WO2020242225A1 - COMPOSITION POUR PRÉVENIR OU TRAITER DES TROUBLES CONFORMATIONNELS PROTÉIQUES, COMPRENANT UN ACTIVATEUR DE LA KINASE IRE1α COMME PRINCIPE ACTIF - Google Patents

COMPOSITION POUR PRÉVENIR OU TRAITER DES TROUBLES CONFORMATIONNELS PROTÉIQUES, COMPRENANT UN ACTIVATEUR DE LA KINASE IRE1α COMME PRINCIPE ACTIF Download PDF

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WO2020242225A1
WO2020242225A1 PCT/KR2020/006933 KR2020006933W WO2020242225A1 WO 2020242225 A1 WO2020242225 A1 WO 2020242225A1 KR 2020006933 W KR2020006933 W KR 2020006933W WO 2020242225 A1 WO2020242225 A1 WO 2020242225A1
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protein
cftr
composition
ire1α
ups
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Korean (ko)
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이민구
박학
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연세대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

Definitions

  • the present invention relates to a composition for the prevention or treatment of abnormal protein form diseases comprising an IRE1 ⁇ 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).
  • 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.
  • ER-associated degradation 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).
  • ESD ER-associated degradation
  • 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).
  • band C 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).
  • the mutation in which the 508th phenylalanine residue is deleted ( ⁇ F508) is the most common disease-causing CFTR mutation (9), causing protein misfolding, ER retention, and degradation by ERAD (10).
  • the ⁇ F508-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).
  • DFNB4 vestibular aqueduct
  • PDS Pendrid syndrome
  • the most common disease-causing mutation of pendrin, p.H723R (His723Arg) induces protein misfolding, ER retention, and degradation by ERAD, like the ⁇ F508-CFTR protein (13).
  • 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.
  • conventional methods of activating the UPS of CFTR and pendrin e.g., overexpression of GRASP55 in ⁇ F508-CFTR and overexpression of MVB12B in p.H723R-fendrin
  • CF, DFNB4 CF, DFNB4
  • It is clinically unsuitable to treat human patients with Pendrid syndrome.
  • 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).
  • UPR unfolded protein response
  • IRE1 ⁇ inositol-desiring enzymes 1 ⁇
  • IRE1 ⁇ RNA-like ER kinase
  • ATF6 ⁇ activating transcription factors 6 ⁇
  • IRE1 ⁇ 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 IRE1 ⁇ does not induce CFTR and Fendrin UPS following ER-Golgi pathway blockade (16, 21). Nevertheless, the clear mechanism of UPS regulation by IRE1 ⁇ 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 IRE1 ⁇ 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.
  • XBP1 transcription factor XBP1
  • UPR-related genes involved in ERAD (22).
  • IRE1-dependent decay (RIDD) process is also recognized as a complementary mechanism to reduce ER loading by degrading the mRNA of various proteins (23).
  • 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 IRE1 ⁇ 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 IRE1 ⁇ kinase-mediated signaling cascade, not by the XBP1- and RIDD-dependent pathways requiring IRE1 ⁇ RNase activity.
  • the IRE1 ⁇ kinase pathway can provide a novel target for the development of therapeutic agents for diseases caused by protein folding and migration defects.
  • 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.
  • the compound of Formula 1 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.
  • UPS Unconventional Protein Secretion
  • 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).
  • UPS atypical protein secretion
  • 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:
  • 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.
  • UPS Unconventional Protein Secretion
  • 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.
  • 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.
  • PCD protein conformational disease
  • 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.
  • ER endoplasmic reticulum
  • ESD vesicle-related degradation mechanisms
  • 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.
  • folding defect is meant to include “misfolding” and “unfolding”.
  • 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.
  • 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.
  • the term “treatment” or “therapeutic agent” in the present specification includes the meaning of “treatment aid” or “treatment aid”.
  • 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.
  • 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”.
  • 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.
  • alkyl refers to a linear or branched saturated hydrocarbon group
  • 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.
  • R 1 to R 3 in the general formula 1 of the present invention are C 1 alkyl.
  • X in the general formula 1 of the present invention is Cl.
  • the concentration of the compound of Formula 1 of the present invention is 5 ⁇ M-50 ⁇ M, more specifically 5 ⁇ M-30 ⁇ M, and most specifically 10 ⁇ M-20.
  • the protein is selected from the group consisting of a cystic fibrosis membrane conduction regulator (CFTR), pendrin, and combinations thereof.
  • CFTR cystic fibrosis membrane conduction regulator
  • pendrin pendrin
  • 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).
  • DFNB4 enlarged vestibular aqueduct
  • PDS Pendrid syndrome
  • 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.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, a talct, a talct, a talct, a stea, stevia, glycerin, glycerin, glycerin, g
  • 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.
  • 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.
  • the present invention provides a method for screening a composition for preventing or treating a protein conformational disorder comprising the following steps:
  • the candidate substance is determined as a composition for preventing or treating abnormal protein morphology.
  • biological sample refers to any sample including cells expressing IRE1 ⁇ obtained from mammals including humans, and includes, but is not limited to, tissues, organs, cells, or cell culture.
  • test substance used while referring to the screening method of the present invention is added to a sample containing IRE1 ⁇ -expressing cells, and is used in screening to test whether it affects the activity or expression level of IRE1 ⁇ kinase.
  • 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 IRE1 ⁇ 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 IRE1 ⁇ kinase is increased, the test substance may be determined as a composition for preventing or treating diseases of abnormal protein form.
  • the term “increased expression” means that the expression level of IRE1 ⁇ 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.
  • 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 IRE1 ⁇ 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.
  • the present invention provides a composition for inhibiting atypical protein secretion (UPS) comprising an inhibitor of IRE1 ⁇ kinase as an active ingredient.
  • UPS atypical protein secretion
  • the term “inhibitor” refers to a substance that causes a decrease in the activity or expression of IRE1 ⁇ kinase, whereby the activity or expression of the IRE1 ⁇ kinase becomes undetectable or exists at an insignificant level, as well as in the IRE1 ⁇ kinase. It refers to a substance that decreases the activity or expression of IRE1 ⁇ kinase to the extent that the UPS pathway induced by it can be significantly reduced.
  • Inhibitors of IRE1 ⁇ 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 IRE1 ⁇ kinase, but are not limited thereto.
  • UPS typically protein secretion
  • UPS inhibition significantly inhibits the UPS pathway induced by IRE1 ⁇ , 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”.
  • the inhibitor of IRE1 ⁇ kinase of the present invention is APY29.
  • 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:
  • the present invention provides a method of inhibiting atypical protein secretion (UPS) in a subject comprising administering an inhibitor of IRE1 ⁇ kinase.
  • UPS atypical protein secretion
  • the present invention provides a composition for preventing or treating a protein conformational disorder, and a screening method thereof.
  • the present invention also provides a composition for inhibiting the atypical protein secretion (UPS) pathway.
  • UPS atypical protein secretion
  • 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.
  • FIG. 1 is a diagram showing that XBP1 splicing and IRE1 ⁇ endonuclease activity are independent of the UPS of ⁇ F508-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 ⁇ M of tapsigargin for 12 hours to induce ER stress. Representative immunoblot results of IRE1 ⁇ , phosphoric acid-IRE1 ⁇ , phosphoric acid-ASK1, and spliced XBP1 are shown in FIG.
  • the cell surface-specific labeling of the protein was confirmed by the absence of the cytoplasmic protein aldolase A in the biotinylated fragment.
  • FIG. 2 is a diagram showing that IRE1 ⁇ kinase activity is required for UPS induction of ⁇ F508-CFTR.
  • APY29 100 ⁇ M or protein synthesis inhibitor cycloheximide (0.1 mg/mL) was treated, and surface biotinylation assay was performed.
  • APY29 selectively reduces the surface ⁇ F508-CFTR without affecting the overall protein level.
  • HeLa cells were transformed with ⁇ F508-CFTR expression plasmids in the presence and absence of Arf1-Q71L, respectively, and APY29 (100 ⁇ M) was added to the medium for a period of time.
  • CFTR was immunostained with anti-M3A7 CFTR antibody (green), and IRE1 ⁇ was labeled with anti-IRE1 ⁇ antibody (red).
  • 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.
  • FIG. 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, ⁇ F508-CFTR and/or Arf1-Q71L.
  • Several cells were treated with MSC2032964A (10 ⁇ M) for a period of time. Representative surface biotinylation results for WT-CFTR and ⁇ F508-CFTR are shown in Figs. 3A and 3B, respectively.
  • FIG. 4 is a diagram showing that activation of IRE1 ⁇ kinase by CSTMP induces UPS of ⁇ F508-CFTR.
  • Surface biotinylation assays were performed in HEK293 cells expressing ⁇ F508-CFTR.
  • 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 ⁇ m. ** P ⁇ 0.01, compared to untreated group (first lane). Data were analyzed for one-way variance and then Tukey multiple comparison test was performed.
  • FIG. 5 is a diagram showing that CSTMP (10 ⁇ M) treatment does not induce cytotoxicity.
  • 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 ⁇ M) on UPS, activation of ASK1 and caspase 3 cleavage of ⁇ F508-CFTR over time.
  • CSTMP (10 ⁇ M) did not cause cleavage of caspase 3 until 72 hours.
  • Camptothecin (2 ⁇ M, 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 ⁇ F508-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 ⁇ M + IBMX 100 ⁇ M) and inhibited by CFTR inh -172 (5 ⁇ M).
  • 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 ⁇ F508-CFTR plasmid.
  • Several cells were treated with CSTMP (10 ⁇ M) for 24 hours, thereby inducing a clear CFTR current in cells expressing ⁇ F508-CFTR. Histogram data are expressed as mean ⁇ standard error. ** P ⁇ 0.01. Data were analyzed by unpaired Student's t -test.
  • FIG. 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 ⁇ M or 30 ⁇ M) for 48 hours.
  • CSTMP (10 ⁇ M or 30 ⁇ M) 10 ⁇ M or 30 ⁇ M
  • FIG. 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 ⁇ F508-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.
  • Figure 8c shows the immunohistochemistry of CFTR.
  • mice colon tissue 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 ⁇ m. 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 ⁇ M) on the tip of the mouse colon.
  • amiloride 100 ⁇ M
  • FIG. 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 ⁇ M) for 12 hours to induce ER stress, and incubated with cross-linking reagent EGS (500 ⁇ M) for 30 minutes ( Fig. 9a).
  • Arrow heads represent cross-linked IRE1 ⁇ polymer (top band) and monomer (bottom band) forms, respectively.
  • 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.
  • tapcigagin not Arf1-Q71L, increased the level of XBP1 mRNA silenced by siRNA against XBP1 .
  • 9C is a diagram showing the effect of IRE1 ⁇ - and XBP1-specific siRNAs on XBP1 splicing.
  • HEK293 cells were treated with tapcigagin (5 ⁇ M) for 12 hours or transformed with a designated siRNA (48 hours).
  • Tapsigagin increased the protein levels of spliced XBP1 (XBP1s) reduced by siRNA against IRE1 ⁇ or XBP1.
  • 9D is a diagram showing that STF-083010 reduces the RNase activity of IRE1 ⁇ .
  • HEK293 cells were treated with tapcigagin (5 ⁇ M, 12 hours) and/or STF-083010 (60 ⁇ M, 12 h), and then immunoblot analysis for XBP1 protein was performed.
  • FIG. 10 is a diagram showing the control experiment results of FIG. 2.
  • 10A and 10B Figure shows that APY29 dose-dependently inhibits phosphorylation of IRE1 ⁇ and ASK1. Phosphorylation of IRE1 ⁇ and ASK1 was induced by transforming with Arf1-Q71L plasmid for 48 hours.
  • the IC 50 value of APY29 for ASK1 phosphorylation is 6.4 ⁇ M.
  • 10C to 10F show the results of analyzing the protein stability of WT- and ⁇ F508-CFTR in HEK293 cells treated with cycloheximide, a protein synthesis inhibitor.
  • the protein stability of ⁇ F508-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.
  • FIG. 11 is a diagram showing that overexpression of IRE1 ⁇ activates the UPS of ⁇ F508-CFTR. Only IRE1 ⁇ overexpression activates the UPS of ⁇ F508-CFTR and reinforces the effect of tarpsigagin.
  • FIG. 12 is a diagram showing that CSTMP further increases the UPS of Arf1-Q71L-induced ⁇ F508-CFTR.
  • FIG. 13 is a diagram showing that apoptosis is induced when Arf1-Q71L overexpression is prolonged.
  • 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 ⁇ m.
  • FIG. 14 is a diagram showing that CSTMP (10 ⁇ M) activates the UPS of CFTR without causing apoptosis.
  • HeLa cells were transformed with ⁇ F508-CFTR and with 10 ⁇ M 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 ⁇ F508-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 ⁇ m.
  • FIG. 15 is a diagram showing the LD 50 value of CSTMP orally administered to a mouse.
  • Vehicle saline solution
  • CSTMP in 4 groups consisting of 4 mice (12 weeks old), 2.59 mg/kg (corresponding to 10 ⁇ M, assuming that CSTMP is evenly distributed throughout the body), 25.9 mg/kg (corresponding to 100 ⁇ M) , Or 259 mg/kg (equivalent to 1,000 ⁇ M) 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.
  • 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 IRE1 ⁇ -pcDNA3.EGFP was purchased from Addgene (gene ID: 2081).
  • Plasmids encoding human pCMV- ⁇ F508-CFTR, pCMV-WT-CFTR (pCMVNot6.2), pCMV-GRASP55-Myc, and extracellular tagged HA- ⁇ F508-CFTR and pcDNA3-HA-Arf1-Q71L have been described in conventional literature. It is described above (15, 29).
  • ON-TARGETplus human ERN1 siRNA (IRE1 ⁇ , gene ID: 2081) and human XBP1 siRNA (gene ID: 7494) were purchased from SMARTpool (Dharmacon, Lafayette, CO, USA).
  • Tapsigagin Sigma Aldrich, T9033
  • STF-083010 (TOCRIS, 4509)
  • APY29 (TOCRIS, 4865)
  • MSC 2032964A (TOCRIS, 5641)
  • cyclohexide (Sigma Aldrich, C4859) were purchased commercially.
  • CSTMP was synthesized by Cayman chemical (Michigan, USA) (CAS registration number 1000672-89-8).
  • anti-CFTR M3A7 (Millipore, Billerica, MA), anti-IRE1 ⁇ (Cell Signaling Technology, 3294), anti-phosphate S724 IRE1 ⁇ (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- ⁇ -actin (Santa Cruz, sc47778) and anti-fendrin (Santa Cruz, sc23779).
  • 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.
  • PCR amplification mix with 100 ng cDNA, 2 ⁇ L primer set, 10 ⁇ L 2x SYBR premix Ex Taq and 0.4 ⁇ L 50 ⁇ ROX standard dye (Takara, RR420L), and then use RNase-free water to reduce the total reaction volume to 20 ⁇ L. 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.
  • Ct comparative threshold cycle
  • the primer sequences used for the qPCR analysis are as follows: hIRE1 ⁇ , 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"nu* primer 5'-TGC TGA GTC CGC AGC AGG TG-3', reverse"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'.
  • PBS phosphate buffered saline
  • 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.
  • BSA bovine serum albumin
  • 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 ⁇ g of total protein was incubated with 200 ⁇ L 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).
  • HEK293 cells were washed three times with PBS and incubated at 37°C for 30 minutes in PBS (pH 8.2) with 500 ⁇ M 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-IRE1 ⁇ antibody made into a sample in a 2 ⁇ SDS sample buffer.
  • Immunofluorescence staining was performed by slightly modifying the previously reported methods (15, 18).
  • mouse tissues were embedded in an optimal incision temperature (OCT) compound (Miles, Elkhart, IN, USA), cooled in liquid nitrogen, and cut into 4- ⁇ m sections. Then, the mouse colon sections were fixed and permeabilized by incubating for 5 minutes at -20°C with cold methanol.
  • OCT optimal incision temperature
  • 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.
  • 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.
  • LSM 780 laser scanning confocal microscope
  • 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 ( ⁇ ) 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 M ⁇ 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.
  • I/V current-voltage
  • CFTR currents were activated by cAMP (5 ⁇ M forskolin and 100 ⁇ M 3-isobutyl-1-methylxanthine [IBMX]). The current generated by CFTR was confirmed by adding CFTR inh -172 (10 ⁇ M), a CFTR inhibitor.
  • PClamp 10.2 and Digidata 1550B 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).
  • 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).
  • the pH i measurement was performed with a standard pH solution containing 150 mM KCl and 5 ⁇ M nigericin. Intrinsic buffer volume ( ⁇ i) was calculated by measuring ⁇ pH i corresponding to 5-40 mM NH 4 Cl pulses in Na + free solution. ⁇ 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 ⁇ pH unit / min Expressed as.
  • 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.
  • Endoribonuclease activity of IRE1 ⁇ is not essential for the UPS of CFTR
  • Mammalian IRE1 ⁇ 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).
  • 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).
  • IRE1 ⁇ 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 IRE1 ⁇ opens the kinase domain and begins to be activated and activates the RNase domain (25).
  • Both Arf1-Q71L and tapcigagin trigger IRE1 ⁇ kinase activity in HEK293 cells, inducing IRE1 ⁇ autophosphorylation and downstream ASK1 phosphorylation (FIGS. 1A and 1B ).
  • 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 ).
  • STF-083010 is a compound targeting the catalytic core of the IRE1 ⁇ RNase domain, and inhibits IRE1 ⁇ endonuclease activity without affecting the kinase activity or the overall oligomerization step (27).
  • STF-083010 60 ⁇ M was treated for 12 hours, tapcigajin-induced XBP1 production was stopped (Fig. 9D).
  • STF-083010 did not affect the Arf1-Q71L-induced UPS of ⁇ F508-CFTR (FIGS. 1E and 1F ).
  • the IRE1 ⁇ kinase-ASK1 pathway is required for UPS of ⁇ F508-CFTR.
  • APY29 a type I kinase inhibitor, inhibits autophosphorylation of IRE1 ⁇ by competitively occupying the ATP-binding pocket of IRE1 ⁇ (28).
  • the IC 50 value of APY29 for ASK1 phosphorylation was calculated to be about 6.4 ⁇ M, and treatment with 100 ⁇ M APY29 for 12 hours almost completely inhibited Arf1-Q71L-induced phosphorylation for IRE1 ⁇ and ASK1 (FIGS. 10A and 10AB. ).
  • ⁇ F508-CFTR surface targeting induced by ectopic expression of Arf1-Q71L by APY29 was abolished (FIGS. 2A and 2B ). Since the stability of ⁇ F508-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 ⁇ F508-CFTR on the cell surface. In fact, treatment with the protein synthesis inhibitor cycloheximide (0.1 mg/mL) drastically reduced the level of ⁇ F508-CFTR on the cell surface (FIGS. 10c-f).
  • APY29 selectively inhibits the recovery of ⁇ F508-CFTR on the cell surface and does not affect protein synthesis.
  • APY29 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 ⁇ F508-CFTR protein remained only within the ER. When ER-Golgi migration was blocked with Arf1-Q71L, a significant amount of ⁇ F508-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 ⁇ F508-CFTR (FIGS. 2C and 2D ).
  • MSC2032964A is a potent and selective ASK1 inhibitor that blocks LPS-induced ASK1 phosphorylation (31).
  • IRE1 ⁇ kinase Activation of IRE1 ⁇ kinase induces UPS of ⁇ F508-CFTR.
  • 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 IRE1 ⁇ -TRAF2-ASK1 complex (32).
  • 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 ⁇ M) (32).
  • HEK293 cells expressing WT- or ⁇ F508-CFTR were incubated with CSTMP (10 ⁇ M) for 48 hours and CFTR-mediated Cl -Measure the current.
  • CSTMP forskolin
  • IBMX 3-isobutyl-1-methylxanthine
  • IRE1 ⁇ is involved in the UPS of ER stress-induced pendrin (16)
  • the present inventors investigated the effect of CSTMP, an IRE1 ⁇ kinase activator, on the recovery of cell surface migration defects induced by p.H723R-pendrin mutations. I did.
  • CSTMP an IRE1 ⁇ kinase activator
  • PANC-1 derived from the pancreatic duct was used (16).
  • 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.
  • CSTMP (30 ⁇ M) was treated, p.H723R-fendrin was strongly expressed on the cell surface.
  • CSTMP In vivo treatment of CSTMP restores cell surface expression and CFTR-mediated anion transport of CFTR in CftrF 508del mouse colon.
  • CSTMP restores epithelial transport defects caused by the ⁇ F508-CFTR mutation in mice.
  • the malfunctioning ⁇ F508-CFTR mutation causes a defect that primarily affects the transport of ions and fluids in the intestine, resulting in intestinal obstruction in mice (33).
  • 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 ⁇ M 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 ⁇ M) (FIG. 15).
  • CSTMP LD 50 2.59 mg/kg/day, equivalent to 10 ⁇ M once a day
  • Cftr WT WT-CFTR
  • Cftr F508del ⁇ F508-CFTR

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Abstract

La présente invention concerne une composition pour prévenir ou traiter des troubles conformationnels protéiques, son procédé de criblage et une composition pour inhiber une voie de sécrétion de protéines non conventionnelle (UPS). La présente invention peut être utilisée utilement comme composition d'agent thérapeutique efficace contre divers troubles conformationnels protéiques provoqués par un défaut de transport de repliement d'une protéine mutante, et concerne également une nouvelle cible hautement fiable pour le développement d'un agent thérapeutique.
PCT/KR2020/006933 2019-05-28 2020-05-28 COMPOSITION POUR PRÉVENIR OU TRAITER DES TROUBLES CONFORMATIONNELS PROTÉIQUES, COMPRENANT UN ACTIVATEUR DE LA KINASE IRE1α COMME PRINCIPE ACTIF WO2020242225A1 (fr)

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US20110319436A1 (en) * 2008-09-15 2011-12-29 The Regents Of The University Of California Methods and Compositions for Modulating IRE1, SRC, and ABL Activity

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