments, Castle Hill, Australia).

Cl ^- / HCO ₃ ^- Measurement of active exchange.

Measurement of pH _i in PANC-1 cells is conventionally performed using a pH-sensitive fluorescent probe, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxy fluorescein (BCECF). It was performed with the reported method (16). BCECF fluorescence was recorded with a resolution of 2/sec at excitation wavelengths of 490 nm and 440 nm (Delta Ram; PTI Inc., Edison, New Jersey, USA). Cl ^- / HCO ₃ ^- exchange activity of HCO ₃ - was estimated from the increase rate of the initial increase in pH _i generated by removing the ^- Cl in buffer containing _{(25 mM HCO 3 -with 5%} CO 2). The pH _i measurement was performed with a standard pH solution containing 150 mM KCl and 5 μM nigericin. Intrinsic buffer volume (βi) was calculated by measuring ΔpH _i corresponding to 5-40 mM NH ₄ Cl pulses in Na ⁺ free solution. β _i value does not operate significantly affected by transfection of a plasmid encoding the WT- gave gave pen or pen H723R-, Cl ^- / HCO ₃ ^- exchange activity without compensating for the buffer capacity ΔpH unit / min Expressed as.

Statistical analysis

Results for multiple experiments were expressed as mean ± standard error. Statistical analysis was performed using GraphPad Prism5 (GraphPad Software, Inc., La Jolla, CA) using a two-sided Student's t -test or a one-way variance analysis and then a Tukey multiple comparison test. If P <0.05, it was considered statistically significant.

Research approval.

All animal experiments were performed under the approval of ILAR (Institutional Laboratory Animal Resources) and Yonsei Life Research Institute Animal Testing Committee (approval number 2018-0139).

Experiment result

Endoribonuclease activity of IRE1α is not essential for the UPS of CFTR

Mammalian IRE1α is maintained as an inactive monomer by binding to the ER chaperone protein BiP (Fig. 9A). Oligomerization of the IRE1 monomer occurs as a response to the accumulation of ER of unfolded proteins in the first step of IRE1 activation (25). When the transport from ER to Golgi is inhibited by the predominant inhibitory form of Arf1 GTPase, Arf1-Q71L induces ER stress as secreted proteins accumulate in the ER lumen (15). ER stress can also be induced by treatment with tapsigargin, a Ca ²⁺ -ATPase inhibitor that depletes calcium in the ER lumen (26). Like this mechanism that induces ER stress, IRE1α in the cross-linking analysis in HEK293 aggregates after Arf1-Q71L expression (48h) or tapsigagin treatment (12h) to become an oligomer (Fig. 9a).

Oligomerization of IRE1α opens the kinase domain and begins to be activated and activates the RNase domain (25). Both Arf1-Q71L and tapcigagin trigger IRE1α kinase activity in HEK293 cells, inducing IRE1α autophosphorylation and downstream ASK1 phosphorylation (FIGS. 1A and 1B ). Interestingly, treatment with tapcigagin, which rapidly induces ER stress (12h), induced XBP1 splicing, whereas ER stress induced by Arf1-Q71L overexpression (48 h) did not (FIGS. 1A and 1B ). Since Arf1-Q71L and tapcigazin both induce the UPS of the membrane protein (15, 16), this suggests that the IRE1α kinase-mediated signal rather than the IRE1α RNase substrate XBP1 is primarily involved in ER stress-related UPS. Surface biotinylation experiments showing depletion of IRE1α rather than XBP1 further support the assumption that ER stress-related UPS is independent of XBP1 (Figs. 1c and 1d). Knockdown of each gene used was confirmed through immunoblot analysis and mRNA quantification for IRE1α and XBP1 (FIGS. 1c, 9b and 9c).

Due to the unexpected discovery that the UPS of ΔF508-CFTR is independent of XBP1 splicing, the present inventors investigated whether IRE1α can mediate UPS in the absence of RNase activity. STF-083010 is a compound targeting the catalytic core of the IRE1α RNase domain, and inhibits IRE1α endonuclease activity without affecting the kinase activity or the overall oligomerization step (27). When STF-083010 (60 μM) was treated for 12 hours, tapcigajin-induced XBP1 production was stopped (Fig. 9D). However, STF-083010 did not affect the Arf1-Q71L-induced UPS of ΔF508-CFTR (FIGS. 1E and 1F ). These results show that the RNase activity of IRE1α is not essential for the UPS of ΔF508-CFTR.

The IRE1α kinase-ASK1 pathway is required for UPS of ΔF508-CFTR.

We focused on the IRE1α kinase-mediated signal in the UPS of membrane proteins. APY29, a type I kinase inhibitor, inhibits autophosphorylation of IRE1α by competitively occupying the ATP-binding pocket of IRE1α (28). In HEK293 cells, the IC ₅₀ value of APY29 for ASK1 phosphorylation was calculated to be about 6.4 μM, and treatment with 100 μM APY29 for 12 hours almost completely inhibited Arf1-Q71L-induced phosphorylation for IRE1α and ASK1 (FIGS. 10A and 10AB. ). Importantly, ΔF508-CFTR surface targeting induced by ectopic expression of Arf1-Q71L by APY29 (100 μM) was abolished (FIGS. 2A and 2B ). Since the stability of ΔF508-CFTR is lower than that of wild-type (WT)-CFTR (FIG. 10c-f), a protein synthesis defect may also have caused a rapid decrease in the amount of ΔF508-CFTR on the cell surface. In fact, treatment with the protein synthesis inhibitor cycloheximide (0.1 mg/mL) drastically reduced the level of ΔF508-CFTR on the cell surface (FIGS. 10c-f). However, while cycloheximide resulted in a rapid decrease in cell-surface and total cell ΔF508-CFTR levels, APY29 only decreased cell surface ΔF508-CFTR without affecting total protein levels (FIGS. 2A and 2B ). From these data, it can be seen that APY29 selectively inhibits the recovery of ΔF508-CFTR on the cell surface and does not affect protein synthesis.

The inhibitory effect of APY29 on the cell surface expression of CFTR was additionally confirmed through immunofluorescence analysis. Morphological analysis was performed using HeLa cells, which are better adsorbed to coverslips and less susceptible to cell loss, instead of HEK293 cells (18, 29). In control cells, the ΔF508-CFTR protein remained only within the ER. When ER-Golgi migration was blocked with Arf1-Q71L, a significant amount of ΔF508-CFTR reached the cell surface as previously reported (arrow, Fig. 2c) (15). Notably, APY29 treatment significantly reduced the cell surface expression of Arf1-Q71L-induced ΔF508-CFTR (FIGS. 2C and 2D ).

Since IRE1α forms the IRE1-TRAF2-ASK1 complex in the ER outer membrane, thereby activating ASK1 and connecting the ER stress signal to the ASK1-JNK signaling pathway (24, 30), the present inventors investigated the role of ASK1 in the secretion of atypical CFTR. . MSC2032964A is a potent and selective ASK1 inhibitor that blocks LPS-induced ASK1 phosphorylation (31). The results in Figure 3A-3C, ASK1 phosphorylation inhibition through MSC2032964A (10 μM) treatment time-dependently reduced the UPS of both Arf1-Q71L-induced WT- and ΔF508-CFTR. Furthermore, inhibition of ASK1 using a specific siRNA results in annihilation of the UPSrk of Arf1-Q71L-induced WT- and ΔF508-CFTR (FIGS. 3D-3G ). Taken together, it can be seen that the kinase activity of IRE1α and ASK1 play an important role in the UPS of core-glycosylated CFTR.

Activation of IRE1α kinase induces UPS of ΔF508-CFTR.

Next, the effect of increasing IRE1 kinase activity on the protein migration defect caused by the F508-CFTR mutation was investigated. Obviously, only overexpression of IRE1α occurred on the surface of ΔF508-CFTR (FIG. 11), which supported the hypothesis that an increase in IRE1α could recover the migration defect of ΔF508-CFTR. Furthermore, IRE1α overexpression showed a synergistic effect on the UPS induction of ΔF508-CFTR when used with Arf1-Q71L or tapcigajin (FIG. 11).

Small molecule therapy is preferred over gene overexpression methods in treating human patients with diseases associated with protein folding and migration defects. The tetramethylpyrazine derivative (E)-2-(2-chlorostyryl)-3,5,6-trimethyl-pyrazine (CSTMP) has been reported to activate the IRE1α-TRAF2-ASK1 complex (32). However, since CSTMP has been reported to induce death of human non-small cell lung cancer A549 cells through JNK activation and mitochondrial dysfunction, it may induce cytotoxicity at high concentrations (>50 μM) (32). As a result of treating HEK293 cells with various concentrations of CSTMP for 48 hours, it was confirmed that even a low concentration of CSTMP (10 μM) can sufficiently activate ASK1 without triggering apoptosis signal, whereas a high concentration (100 μM) of CSTMP The death marker proteins PARP and caspase 3 were cleaved (FIGS. 4A and 4B ). Therefore, the present inventors investigated whether cells were treated with 10 μM CSTMP and the migration defect of ΔF508-CFTR was recovered. As a result, 10 μM CSTMP was sufficient to induce cell surface expression of ΔF508-CFTR (Figs. 4c and 4d), synergistic effect when combined with overexpression of GRASP55 (Figs. 4c and 4d) or Arf1-Q71L (Fig. 12). Showed.

Next, the cytotoxicity of 10 μM CSTMP and Arf1-Q71L overexpression was compared. 5A and 5B, when transformed with the Arf1-Q71L-expressing plasmid, the cell surface expression of ΔF508-CFTR was recovered and reached its peak at 48 hours. Notably, when transforming with the Arf1-Q71L-expressing plasmid for 72 hours, it induces caspase 3 cleavage while prolonging ER-Golgi blockade (Figs. 5A and 5B). On the other hand, CSTMP (10 μM) treatment according to time (0, 6, 12, 24, 48, and 72 hours) continuously recovered the surface expression of ΔF508-CFTR without triggering apoptosis signal (FIGS. 5C and 5D ). As a result of immunostaining with Annexin V, an apoptosis marker, it was further confirmed that prolonged ectopic Arf1-Q71L expression (72 hours) induced cytotoxicity, but not CSTMP (10 μM) (FIGS. 13 and 14 ).

In HEK293 cells, the function of ΔF508-CFTR is restored by the IRE1α kinase activator CSTMP.

To investigate whether ΔF508-CFTR, in which cell surface expression was induced by CSTMP, maintains functional activity, HEK293 cells expressing WT- or ΔF508-CFTR were incubated with CSTMP (10 μM) for 48 hours and CFTR-mediated Cl ^{-Measure the} current. Even treatment with forskolin (forskolin) and 3-isobutyl-1-methylxanthine (IBMX), which increases intracellular cAMP, does not induce Cl ^- current in control HEK293 cells treated with only the transformation reagent (Figs. 6A and 6B. ). After applying a ramp pulse (-100 to +100 mV, 0.8 mV/ms), cells expressing WT-CFTR exhibited a typical CFTR Cl ^- current, which was (1) activated by cAMP treatment (Forskolin And IBMX), (2) CFTRinh-172, which is a CFTR inhibitor, (3) has a linear IV relationship (Fig. 6c). Treatment with CSTMP (10 μM) for 48 hours did not significantly change the WT-CFTR current (FIG. 6D ). Next, the effect of CSTMP on ΔF508-CFTR-expressing cells was investigated. As shown in Figure 6e, cells expressing ΔF508-CFTR did not clearly show a CFTR current activated by cAMP under normal conditions. After treatment with CSTMP (10 μM) for 48 hours, a typical CFTR current was measured in cells expressing ΔF508-CFTR. As a result, the current magnitude of ΔF508-CFTR cells was 32% of the value measured in wild-type cells (FIG. 6A and 6f).

CSTMP biochemically and functionally restores p.H723R-fendrin.

Since IRE1α is involved in the UPS of ER stress-induced pendrin (16), the present inventors investigated the effect of CSTMP, an IRE1α kinase activator, on the recovery of cell surface migration defects induced by p.H723R-pendrin mutations. I did. To investigate the cell surface expression of pendrin, PANC-1 derived from the pancreatic duct was used (16). First, the surface expression of p.H723R-fendrin was investigated through surface biotinylation analysis. 7A and 7B, p.H723R-fendrin is hardly detected in the plasma membrane of the control cells. In contrast, when CSTMP (30 μM) was treated, p.H723R-fendrin was strongly expressed on the cell surface. Furthermore, CSTMP processing p.H723R- Pen Pen gave gave expression in cell-mediated Cl ^- was induced activation / HCO ³ exchange, was approximately 35% of the transport function of the cells expressing WT- Pen gave a recovery, which Arf1- It is at a level similar to the effect of Q71L overexpression (Figs. 7C and 7D). These results show that IRE1α kinase activation exerts a therapeutic effect on diseases caused by not only the migration defect mutation of CFTR, but also other membrane proteins including p.H723R-fendrin.

In vivo treatment of CSTMP ^restores cell surface expression and CFTR-mediated anion transport of CFTR in CftrF ^508del mouse colon.

Finally, it was investigated whether in vivo administration of CSTMP restores epithelial transport defects caused by the ΔF508-CFTR mutation in mice. The malfunctioning ΔF508-CFTR mutation causes a defect that primarily affects the transport of ions and fluids in the intestine, resulting in intestinal obstruction in mice (33). To analyze the in vivo effect of CSTMP, toxicity was evaluated by measuring half the lethal dose (LD ₅₀ ) of CSTMP in mice. Mice were orally administered 3 different doses of CSTMP corresponding to 10-1,000 μM for 5 days (assuming that CSTMP is evenly distributed throughout the body). The LD ₅₀ value of the mouse was calculated as 25.9 mg/kg/day (corresponding to 100 μM) (FIG. 15). Thereafter, ^1/10 of CSTMP LD ₅₀ (2.59 mg/kg/day, equivalent to 10 μM once a day) was applied to 6-week-old mice homozygous for WT-CFTR (Cftr ^WT ) or ΔF508-CFTR (Cftr ^F508del ). Treated for 5 days. During the LD ₅₀ evaluation experiment, no mice died at this dose (Fig. 15).

Notably, as a result of surface biotinylation experiments using samples taken from the mouse colon mucosa, it was confirmed that CSTMP restores the cell surface expression of core-glycosylated ΔF508-CFTR (FIGS. 8A and 8B ). Through immunohistochemical analysis, the CSTMP recovery effect in Cftr ^F508del mice was confirmed again (Fig. 8c). In Cftr ^WT mice, CFTR was expressed in the apical membrane of colon crypt cells, and the distribution of WT-CFTR was not affected by CSTMP treatment. As previously reported (15, 34), apical expression of ΔF508-CFTR was not observed in the colon ^gland of Cftr ^F508del mice. In particular, oral administration of CSTMP in Cftr ^F508del mice restored ΔF508-CFTR expression at the tip of colon crypt cells (Fig. 8c). Furthermore, CSTMP treatment resulted in a functional recovery of CFTR-mediated anion transport in Cftr ^F508del mouse colon. Increased intracellular cAMP levels due to forskolin treatment induced typical CFTR-dependent short-circuit current (Isc) in the colon of Cftr ^WT mice, which were (1) lumen-negative and (2) Na ⁺ / K ⁺ /2Cl ^- is inhibited by blocking the basal side Cl ^- absorption using bumetanide, a cotransporter inhibitor, (3) Cftr ^F508del does not exist in the mouse colon (Figs. 8D and 8E). Obviously, CSTMP treatment causes forskolin-activated, lumen-negative and bumetanide-inhibitable Isc with current magnitudes reaching 89% of the levels measured in Cftr ^WT mice (FIGS. 8D and 8E ). Taken together, these results provide in vivo evidence that CSTMP may be an effective therapeutic agent for migration-defective CFTR mutations.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

references

1. Thomas PJ et al. Trends Biochem Sci. 1995;20(11):456-9.

2. Park HJ et al. J Med Genet. 2003;40(4):242-8.

3.Bullock AN, and Fersht AR. Nat Rev Cancer. 2001;1(1):68-76.

4. Hammond C, and Helenius A. Curr Opin Cell Biol. 1995;7(4):523-9.

5.Meusser B et al. Nat Cell Biol. 2005;7(8):766-72.

6. Lee MG et al. Physiol Rev. 2012;92(1):39-74.

7. Chang XB et al. J Cell Sci. 2008;121(Pt 17):2814-23.

8.Amaral MD. J Mol Neurosci. 2004;23(1-2):41-8.

9. Meng X et al. Cell Mol Life Sci. 2017;74(1):23-38.

10. Ward CL et al. Cell. 1995;83(1):121-7.

11. Cheng SH et al. Cell. 1990;63(4):827-34.

12. Alper SL, and Sharma AK. Mol Aspects Med. 2013;34(2-3):494-515.

13.Yoon JS et al. J Med Genet. 2008;45(7):411-9.

14.Gee HY et al. Semin Cell Dev Biol. 2018;83(59-66).

15. Ge HY et al. Cell. 2011;146(5):746-60.

16. Jung J et al. Nat Commun. 2016;7(11386).

17. Noh SH et al. Autophagy. 2018;14(10):1761-78.

18. Piao H et al. Sci Rep. 2017;7(39887).

19. Giuliani F et al. Curr Opin Cell Biol. 2011;23(4):498-504.

20. Hetz C, and Papa FR. Mol Cell. 2018;69(2):169-81.

21.Gee HeonY et al. Cell. 2011;146(5):746-60.

22. Calfon M et al. Nature. 2002;415(6867):92-6.

23. Holleyn J, and Weissman JS. Science. 2006;313(5783):104-7.

24. Nishitoh H et al. Genes Dev. 2002;16(11):1345-55.

25. Korennykh AV et al. Nature. 2009;457(7230):687-93.

26.Pahl HL. Physiol Rev. 1999;79(3):683-701.

27. Hetz C et al. T Nat Rev Drug Discov. 2013;12(9):703-19.

28. Wang L et al. Nat Chem Biol. 2012;8(12):982-9.

29. Kim J et al. Traffic. 2016;17(7):733-53.

30. Urano F et al. Science. 2000;287(5453): 664-6.

31. Guo X et al. EMBO Mol Med. 2010;2(12):504-15.

32. Zhang J et al. Biomed Pharmacother. 2016;82(281-9).

33. Zeiher BG et al. A mouse model for the delta F508 allele of cystic fibrosis. J Clin Invest. 1995;96(4):2051-64.

34. Namkung W et al. Gastroenterology. 2005;129(6):1979-90.

35. Rabouille C. Trends Cell Biol. 2017;27(3):230-40.

36. Kim J et al. Unconventional protein secretion -new insights into the pathogenesis and therapeutic targets of human diseases. 2018;131(12).

37. Xu Y et al. Cell Discov. 2018;4(11).

38. Shamu CE, and Walter P. Embo j. 1996;15(12):3028-39.

39. Karagoz GE et al. IRE1. 2017;6

40. Hetz C, and Glimcher LH. Mol Cell. 2009;35(5):551-61.

41. Pattingre S et al. J Biol Chem. 2009;284(5):2719-28.

42. Barrett KE, and Keely SJ. Annu Rev Physiol. 2000;62(535-72).

Claims (12)

1. A composition for preventing or treating a protein conformational disorder comprising a compound represented by the following general formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

   General Formula 1

   

   In the above general formula, R ₁ to R ₃ are each independently C ₁ -C ₃ alkyl, and X is halogen.
2. The composition of claim 1, wherein R ₁ to R ₃ in the general formula 1 are C ₁ alkyl.
3. The composition of claim 1, wherein X in Formula 1 is Cl.
4. The composition of claim 1, wherein the protein morphology disorder is a disease caused by unfolding or misfolding of proteins due to amino acid mutations.
5. The composition of claim 4, wherein the protein is selected from the group consisting of cystic fibrosis membrane conduction regulator (CFTR), pendrin, and combinations thereof.
6. The composition of claim 4, wherein the protein morphological disorder is cystic fibrosis or congenital hearing impairment.
7. A method for screening a composition for preventing or treating protein conformational disorders comprising the following steps:

   (1) contacting a test substance with a biological sample containing IRE1α kinase; And

   (2) measuring the activity or expression of IRE1α kinase in the sample,

   When the activity or expression of the IRE1α kinase is increased, the candidate substance is determined as a composition for preventing or treating abnormal protein morphology.
8. The method of claim 7, wherein the protein morphology disorder is a disease caused by unfolding or misfolding of the protein due to amino acid mutation.
9. 9. The composition of claim 8, wherein the protein is selected from the group consisting of cystic fibrosis membrane conduction regulator (CFTR), pendrin, and combinations thereof.
10. 9. The composition of claim 8, wherein the protein morphological disorder is cystic fibrosis or congenital hearing impairment.
11. A composition for inhibiting atypical protein secretion (UPS) comprising an inhibitor of IRE1α kinase as an active ingredient.
12. 12. The composition of claim 11, wherein the inhibitor of IRE1α kinase is APY29.

Images