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WO2018168777A1 - Agent prophylactique ou thérapeutique pour la fibrose kystique - Google Patents

Agent prophylactique ou thérapeutique pour la fibrose kystique Download PDF

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Publication number
WO2018168777A1
WO2018168777A1 PCT/JP2018/009524 JP2018009524W WO2018168777A1 WO 2018168777 A1 WO2018168777 A1 WO 2018168777A1 JP 2018009524 W JP2018009524 W JP 2018009524W WO 2018168777 A1 WO2018168777 A1 WO 2018168777A1
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amino acid
rffl
acid sequence
cftr
nucleic acid
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PCT/JP2018/009524
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English (en)
Japanese (ja)
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司 沖米田
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学校法人関西学院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • 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
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • 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

Definitions

  • the present invention relates to a preventive or therapeutic agent for cystic fibrosis, a screening method thereof, and the like.
  • Cystic fibrosis is a recessive hereditary disease whose main feature is chronic obstructive pulmonary disease, which is common in white people. There are 70,000 CF patients worldwide, but there is no fundamental cure, and many patients die by the age of 40.
  • the cause of CF is a gene mutation of a chloride ion channel (cytic fibrosis transmembrane conductance regulator, CFTR) expressed in epithelial cells.
  • CFTR chloride ion channel
  • the ⁇ F508 CFTR mutant deletion of the 508th phenylalanine in the CFTR amino acid sequence found in the majority of CF is normal in transcription and translation, but causes steric structure abnormality of the protein. Received. As a result, the ⁇ F508 CFTR mutant is degraded by the proteasome and cannot be expressed on the plasma membrane.
  • a CFTR collector developed as a CF therapeutic drug partially improves the plasma membrane expression and chloride ion channel function of ⁇ F508 CFTR, but its clinical therapeutic effect is insufficient. This is because ⁇ F508 CFTR expressed in the plasma membrane by a collector or the like is quickly ubiquitinated by the plasma membrane quality control mechanism and is degraded by lysosomes after endocytosis.
  • ⁇ F508 CFTR expressed in the plasma membrane can inhibit the lysosomal degradation cascade, ⁇ F508 CFTR plasma membrane expression can be maintained, and clinical therapeutic effects can be further enhanced.
  • the clinical therapeutic effect can be dramatically enhanced by using the cascade inhibitor in combination with a CFTR collector.
  • ⁇ F508 CFTR expressed in the plasma membrane undergoes ubiquitination by the plasma membrane quality control mechanism and inhibits lysosomal degradation after endocytosis, thereby preventing or treating cystic fibrosis. It is an object to provide a method for obtaining the above.
  • the present inventors analyzed various types of ubiquitin ligases, and found that RFFL (Ring Finger and FYVE-like Containing E3 Ubiquitin Protein Ligase), which is a kind of ubiquitin ligase, was involved in decomposing mutant CFTR.
  • RFFL Ring Finger and FYVE-like Containing E3 Ubiquitin Protein Ligase
  • ⁇ F508 CFTR ubiquitination can be inhibited by inhibiting the expression of RFFL, and that when CFTR has no ⁇ F508 mutation (such as wild type), it does not interact with RFFL.
  • RFFL Fring Finger and FYVE-like Containing E3 Ubiquitin Protein Ligase
  • Item 1 In the system to which the test substance is applied, (1) a step of measuring the interaction between CFTR having a mutation in which the 508th phenylalanine is deleted or a peptide fragment containing the mutation and RFFL or a peptide fragment containing the fragment, or (2) ubiquitin of RFFL Measuring the ligase activity, A screening method for an agent for preventing or treating cystic fibrosis.
  • Item 2. Item 2. The method according to Item 1, wherein the CFTR having a mutation in which the 508th phenylalanine is deleted is the CFTR described in (i-1a) or (i-2a) below.
  • the peptide fragment according to Item 1 or 2, wherein the peptide fragment containing a mutation in which the 508th phenylalanine of CFTR is deleted is the polypeptide described in (i-1b), (i-2b), or (i-3b) below: the method of.
  • Item 4. The method according to any one of Items 1 to 3, wherein the RFFL in the step (1) is the RFFL described in (ii-1a) or (ii-2a) below.
  • Item 5 The method according to any one of Items 1 to 4, wherein the peptide fragment containing a fragment of RFFL is the polypeptide according to (ii-1b), (ii-2b), or (ii-3b).
  • a preventive or therapeutic agent for cystic fibrosis comprising an expression vector in which a nucleic acid is incorporated. Item 8.
  • the preventive or therapeutic agent for cystic fibrosis according to Item 7, comprising at least one selected from the group consisting of: Item 9. At least one selected from the group consisting of a nucleic acid complementary to a nucleic acid encoding a peptide fragment containing RFFL or a fragment thereof, a nucleic acid comprising the complementary nucleic acid or a fragment thereof, or a nucleic acid and a fragment thereof
  • a preventive or therapeutic agent for cystic fibrosis comprising a nucleic acid donor capable of donating a nucleic acid into a cell.
  • the preventive or therapeutic agent for cystic fibrosis according to Item 9 comprising at least one selected from the group consisting of nucleic acid donors capable of donating at least one nucleic acid selected from the group consisting of: Item 11.
  • the screening method of the present invention can be used to screen for a candidate substance for the prevention or treatment of cystic fibrosis that can inhibit the lysosomal degradation cascade of ⁇ F508 CFTR expressed in the plasma membrane.
  • the present invention also provides a preventive or therapeutic agent for cystic fibrosis comprising a specific polypeptide or nucleic acid (and its expression vector) as an active ingredient.
  • the prophylactic or therapeutic agent can maintain the plasma membrane expression of ⁇ F508 CFTR, and can further enhance the clinical therapeutic effect.
  • the cascade inhibitor in combination with a CFTR collector, the clinical therapeutic effect can be dramatically enhanced.
  • the examination result of intracellular localization of RFFL is shown.
  • the examination result of plasma membrane expression of rescued ⁇ F508 CFTR (r ⁇ F508) at the time of RFFL knockdown is shown.
  • the examination result of the expression level of rescued ⁇ F508 CFTR (r ⁇ F508) at the time of RFFL knockdown is shown.
  • the examination result of the influence on the plasma membrane protein level by RFFL knockdown is shown.
  • the result of examination of the influence of RFFL knockdown on ⁇ F508 CFTR protein stability is shown.
  • the examination result of the influence of RFFL knockdown with respect to endocytosis of a plasma membrane protein is shown.
  • the examination result of localization to the endosome of r (DELTA) F508 CFTR by RFFL knockdown is shown.
  • r ⁇ F508 shows the interaction analysis results of CFTR and RFFL.
  • mold r (DELTA) F508 CFTR and RFFL by a pull-down experiment is shown.
  • the interaction analysis result of RFFL and r ⁇ F508 CFTR by BiFC method is shown.
  • mold r (DELTA) F508 CFTR ubiquitination is shown.
  • mold r (DELTA) F508 CFTR polyubiquitin modification is shown.
  • the examination result of mature type r ⁇ F508 CFTR in vitro polyubiquitination by RFFL is shown.
  • the result of having examined the influence when CFTR collector is administered to RFFL knockdown cells is shown.
  • the result of having examined the influence when CFTR collector is administered to RFFL knockdown cells is shown.
  • the result of examination of the influence on the CFTR channel function by the knockdown of RFFL and CFTR collector is shown.
  • summary estimated from the result of FIG.14 and FIG.15 is shown.
  • the examination result of interaction with RFFL and (DELTA) F508 CFTR-NBD1 (The peptide fragment which consists of the amino acid sequence 433-583 of (DELTA) F508 CFTR) is shown.
  • the present invention includes a screening method for an agent for preventing or treating cystic fibrosis.
  • a screening method for an agent for preventing or treating cystic fibrosis.
  • the screening method in a system to which a test substance is applied, (1) interaction between CFTR having a mutation in which the 508th phenylalanine is deleted or a peptide fragment containing the mutation and RFFL or a peptide fragment containing the fragment Or (2) a step of measuring the ubiquitin ligase activity of RFFL. Steps (1) and (2) can be performed as independent steps, and (1) and (2) do not represent the order of execution.
  • the screening method preferably includes a method including only step (1), a step including only step (2), and a method including steps (1) and (2).
  • the test substance here is not particularly limited.
  • it can be a compound and a composition.
  • the compound may be, for example, a low molecular compound, a high molecular compound such as a nucleic acid (for example, DNA or RNA), a protein (for example, an antibody or a part thereof), or a polymer.
  • the composition may be an extract obtained from a living organism (eg, animal, plant, microorganism, etc.), or may be a combination of two or more compounds.
  • step (1) the interaction between CFTR having a mutation in which the 508th phenylalanine is deleted or a peptide fragment containing the mutation and RFFL or a fragment thereof is measured.
  • CFTR Crystal Fibrosis Transmembrane Conductor Regulator
  • Versatile, particularly mammalian known CFTRs such as humans, monkeys, dogs, cats, mice, rats, sheep, cows, horses, etc. are preferably included. Humans, monkeys, mice and rats are more preferred.
  • the amino acid sequence of the human CFTR is shown in SEQ ID NO: 1.
  • such a CFTR having a mutation in which the 508th phenylalanine is deleted is used.
  • the CFTR used in the present invention preferably also includes the CFTRs defined in the following (i-1a) and (i-2a).
  • the CFTR defined in (i-1a) may be expressed as “ ⁇ F508-CFTR”, “ ⁇ F508 CFTR”, or the like.
  • amino acid sequence from 433 to 584 of the amino acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2. Therefore, deletion of phenylalanine at amino acid sequence 508 of ⁇ F508-CFTR corresponds to deletion of phenylalanine at amino acid sequence 76 in SEQ ID NO: 2.
  • “1 or 2 or more” in (i-2a) is preferably 1 to 200, 1 to 150, 1 to 100, or 1 to 50, more preferably 1 to 40, still more preferably 1 to 30, More preferably 1 to 20, particularly preferably 1 to 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), particularly preferably. 1-10. This number is the sum of the number of amino acids deleted, substituted or added.
  • the peptide fragment containing the ⁇ F508 displacement of CFTR used in the present invention preferably includes a polypeptide defined in the following (i-1b), (i-2b) or (i-3b).
  • “1 or 2 or more” in (i-2b) is preferably 1 to 50, 1 to 40, or 1 to 30, more preferably 1 to 20, and further preferably 1 to 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), and more preferably 1-10. This number is the sum of the number of amino acids deleted, substituted or added.
  • the RFFL in (i-2b) is particularly preferably an RFFL consisting of the amino acid sequence of the following (ii-1b) SEQ ID NO: 3.
  • the polypeptide of (i-3b) is composed of an amino acid sequence including the amino acid sequence of the polypeptide of (i-1b) or (i-2b), so that it can interact with RFFL.
  • the polypeptide (i-3b) has a structure in which one or two or more amino acids are bonded to the N-terminus and / or C-terminus of the polypeptide (i-1b) or (i-2b) by a peptide bond.
  • the number of amino acids bound to the polypeptide of (i-1b) or (i-2b) is relatively large, the ability of the polypeptide of (i-3b) to interact with RFFL decreases due to steric hindrance or the like.
  • the number of amino acids bound to the polypeptide (i-1b) or (i-2b) is, for example, preferably about 1 to 100, more preferably about 1 to 80, and further about 1 to 60.
  • about 1 to 40 is more preferable, and about 1 to 20 is particularly preferable.
  • 1 to 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 Or about 15) is particularly preferable.
  • the number of amino acids bound to the N-terminus is, for example, preferably about 1 to 50, more preferably about 1 to 40, still more preferably about 1 to 30, and still more preferably about 1 to 20,
  • About 1 to 10 is preferable, and about 1 to 8 or about 1 to 7 (1, 2, 3, 4, 5, 6, or 7) is particularly preferable.
  • the number of amino acids bonded to the C-terminal is, for example, preferably about 1 to 50, more preferably about 1 to 40, still more preferably about 1 to 30, and still more preferably about 1 to 20, About 1 to 10 is preferable, and about 1 to 8 or about 1 to 7 (1, 2, 3, 4, 5, 6, or 7) is particularly preferable.
  • CFTR of (i-2a) or the above polypeptide can interact with RFFL can be confirmed as follows. That is, a host (preferably human cells) that stably expresses tagged ⁇ F508 CFTR or the above polypeptide is prepared by genetic engineering techniques, and the tagged RFFL expression vector is transfected into the host and cultured for several days. When a host lysate (for example, cell lysate) is prepared and protein is recovered using ⁇ F508 CFTR or polypeptide tag and RFFL tag as an index, the other tag is identified from the recovered product. If it can be detected automatically, it is understood that interaction is possible.
  • a host preferably human cells
  • tagged RFFL expression vector is transfected into the host and cultured for several days.
  • a host lysate for example, cell lysate
  • protein is recovered using ⁇ F508 CFTR or polypeptide tag and RFFL tag as an index
  • the other tag is identified from the recovered product. If it can
  • tags known in the art preferably peptide detection tags; HA tags, HB tags, etc.
  • affinity chromatography using an antibody that specifically binds to a tag
  • tag detection Western blotting, ELISA, or the like using an antibody that specifically binds to a tag can be used.
  • the RFFL used here is preferably an RFFL consisting of the amino acid sequence of SEQ ID NO: 3 described below (ii-1a).
  • step (1) interaction between CFTR having a mutation in which the 508th phenylalanine is deleted or a peptide fragment containing the mutation and RFFL or a peptide fragment containing the fragment
  • the interaction in step (1) is measured by the method. can do.
  • the strength of interaction can also be measured.
  • RFFL Ring Finger and FYVE-like domain containing E3 ubiquitin protein Ligase
  • the RFFL used in the present invention preferably includes known RFFLs of vertebrates, particularly mammals, such as RFFLs of humans, monkeys, dogs, mice, rats and the like. Humans, monkeys, mice and rats are more preferred.
  • the accession no. Is NP_001017368.1 and the accession number of the amino acid sequence of mouse RFFL Is NP_001007466.1 or the like.
  • the amino acid sequence of the human RFFL is shown in SEQ ID NO: 3.
  • the RFFL used in the step (1) of the present invention preferably includes RFFLs defined in the following (ii-1a) and (ii-2a).
  • (i-1a) the amino acid sequence of SEQ ID NO: 1 RFFL capable of interacting with CFTR consisting of the amino acid sequence lacking the 508th phenylalanine
  • “1 or 2 or more” in (ii-2a) is preferably 1 to 50, more preferably 1 to 40, still more preferably 1 to 30, even more preferably 1 to 20, and particularly preferably 1 to 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), particularly preferably 1 to 10. This number is the sum of the number of amino acids deleted, substituted or added.
  • the peptide fragment containing the RFFL fragment used in the step (1) of the present invention preferably includes a polypeptide defined in the following (ii-1b), (ii-2b) or (ii-3b).
  • the amino acid sequence of the polypeptide of (ii-1b) consists of an amino acid sequence in which one or more amino acids are deleted, substituted or added, and (i-1a) in the amino acid sequence of SEQ ID NO: 1 From the amino acid sequence including the amino acid sequence of the polypeptide (ii-3b): (ii-1b) or (ii-2b) that can interact with CFTR ( ⁇ F508 CFTR) consisting of an amino acid sequence lacking phenylalanine
  • a polypeptide A polypeptide consisting of an amino acid sequence in
  • the polypeptide of (ii-1b) is a polypeptide consisting of an amino acid sequence in which one or more amino acids are deleted in the state where the amino acid sequence of 2 to 88 is maintained in the amino acid sequence of SEQ ID NO: 3. It is more preferable. From the definition, the length of the polypeptide of (ii-1b) is 42 to 362 amino acids long, and any integer value can be taken as long as it is an integer within this range. For example, a range of 45 to 360, 50 to 350, 100 to 300, 150 to 250 or the like is preferably exemplified.
  • “1 or 2 or more” in (ii-1b) is defined from the description of the polypeptide length, preferably 1 to 321, 1 to 300, 1 to 250, 1 to 200, 1 to 150, 1 to 100, or 1 to 50, more preferably 1 to 40, further preferably 1 to 30, even more preferably 1 to 20, and particularly preferably 1 to 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), particularly preferably 1 to 10.
  • the polypeptide of (ii-2b) has one or more amino acids in the amino acid sequence of the polypeptide of (ii-1b) with the 2nd to 88th sequences of the amino acid sequence of SEQ ID NO: 3 maintained.
  • (I-1a) consisting of an amino acid sequence deleted, substituted or added, and capable of interacting with CFTR ( ⁇ F508 CFTR) consisting of an amino acid sequence in which the 508th phenylalanine is deleted in the amino acid sequence of SEQ ID NO: 1. More preferably, it is a polypeptide.
  • “1 or 2 or more” in (ii-2b) is preferably 1 to 50, more preferably 1 to 40, still more preferably 1 to 30, even more preferably 1 to 20, and particularly preferably 1 to 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), particularly preferably 1 to 10. This number is the sum of the number of amino acids deleted, substituted or added.
  • the polypeptide of (ii-3b) consists of an amino acid sequence including the amino acid sequence of the polypeptide of (ii-1b) or (ii-2b), and thus can interact with ⁇ F508 CFTR.
  • the polypeptide of (ii-3b) has a structure in which one or two or more amino acids are bonded to the N-terminal and / or C-terminal of the polypeptide of (ii-1b) or (ii-2b) by a peptide bond.
  • the number of amino acids bound to the polypeptide of (ii-1b) or (ii-2b) is relatively large, the ability of the polypeptide of (ii-3b) to interact with CFTR decreases due to steric hindrance or the like.
  • the number of amino acids bound to the polypeptide of (ii-1b) or (ii-2b) is, for example, preferably about 1 to 100, more preferably about 1 to 80, and further about 1 to 60
  • about 1 to 40 is more preferable, and about 1 to 20 is particularly preferable.
  • 1 to 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 Or about 15) is particularly preferable.
  • the number of amino acids bound to the N-terminus is, for example, preferably about 1 to 50, more preferably about 1 to 40, still more preferably about 1 to 30, and still more preferably about 1 to 20,
  • About 1 to 10 is preferable, and about 1 to 8 or about 1 to 7 (1, 2, 3, 4, 5, 6, or 7) is particularly preferable.
  • the number of amino acids bonded to the C-terminal is, for example, preferably about 1 to 50, more preferably about 1 to 40, still more preferably about 1 to 30, and still more preferably about 1 to 20, About 1 to 10 is preferable, and about 1 to 8 or about 1 to 7 (1, 2, 3, 4, 5, 6, or 7) is particularly preferable.
  • RFFL of (ii-2a) and the peptide fragment containing the above RFFL fragment can interact with ⁇ F508 CFTR can be confirmed as follows. That is, a host (preferably a human cell) that stably expresses tagged ⁇ F508 CFTR is prepared by genetic engineering techniques, and the host is transfected with an expression vector of a peptide fragment containing tagged RFFL or a fragment thereof for several days.
  • a host lysate (eg cell lysate) is prepared, and when a protein is recovered using one of the CFTR tag and the peptide fragment tag containing RFFL or a fragment thereof as an index, If the other tag can be specifically detected, it can be seen that the interaction is possible.
  • tags not the same tags but tags known in the art (preferably peptide detection tags; HA tags, HB tags, etc.) can be used.
  • affinity chromatography using an antibody that specifically binds to a tag can be used.
  • tag detection Western blotting, ELISA, or the like using an antibody that specifically binds to a tag can be used.
  • the interaction in step (1) is performed.
  • the strength of interaction can also be measured.
  • test substance Since the degradation of ⁇ F508 CFTR is suppressed as the interaction is inhibited by the test substance, the test substance can be selected as a more promising candidate for the prevention or treatment of cystic fibrosis.
  • the RFFL used in the step (2) of the present invention preferably includes the RFFLs defined in the following (ii-1c) and (ii-2c).
  • the RFFL of (ii-1c) is the same as the RFFL of (ii-1a).
  • (ii-2c) is preferably 1 to 50, more preferably 1 to 40, still more preferably 1 to 30, even more preferably 1 to 20, and particularly preferably 1 to 15 ( 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), particularly preferably 1 to 10. This number is the sum of the number of amino acids deleted, substituted or added.
  • the ubiquitin ligase activity can be confirmed as follows. That is, a host (preferably a human cell) that stably expresses tagged ⁇ F508 CFTR is prepared by genetic engineering techniques, and a peptide fragment containing RFFL or a fragment thereof is expressed in the host (for example, an expression vector is transfected, After culturing for several days, a host lysate (eg, cell lysate) is prepared, and when the protein is recovered using the ⁇ F508 CFTR tag as an index, the amount of ubiquitin bound to the recovered protein ( ⁇ F508 CFTR) is analyzed.
  • a host preferably a human cell
  • a host lysate eg, cell lysate
  • the ubiquitin ligase activity of a peptide fragment containing RFFL or a fragment thereof can be confirmed.
  • Western blotting using an ubiquitin-specific antibody, ELISA, or the like can be used.
  • the ubiquitin ligase activity of RFFL in step (2) can be measured by the method. By analyzing the intensity of the signal detected by the Western blot method or ELISA method, the strength of ubiquitin ligase activity can also be measured.
  • the test substance As the ubiquitin ligase activity is inhibited by the test substance, the degradation of ⁇ F508 CFTR is suppressed, so that the test substance can be selected as a more promising candidate for the prevention or treatment of cystic fibrosis.
  • the screening method of the present invention can be used to screen for substances useful as preventive or therapeutic agents for cystic fibrosis.
  • the cystic fibrosis here is particularly preferably cystic fibrosis that develops due to a mutation in which the 508th phenylalanine of CFTR is deleted.
  • the system to which the test substance is applied is not particularly limited as long as the step (1) or (2) can be performed.
  • it can be cell-based or cell-free.
  • CFTR having a mutation in which the 508th phenylalanine is deleted or a peptide fragment containing the mutation and a peptide fragment containing RFFL or a fragment thereof are expressed.
  • cell systems include mammalian cells (preferably human cultured cells).
  • Step (1) If the cell line expresses CFTR having a mutation lacking the 508th phenylalanine or a peptide fragment containing the mutation and a peptide fragment containing RFFL or a fragment thereof, and the interaction between them can be measured If the ubiquitin ligase activity of RFFL can be measured (Step (1)) (Step (2)), it can be said that the system can be preferably used in the present invention.
  • CFTR having a mutation in which the 508th phenylalanine is deleted or a peptide fragment containing the mutation and RFFL or a peptide fragment containing the fragment exist
  • the system can be preferably used in the present invention.
  • the present invention includes a preventive or therapeutic agent for cystic fibrosis containing a specific polypeptide.
  • specific polypeptide include the following polypeptides. That is, CFTR having a mutation in which the 508th phenylalanine is deleted or a peptide fragment containing the mutation, and a peptide fragment containing RFFL or a fragment thereof. These can be used individually by 1 type or in combination of 2 or more types.
  • Examples of CFTR having a mutation in which the 508th phenylalanine is deleted include, for example, the polypeptides defined in (i-1a) and (i-2a) above, and peptide fragments including the ⁇ F508 displacement of CFTR include, for example, the above The polypeptides defined in (i-1b), (i-2b), and (i-3b) are preferably mentioned. Further, as RFFL, for example, RFFL defined in the above (ii-1a) and (ii-2a), and as peptide fragments containing the fragment of RFFL, for example, the above (ii-1b), (ii-2b) ) And (ii-3b) are preferably mentioned.
  • the present invention also includes an agent for preventing or treating cystic fibrosis, which contains an expression vector in which a nucleic acid encoding the specific polypeptide is incorporated.
  • the polypeptide encoded by the nucleic acid to be incorporated can be one or a combination of two or more of the aforementioned polypeptides.
  • an expression vector a known expression vector can be used although it depends on the target species. For example, a plasmid equipped with an expression promoter can be exemplified.
  • these polypeptides interact with ⁇ F508 CFTR in the same manner as RFFL, they inhibit the action of RFFL in an antagonistic manner, thereby suppressing the degradation of ⁇ F508 CFTR and exhibiting the effect of preventing or treating cystic fibrosis. Can do.
  • the present invention includes a preventive or therapeutic agent for cystic fibrosis containing a specific nucleic acid.
  • the specific nucleic acid includes the following nucleic acids. That is, a nucleic acid complementary to a nucleic acid encoding a peptide fragment containing RFFL or a fragment thereof, or a nucleic acid comprising the complementary nucleic acid or a fragment thereof.
  • the agent for preventing or treating cystic fibrosis may contain a nucleic acid donor capable of donating these nucleic acids or fragments thereof into cells instead of (or together with) these nucleic acids or fragments thereof. These can be used individually by 1 type or in combination of 2 or more types.
  • nucleic acid examples include DNA, RNA, and PNA (peptide nucleic acid), but DNA or RNA (polynucleotide) is preferable. Further, these nucleic acids may be subjected to modification (chemical modification or the like) for suppressing degradation.
  • nucleic acid complementary to the nucleic acid encoding the peptide fragment containing RFFL or a fragment thereof include the following nucleic acids (I) to (III).
  • nucleic acids (I) to (III) can suppress the expression of the RFFL gene as an antisense nucleic acid. Thereby, the decomposition
  • examples of the nucleic acid preferably a double-stranded nucleic acid or a fragment thereof comprising these complementary nucleic acids preferably include the following (I-ds) to (IV-ds) nucleic acids or fragments thereof.
  • a nucleic acid comprising a base sequence having a base sequence of 80% or more (preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and still more preferably 98% or more), and the nucleic acid A double-stranded nucleic acid or a fragment thereof (III-ds) that suppresses the expression of RFFL: a nucleic acid that hybridizes with the nucle
  • Stringent conditions means hybridization in a 5 ⁇ SSC solution at 65 ° C. (composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate), and further 0.1% SDS. Means a condition of washing at 65 ° C. with a 0.5 ⁇ SSC solution containing Each hybridization operation under stringent conditions is described in “Molecular Cloning (Third Edition)” (J. Sambrook & D. W. Russell, Cold Spring Harbor Press, 2001, etc.). It can be performed by a known method.
  • the double-stranded nucleic acid or a fragment thereof is preferably a ds nucleic acid and can suppress the expression of the RFFL gene.
  • the nucleic acid is preferably RNA (double-stranded RNA; dsRNA).
  • dsRNA double-stranded RNA
  • the fragment here is also preferably a double-stranded nucleic acid, more preferably a double-stranded nucleic acid having a base length of about 15 to 50, more preferably a double-stranded nucleic acid having a base length of about 20 to 35. . It is particularly preferable that the fragment is also a dsRNA.
  • the (IV-ds) nucleic acid is a nucleic acid having a partially double-stranded nucleic acid structure, for example, by taking a hairpin structure, and the double-stranded nucleic acid moiety is (I-ds), (II-ds) Or a nucleic acid corresponding to (III-ds) double-stranded nucleic acid or a fragment thereof.
  • double-stranded nucleic acid include RNA having a hairpin structure (hairpin RNA).
  • the double-stranded nucleic acid portion when the double-stranded nucleic acid portion is the above fragment, the whole becomes a hairpin RNA having a relatively short base length, that is, shRNA (short hairpin RNA), which is also preferably included in the nucleic acid of (IV-ds).
  • shRNA short hairpin RNA
  • nucleic acid donor capable of donating the above-described nucleic acid or a fragment thereof into a cell
  • a nucleic acid obtained by incorporating the nucleic acid or a fragment thereof into a known vector can be preferably used.
  • the incorporated nucleic acid or a fragment thereof may be one or a combination of two or more of the aforementioned nucleic acids and fragments thereof.
  • the vector include plasmids, viruses (for example, adeno-associated virus, adenovirus, lentivirus, retrovirus, etc.), phages, liposomes and the like.
  • the above-mentioned agent for preventing or treating cystic fibrosis included in the present invention may contain other components as long as the effects of the present invention are not impaired.
  • examples of such other components include pharmaceutically acceptable carriers and other cystic fibrosis preventive or therapeutic agents (for example, CFTR collectors, more specifically, Ivacaftor and Lumacabtor (VX-809)) and the like. Illustrated.
  • SEQ ID NO: 4 An example of a preferable sequence is shown in SEQ ID NO: 4 as the base sequence of the nucleic acid encoding RFFL consisting of the amino acid sequence of SEQ ID NO: 3.
  • the base sequence of SEQ ID NO: 4 is the base sequence of human RFFL gene.
  • the base sequence of the nucleic acid encoding RFFL consisting of the amino acid sequence of SEQ ID NO: 3 can be easily obtained by using a codon reverse control table. (In the case of DNA, U is read as T in the codon.)
  • the polypeptide or nucleic acid according to the present invention can be produced by a known method.
  • the nucleic acid may be produced by extracting DNA or RNA encoding CFTR or RFFL from an organism and artificially mutating it, or may be chemically synthesized.
  • a method of artificially mutating, site-specific mutagenesis Method of artificially mutating, site-specific mutagenesis [Methods in Enzymology, 154, 350, 367-382 (1987); 100, 468 (1983); Nucleic Acids Res.
  • these proteins or a part thereof can be obtained by genetic engineering techniques.
  • the present invention also includes a preventive or therapeutic agent for cystic fibrosis containing a specific antibody or fragment thereof.
  • the specific antibody or fragment thereof is an antibody that recognizes RFFL or a fragment thereof (one that recognizes RFFL, for example, Fab).
  • the antibody may be a polyclonal antibody or a monoclonal antibody. Moreover, it is preferable that it is a humanized antibody.
  • the antibody is preferably an antibody that recognizes human RFFL, particularly an antibody that specifically recognizes human RFFL. Furthermore, an antibody that recognizes a peptide portion consisting of amino acid sequences 1 to 44 of human RFFL is preferable.
  • Such an antibody can be produced by a known technique using, for example, RFFL or a peptide consisting of the 1st to 44th amino acid sequences of RFFL as an immunogen.
  • an appropriate mammal mammal (mouse, rat, Chinese hamster, rabbit, monkey, etc.) is immunized with a human RFFL protein or a fragment thereof (combined with an appropriate conjugate as necessary) as an immunogen, and a polyclonal antibody is obtained.
  • a hybridoma that produces an antibody with high characteristics in human RFFL is selected, and an antibody produced by the hybridoma is isolated and purified to prepare a monoclonal antibody.
  • a hybridoma that produces an antibody with high characteristics in human RFFL is selected, and an antibody produced by the hybridoma is isolated and purified to prepare a monoclonal antibody. Can do.
  • the specific antibody or a fragment thereof can suppress the interaction of RFFL with ⁇ F508 CFTR (in particular, an antibody or a fragment thereof that recognizes a peptide portion consisting of amino acid sequences 1 to 44 of human RFFL has a high effect) And / or may suppress the ubiquitin ligase activity of RFFL.
  • degradation of (DELTA) F508 CFTR can be suppressed and the effect which prevents or treats cystic fibrosis can be exhibited.
  • GFP Green Fluorescent Protein
  • HRP Horseradish Peroxidase
  • 3HA, V5, VN155, VC155, etc. are tags.
  • the CFTR and RFFL used were derived from humans (the amino acid sequence of human CFTR is shown in SEQ ID NO: 1, and the amino acid sequence of RFFL is shown in SEQ ID NO: 3, respectively).
  • RFFL-GFP RFFL-mCherry
  • RFFL- ⁇ NT-GFP RFFL- ⁇ NT-GFP by lipofection.
  • cells were fixed with 4% paraformaldehyde, and immunofluorescent staining was performed using anti-Rab5 (early endosomal marker), anti-Rab7 (late endosomal marker), or anti-Rab9 (late late endosomal marker) antibody. It was.
  • the plasma membrane was stained with Alexa594-WGA (Wheat Germ Agglutinin), and the nucleus was stained with DAPI. After staining, the cells were observed with a confocal laser microscope.
  • RFFL- ⁇ NT means that the N-terminal site (amino acid 2-88) of RFFL has been deleted.
  • FIG. 2 shows that RFFL knockdown increases the plasma membrane expression and stability of r ⁇ F508 CFTR.
  • the cells were incubated at 37 ° C. for 1 hour, and the cell lysate was added to RIPA buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Triton-X100, 0.5% sodium deoxycholate, 5 ⁇ g). / Ml pepstatin, 5 ⁇ g / ml leupeptin, 1 mM PMSF).
  • Anti-HA (16B12, BioLegend), anti-RFFL (N2C3, GeneTex), anti-Na + / K + ATPase (H-3, santa cruz biotechnology) antibodies were used for Western blotting.
  • ⁇ means “anti” and is a description for indicating the antibody species used. From the results, it was found that RFFL knockdown increases the expression level of mature r ⁇ F508 CFTR.
  • T70 CFTR is a kind of known CFTR variant different from ⁇ F508 CFTR.
  • CD4T is one of plasma membrane expressed proteins, and was used as a model protein for comparison with CFTR.
  • CD4Tl-Lamp, CD4TccUbAllR ⁇ G, and CD4Tl- ⁇ m are CD4T modified proteins (chimeras).
  • CD4TccUbAllR ⁇ G and CD4Tl- ⁇ m are known (Apaja PM, J Cell Biol. 2010 Nov 1; 191 (3): 553-70., Barriere H. Mol Biol Cell. 2007 Oct; 18 (10): 3952-65: 3952-65. H., Traffic.2006 Mar; 7 (3): 282-97.)
  • Cc is coiled-coiled (tetramer forming sequence)
  • UbAllR ⁇ G is a ubiquitin variant that is not polyubiquitinated
  • ⁇ m is a bacteriophage. Lambda mutants (L57C) are shown respectively.
  • CD4Tl-lamp is a modified protein in which the cytoplasmic region of Lamp1 (lysosomal protein) is fused to CD4.
  • r ⁇ F508 CFTR-HRP, WT CFTR-HRP, T70 CFTR-HRP After washing the cells twice with PBS, HRP substrate (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific) was added to the extracellular fluid, and the chemiluminescence was quantified with a plate reader (Varioscan, Thermo Scientific. Quantitative).
  • the plasma membrane expression of ⁇ F508 CFTR-HRP was induced by culture at 26 ° C. for 2 days (rescue treatment), and then the plasma membrane expression level of r ⁇ F508 CFTR-HRP after 1 hour incubation at 37 ° C. was expressed as r ⁇ F508 CFTR PM density. Quantified.
  • CD4Tl-Lamp, CD4TccUbAllR ⁇ G, CD4Tl- ⁇ m Quantification was performed by a cell surface ELISA method using an anti-CD4 antibody (OKT4) (Okiyoneda et al, Science 2010).
  • TfR Transferrin Receptor
  • ⁇ F508 CFTR-3HA was cultured at 26 ° C. for 2 days, and then incubated at 37 ° C. for 1 hour and used in the experiment.
  • anti-HA (16B12, BioLegend) antibody was used, and mature and immature bands were quantitatively analyzed by densitometry.
  • CHX is a protein synthesis inhibitor.
  • r ⁇ F508 CFTR, TfR and CD4TccUbAllR ⁇ G present in the plasma membrane were transformed into anti-HA antibody (16B12, BioLegend), Biotin-Tf (Transferrin Biotin-XX conjugate, ThermoT4, ThermoT4, Anti-HA4).
  • the amount of plasma membrane protein that was labeled with BioLegend) and disappeared from the plasma membrane upon incubation at 37 ° C. for 5 minutes was quantified by ELISA as the amount of endocytosis.
  • ⁇ F508 CFTR-3HA was used for experiments by inducing plasma membrane expression by culturing at 26 ° C. for 2 days, and then incubating at 37 ° C. for 1 hour.
  • extracellular anti-HA antibody was washed out and incubated at 37 ° C. for 2 hours (chase 2h).
  • immunofluorescence staining was performed, and the intracellular localization of r ⁇ F508 CFTR-3HA was observed with a confocal laser microscope.
  • ⁇ F508 CFTR-3HA was cultured at 26 ° C. for 2 days and then incubated at 37 ° C. for 1 hour for use in the experiment. Nuclei were stained with DAPI.
  • EEA1 which is an early endosome marker, was immunofluorescently stained with an anti-EEA1 antibody (MBL, PM062).
  • COS-7 cells were co-transfected with ⁇ F508 CFTR-3HA-VN155 ( ⁇ F508-VN) and RFFL-VC155 (RFFL-VC) by lipofection, and the cells were washed 3 days later. After fixation with 4% paraformaldehyde, immunofluorescence staining was performed using an anti-HA antibody (16B12, BioLegend) or an anti-RFFL antibody (N2C3, GeneTex).
  • ⁇ F508 CFTR-3HA was cultured at 26 ° C. (r ⁇ F508) or 37 ° C. ( ⁇ F508) for 36 hours and then incubated at 37 ° C.
  • the cells were observed with a confocal laser microscope.
  • the initial endosome marker mRFP-Rab5 was co-expressed, followed by immunofluorescence staining in the same manner as described above, followed by observation with a focused laser microscope. .
  • ubiquitinated CFTR was immobilized on Neutravidin coated 96 well white plate (ThermoFisher) under denaturing conditions, followed by anti-HA (16B12, BioLegend), anti-K48-Ub (DuM) CFTR, K48 by ELISA using an anti-K63-Ub (Apu3, EMD Millipore) antibody. Quantified re ubiquitin chains and K48 polyubiquitin chain modification, the ubiquitination CFTR amount corrected by CFTR amounts each ubiquitin chains weights were comparative analysis.
  • Tris-NP-40 solubilization buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 2 mM MgCl2, 10% glycerol, 5 mM imidazole, 0.1% NP-40, 5 mM TCEP, 1 mM TCEP, 1 mM Cell lysate was prepared using PMSF, 5 ⁇ g / ml leupeptin, 5 ⁇ g / ml pepstatin A and 2.5 mM ATP, and then matured into NeutrAvidin agarose (Thermo Scientific).
  • the mature r ⁇ F508 CFTR immobilized on NeutrAvidin agarose was mixed with 2M urea wash buffer (2M urea, 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Triton-X100, 0.5%). After washing with sodium deoxycholate, ubiquitinated r ⁇ F508 CFTR was isolated with an elution buffer (8 Murea, 2% SDS, 3 mM biotin) and analyzed by Western blot.
  • E1 indicates ubiquitin activating enzyme (E1), UbcH5c and Ubc13 / Uev1a are one of ubiquitin-binding enzymes (E2), UbcH5c is non-selective ubiquitin binding, and Ubc13 / Uev1a is specific for K63.
  • Ub represents ubiquitin
  • Ub-63R represents ubiquitin in which K63 is changed to R
  • Ub-63K represents ubiquitin in which all K except K63 has been changed to R. From these results, it was found that RFFL directly promotes ubiquitination of r ⁇ F508 CFTR, in particular, K63-linked polyubiquitination.
  • cell lysate was added to RIPA buffer (20 mM Tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Triton-X100, 0.5% sodium deoxycholate, 5 ⁇ g / ml pepstatin, 5 ⁇ g / ml leupeptin, 1 mM PMSF) and the expression level of ⁇ F508 CFTR-3HA was quantified by Western blotting.
  • Anti-HA (16B12, BioLegend), anti-RFFL (N2C3, GeneTex), and anti-Na + / K + ATPase (H-3, santa cruz biotechnology) antibodies were used for Western blotting. The result is shown in FIG. 14a.
  • AllStars Netative Control siRNA siNT, Qiagen
  • RFFL siRNA Hs_RFFL_1 (SI00148232), Hs_RFFL_3 (SI00148246), a mixture of Hs_RFFL_5 (SI03089653), Qiagen
  • Lipofectamine RNAiMAX Transfection Reagent The cells were transfected using Thermo Scientific).
  • VX-809 is a compound represented by the following structural formula and is said to have an effect of promoting normalization of misfolding of ⁇ F508 CFTR.
  • VX-809 in combination with C4 and glycerol referred to as Trio
  • the activation of the CFTR channel was performed using 20 ⁇ M forskolin, 0.5 mM 3-isobutyl-1-mthyl-xanthine (IBMX), 0.5 mM 8- (4-chlorophenylthio) -adenosine-3 ′, 5′-cyclic monophosphate ( cpt-cAMP) and 0.1 mM genistein.
  • FIG. 16 shows an outline estimated from the results of FIG. 14 and FIG.
  • ⁇ F508 CFTR-NBD1-VN155 ⁇ F-NBD1-VN
  • RFFL-VC155 RFFL-VC155
  • the cells were later fixed with 4% paraformaldehyde, and then immunofluorescent staining was performed using an anti-GFP (VC) antibody (mFX75, wako). After nuclear staining with DAPI, the cells were observed with a confocal laser microscope.
  • ⁇ F508 CFTR-NBD1 is a peptide fragment consisting of the amino acid sequence of amino acid sequence 433-583 of ⁇ F508 CFTR.

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Abstract

L'invention concerne un procédé permettant la prévention ou le traitement de la fibrose kystique par l'inhibition d'un mécanisme dans lequel un CFTR ΔF508 exprimé dans la membrane de plasma est ubiquitiné par un système de contrôle de qualité de membrane de plasma puis soumis à une endocytose suivie d'une dégradation lysosomale, etc. Plus particulièrement, l'invention concerne : un agent prophylactique ou thérapeutique pour la fibrose kystique comprenant un polypeptide ou un acide nucléique spécifiques ; et un procédé de dépistage d'un agent prophylactique ou thérapeutique pour la fibrose kystique, ledit procédé comprenant l'étape suivante (1) ou (2). (1) Une étape de mesure d'une interaction entre un CFTR présentant une mutation de manque de phénylalanine en position 508 ou un fragment peptidique contenant la mutation précitée et une RFFL ou un fragment peptidique contenant un fragment de cette dernière. (2) Une étape de mesure de l'activité de l'ubiquitine ligase de RFFL.
PCT/JP2018/009524 2017-03-13 2018-03-12 Agent prophylactique ou thérapeutique pour la fibrose kystique WO2018168777A1 (fr)

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