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WO2021256200A1 - Cathepsin k inhibitor - Google Patents

Cathepsin k inhibitor Download PDF

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Publication number
WO2021256200A1
WO2021256200A1 PCT/JP2021/019927 JP2021019927W WO2021256200A1 WO 2021256200 A1 WO2021256200 A1 WO 2021256200A1 JP 2021019927 W JP2021019927 W JP 2021019927W WO 2021256200 A1 WO2021256200 A1 WO 2021256200A1
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Prior art keywords
cathepsin
vitronectin
hvn
collagen
fragment
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PCT/JP2021/019927
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French (fr)
Japanese (ja)
Inventor
公恵 伊達
敬 由良
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国立大学法人お茶の水女子大学
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Priority to JP2022532440A priority Critical patent/JPWO2021256200A1/ja
Publication of WO2021256200A1 publication Critical patent/WO2021256200A1/en

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    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention relates to a cathepsin K inhibitor comprising vitronectin or a fragment thereof that binds to cathepsin K.
  • Bone osteoporosis is a disease in which the balance between bone decomposition and resorption (bone resorption) by osteoblasts and bone formation by osteoblasts is lost, and bone resorption becomes excessive and the bone becomes brittle.
  • Bone resorption inhibitors for example, calcitonin preparations, estrogen preparations, vitamin K preparations, bisphosphonate preparations, etc.
  • bone resorption inhibitors also have the problem of inhibiting bone formation. Therefore, there is a need for the development of a drug that does not inhibit bone formation but inhibits only bone resorption.
  • Cathepsin K is one of the enzymes of the cysteine cathepsin family, which is part of the papain superfamily of cysteine proteases.
  • Cathepsin K is a protease that is secreted from osteoclasts as a precursor procatehepsin K, self-activates under acidic conditions, and decomposes type I collagen, which is the main component of bone.
  • Cathepsin K inhibitors are promising as therapeutic agents for osteoporosis because they inhibit only bone decomposition and hardly inhibit bone formation.
  • VN Vitronectin
  • Human VN has three complex N-type sugar chains, and the N-type sugar chain binding site is highly conserved among mammals (Non-Patent Documents 1 to 4). VN strongly interacts with collagen (Non-Patent Documents 5 and 6). Since the sugar chain of VN affects the adhesion progress of cells, it is considered that it affects the adhesion progress of osteoblasts and osteoclasts (Non-Patent Document 7), and VN plays an important role in osteoporosis. Conceivable.
  • Non-Patent Document 8 It has been reported that the VN peptide promotes the adhesion and extension of bone-forming cells and is effective in ovariectomized osteoporosis model rats. However, the effect of VN on cathepsins is unknown.
  • the present invention is a problem to be solved to provide a novel cathepsin K inhibitor.
  • vitronectin has a cathepsin K activity inhibitory action, a cathepsin K self-activation inhibitory action, and a collagen acid lysis promoting action, which are bones in osteoclasts. It has been found to have three actions of suppressing absorption. Furthermore, the present inventors have found that vitronectin has an action of promoting the fibrosis of collagen produced in bone-forming cells. Furthermore, we present that the binding site of vitronectin to cathepsin K is disordered regions (amino acid SEQ ID NOs: 100-130, 292-296, 364-397 of human vitronectin (hVN)) that do not normally have a fixed structure. I found that. The present invention has been completed based on the above findings.
  • a cathepsin K inhibitor comprising vitronectin or a fragment thereof that binds to cathepsin K.
  • a self-activation inhibitor of procaptesin K which comprises vitronectin or a fragment thereof that binds to cathepsin K.
  • a lysis promoter for fibrotic collagen which comprises vitronectin or a fragment thereof that binds to cathepsin K.
  • An agent for promoting collagen fibrosis which comprises a fragment thereof that binds to vitronectin or cathepsin K.
  • ⁇ 5> The agent according to any one of ⁇ 1> to ⁇ 3>, which is used for suppressing bone resorption of osteoclasts.
  • ⁇ 6> The agent according to any one of ⁇ 1> to ⁇ 5>, wherein the vitronectin is human vitronectin or porcine vitronectin.
  • ⁇ 7> The agent according to any one of ⁇ 1> to ⁇ 6>, wherein the vitronectin has an N-type sugar chain.
  • Methods of inhibiting cathepsin K comprising administering to the subject a fragment of vitronectin, or a fragment thereof that binds to cathepsin K; Methods of Inhibiting Self-Activation of Procaptesin K, Containing Administration of Vitronectin, or Fragments thereof that Bind Cathepsin K, to Subjects; Methods of promoting the lysis of fibrotic collagen, including administering to the subject a fragment of vitronectin, or a fragment thereof that binds to cathepsin K; Methods of promoting collagen fibrosis, comprising administering to the subject a fragment of vitronectin, or a fragment thereof that binds to cathepsin K; Is provided.
  • Vitronectin for use in treatments that inhibit cathepsin K, or a fragment thereof that binds to cathepsin K; Vitronectin, or a fragment thereof that binds to cathepsin K, for use in treatments that inhibit the self-activation of procaptesin K; Vitronectin or a fragment thereof that binds to cathepsin K for use in a procedure that promotes lysis of fibrotic collagen; and a fragment thereof that binds to vitronectin or cathepsin K for use in a procedure that promotes fibrosis of collagen; Is provided.
  • vitronectin, or a fragment thereof that binds to cathepsin K, for the production of cathepsin K inhibitors Use of vitronectin, or a fragment thereof that binds to cathepsin K, for the production of self-activation inhibitors of procaptesin K; Use of a fragment thereof that binds to vitronectin or cathepsin K for the production of a lysis promoter of fibrotic collagen; and a fragment thereof that binds to vitronectin or cathepsin K for the production of a promoter of collagen fibrosis. Use of; Is provided.
  • a novel cathepsin K inhibitor is provided.
  • the cathepsin K inhibitor of the present invention is useful as a therapeutic agent for osteoporosis.
  • FIG. 1 shows inhibition of VN cathepsin K activity at various pHs.
  • FIG. 2 shows inhibition of VN cathepsin K activity (pH,%) (data with a reaction time of 30 min).
  • FIG. 3 shows inhibition of VN cathepsin K activity (concentration-dependent) (data with a reaction time of 30 min).
  • FIG. 4 shows inhibition of cathepsin K activity (competitive inhibition) of pVN (data with a reaction time of 30 min).
  • FIG. 5 shows the inhibition of self-activation of hVN procathepsin K at various pHs.
  • FIG. 6 shows the effect of VN on collagen acid dissolution.
  • FIG. 1 shows inhibition of VN cathepsin K activity at various pHs.
  • FIG. 2 shows inhibition of VN cathepsin K activity (pH,%) (data with a reaction time of 30 min).
  • FIG. 3 shows inhibition of VN cathepsin K activity (concentration-dependent) (data with a
  • FIG. 7 shows the effect of pVN / hVN on collagen acid dissolution (data with a reaction time of 20 min).
  • FIG. 8 shows the effect of pVN / hVN on collagen acid dissolution (data with a reaction time of 20 min).
  • FIG. 9 shows the promotion of collagen fiber formation in VN (difference in collagen type (pig / bovine)).
  • FIG. 10 shows the promotion of collagen fiber formation (concentration dependence) of VN.
  • FIG. 11 shows a transmission electron micrograph of collagen fibers formed in the presence of VN.
  • FIG. 12 shows the effect of hVN sugar chains on cathepsin K activity.
  • FIG. 13 shows the effect of hVN sugar chains on the self-activation of procathepsin K.
  • FIG. 14 shows the degradation of VN by cathepsin K (effect of VN sugar chain).
  • FIG. 15 shows the acid dissolution promoting action of VN (effect of VN sugar chain).
  • FIG. 16 shows the promotion of collagen fiber formation in VN (effect of VN sugar chain).
  • FIG. 17 shows a diagram of disordered regions prediction that does not normally have a fixed structure with hVN.
  • FIG. 18 shows a multiple alignment of hVN and pVN.
  • the present invention relates to a cathepsin K inhibitor, a self-activation inhibitor of procaptesin K, a fibrotic collagen lysis promoter, and a collagen fibrosis promoter, which comprises a fragment thereof that binds to vitronectin or cathepsin K.
  • vitronectin has been demonstrated to have an inhibitory effect on cathepsin K, an inhibitory effect on self-activation of procapthecin K, and a lysis promoting effect on fibrotic collagen, and particularly selectively cathepsin K. Can be inhibited. Since vitronectin has these effects, the above-mentioned agent of the present invention can be used to suppress bone resorption of osteoclasts.
  • the origin of the vitronectin used in the present invention is not particularly limited, but a mammalian-derived vitronectin can be preferably used, and for example, human vitronectin or porcine vitronectin can be used.
  • Human vitronectin is composed of 478 amino acids, and is composed of 388 to 655 amino acids depending on the animal species.
  • the amino acid sequence of human vitronectin is registered in NCBI Reference Sequence: NP_000629.3
  • the amino acid sequence of porcine vitronectin is registered in NCBI Reference Sequence: NP_999269.1.
  • Natural vitronectin produced in a living body such as human is a glycoprotein in which a sugar chain (N-type sugar chain) is bound to a part of amino acids.
  • vitronectin it is preferable to use vitronectin to which an N-type sugar chain is bound. Further, it is preferable to use vitronectin to which a plurality of complex N-type sugar chains to which sialic acid is added at the end are bound.
  • Vitronectin is a protein consisting of 478 amino acid residues having adhesive activity to cells carrying the vitronectin receptor. Vitronectin may be a partial sequence deletion as long as it exhibits the effect of the present invention. Vitronectin is composed of a somatomedin B region, a connecting region, and a hemopexin region from the N-terminal, and may be a fragment consisting of any one or more of the regions or a peptide fragment as long as the effect of the present invention is exhibited (Yoneda A et). al., Biochemistry 1998, 37, 18, 6351-6360).
  • recombinant vitronectin in which the signal domain consisting of 20 amino acids on the N-terminal side is deleted recombinant vitronectin in which the C-terminal portion (the 399th amino acid to the 478th amino acid sequence portion) is deleted may be used.
  • the vitronectin may be either naturally-derived vitronectin, recombinant vitronectin produced by genetically modified technology, or chemically synthesized vitronectin.
  • vitronectin human plasma-derived (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Inc.), Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific), and the like can also be used. can.
  • a fragment of vitronectin that binds to cathepsin K can also be used.
  • Fragments of vitronectin that bind to cathepsin K include, for example, in the case of human vitronectin (hVN), amino acid SEQ ID NOs: 100-130 (SEQ ID NO: 3), 292-296 (SEQ ID NO: 4), or 364 of hVN. Fragments containing at least one of 397 (SEQ ID NO: 5) can be mentioned.
  • the catepsin K inhibitor, the self-activation inhibitor of procaptesin K, the lysis promoter of fibrotic collagen, and the fibrosis promoter of collagen are, for example, locomotive syndrome (exercise). Treatment or prevention of organ syndrome), osteoporosis, osteoarthritis osteoarthritis, rheumatoid arthritis, bone Paget's disease, hypercalcemia, bone metastasis of cancer, bone pain, fibrous disease, arteriosclerosis, or cutaneous muscle inflammation Can be used for.
  • the agent of the present invention is a reagent composition or a pharmaceutical composition comprising the active ingredient vitronectin or a fragment thereof that binds to cathepsin K and one or more pharmaceutically acceptable pharmaceutical additives. It may be provided in the form.
  • the type of pharmaceutical additive used for producing the drug of the present invention is not particularly limited and can be appropriately selected by those skilled in the art.
  • Agents, tonicity agents, pH regulators, solubilizers, stabilizers and the like can be used, and the individual specific ingredients used for these purposes are well known to those skilled in the art.
  • the injectable or drip-form may be a solution in which a fragment thereof that binds to vitronectin or catepsin K is dissolved, suspended or emulsified in a solvent, or is frozen in which it is dissolved, suspended or emulsified in a solvent at the time of use.
  • the solvent which may be a solid agent such as a dry preparation, include distilled water for injection, physiological saline, vegetable oil, alcohols such as propylene glycol, polyethylene glycol, and ethanol, or a combination thereof. ..
  • the agent of the present invention can be administered in various forms, and suitable administration forms include oral administration and parenteral administration (for example, intravenous, intramuscular, subcutaneous or intradermal injection, intrarectal injection, etc.). Administration, transmucosal administration, etc.) may be used, but parenteral administration is preferable.
  • parenteral administration for example, intravenous, intramuscular, subcutaneous or intradermal injection, intrarectal injection, etc.
  • parenteral administration for example, intravenous, intramuscular, subcutaneous or intradermal injection, intrarectal injection, etc.
  • parenteral administration may be used, but parenteral administration is preferable.
  • Examples of the pharmaceutical composition suitable for oral administration include tablets, granules, capsules, powders, solutions, suspensions, syrups and the like, and examples of the pharmaceutical composition suitable for parenteral administration include tablets, granules, capsules, powders, solutions and syrups. , Injections, infusions, suppositories, transdermal absorbents and the like, but are not limited thereto.
  • the administration target of the agent of the present invention includes mammals, for example, humans and non-human animals such as monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, and mice.
  • the administration target of the agent of the present invention is a human (preferably a patient).
  • the dose of the agent of the present invention should be appropriately increased or decreased according to conditions such as the age, sex, weight, symptoms, and route of administration of the patient, but generally, the amount of the active ingredient per adult day. It is in the range of about 1 ⁇ g / kg to about 1,000 mg / kg, preferably in the range of about 10 ⁇ g / kg to about 100 mg / kg.
  • the drug having the above dose may be administered once a day, or may be administered in several divided doses (for example, about 2 to 4 doses).
  • CatK Cathepsin K
  • hVN Human vitronectin (purified from plasma by heparin affinity chromatography)
  • Example 1 Assay for Cathepsin K Activity CatK activity was measured using a fluorescent substrate in a 96-well plate (Adeleke Het al., PNAS, 111 (49) 2014, 17474-79, Peter A., J. Biol Chem. 289 (31) 21562-21572). Z-Phe-Arg-AMC (Enzo Life Science) as a substrate was dissolved in DMSO at 2 mg / ml and stored at -80 ° C before use. Procathepsin K (5 ⁇ M) (Enzo Life Science) was self-activated in 100 mM acetate buffer (pH 4.0) containing 2.5 mM EDTA / DTT at 37 ° C.
  • FIG. 1 shows inhibition of VN cathepsin K activity at various pHs.
  • pVN (+) markedly inhibited cathepsin K activity at any pH.
  • (-) Indicates no pVN.
  • FIG. 2 shows inhibition of VN cathepsin K activity (pH,%) (data with a reaction time of 30 min).
  • pVN and hVN inhibited cathepsin K activity at pH 4-7.2.
  • FIG. 3 shows inhibition of VN cathepsin K activity (concentration-dependent) (data with a reaction time of 30 min). The VN concentration was measured at 0 to 2 nM (pH 5.5).
  • pVN and hVN inhibited cathepsin K activity at pH 5.5 in a concentration-dependent manner.
  • FIG. 4 shows inhibition of cathepsin K activity (competitive inhibition) of pVN (data with a reaction time of 30 min).
  • the VN concentration was measured at 15.63, 31.25, 62.5, 125, 250 nM (pH 5.5).
  • S indicates the Z-Phe-Arg-AMC concentration ( ⁇ M).
  • Inhibition of cathepsin K activity at pH 5.5 by pVN and hVN was shown to be competitive inhibition.
  • Example 2 Self-activation assay for proCatK ProCatK (3 ⁇ M) in 100 mM acetate buffer (pH 4.0-5.5) containing 2.5 mM EDTA / DTT at 37 ° C. in the presence or absence of VN. Incubated in. After incubation at 37 ° C. for 10 or 30 minutes, 2xSDS-PAGE sample buffer was immediately added and heated at 98 ° C. for 10 minutes. Protein samples were electrophoresed using a 15% Tris-glycine gel containing 2-mercaptoethanol and stained with EzStain Silver (AE-1360, ATTO). The total band intensities of both proCatK and CatK were quantified by ImageJ (NIH).
  • FIG. 5 shows the inhibition of self-activation of hVN procathepsin K at various pH.
  • cathepsin K Pro-CatK (inactive form)
  • cathepsin K Melt (active form)
  • hVN inhibited self-activation from procathepsin K to cathepsin K (+ hVN lane).
  • Example 3 Collagen Acid Solubilization Assay In an Iwaki 96-well assay plate (AGC TECHNO GLASS co., Ltd), solubilized type I collagen in the presence or absence of 0.1 mg / ml VN in 100 ⁇ l TBS. The solution (1 mg / ml) (Nippi Co., Ltd.) was treated at 37 ° C. for 2 hours to form collagen fibers. 1 M pH buffer (1 M acetate buffer (pH 4.0-6.0) or Tris-HCl buffer (pH 7.2), 10 ⁇ l) was added to the formed collagen fibers. Immediately, the turbidity of the sample in each well was measured by Cytation 3 (BioTek) at 37 ° C. every minute at 350 nm (Glycobiology, Tatara et al., 2017).
  • FIG. 6 shows the effect of VN on collagen acid dissolution. Fibrotic collagen was lysed at pH 4-4.5 (Control). hVN promoted the dissolution of fibrotic collagen at pH 4.5-5.5 (hVN).
  • FIG. 7 shows the effect of pVN / hVN on collagen acid dissolution (data with a reaction time of 20 min). Both hVN and pVN promoted the dissolution of fibrotic collagen at pH 4.5-5.5.
  • FIG. 8 shows the effect of pVN / hVN on collagen acid dissolution (data with a reaction time of 20 min). The VN concentration was measured at 0-100 ⁇ g / ml at pH 5.0. hVN and pVN promoted acid dissolution of fibrotic collagen in a concentration-dependent manner.
  • Example 4 Collagen Fibrosis Assay
  • Collagen (2 mg / ml, 50 ⁇ l) and VN (0.1 mg / ml, 50 ⁇ l) in TBS were added to the wells of the Iwaki 96-well assay plate. After stirring, the collagen solution in the presence or absence of VN was incubated at 37 ° C. for 2 hours to form collagen fibers. The turbidity of each well was measured every 5 minutes at 350 nm by Cytation3 (BioTek).
  • FIG. 9 shows the promotion of collagen fiber formation in VN (difference in collagen type (pig / bovine)).
  • hVN promoted the fibrosis of type I collagen derived from pigs and cattle
  • pVN promoted the fibrosis of type I collagen derived from pigs but suppressed the fibrosis of type I collagen derived from cattle.
  • FIG. 10 shows the promotion of collagen fiber formation (concentration dependence) of VN. The VN concentration was 0-100 ⁇ g / ml. Both hVN and pVN promoted the fibrosis of type I collagen derived from pigs in a concentration-dependent manner.
  • Example 5 Transmission electron micrograph of collagen fibers Collagen (2 mg / ml, 50 ⁇ l) in TBS and 0.1 mg / ml VN (labeled concentration, 50 ⁇ l) were added to the wells of the Iwaki 96-well assay plate. After stirring, the collagen solution in the presence or absence of VN was incubated at 37 ° C. for 2 hours to form fibril.
  • Collagen samples were fixed overnight at 4 ° C. with 2% paraformaldehyde (PFA) and 2% glutaraldehyde (GA) in 0.1 M cacodylic acid buffer (pH 7.4). Samples were fixed at 4 ° C. for an additional 2 hours with 1% tannic acid in 0.1 M cacodylic acid buffer (pH 7.4). After fixation, the sample was washed 4 times with 0.1 M cacodylic acid buffer for 30 minutes each, and post-fixed with 2% osmium tetroxide (OsO4) in 0.1 M cacodylic acid buffer at 4 ° C. for 3 hours.
  • PFA paraformaldehyde
  • GA glutaraldehyde
  • OsO4 osmium tetroxide
  • Samples were sampled once at 4 ° C for 30 minutes with 50% ethanol, once at 4 ° C for 30 minutes with 70% ethanol, once at room temperature for 30 minutes with 90% ethanol, and then 4 times at room temperature for 30 minutes with 100% ethanol. Dehydrated. After these dehydration operations, the samples were continuously dehydrated overnight at room temperature with 100% ethanol. The sample was replaced with 50% tert-butyl alcohol / ethanol for 1 hour and 100% tert-butyl alcohol at room temperature for 1 hour 3 times. After the replacement, the sample was frozen at 4 ° C. and vacuum dried.
  • Samples were coated with a thin layer of osmium (30 nm) using an osmium plasma coater (NL-OPC80A; Nippon Laser & Electricals Laboratory, Nagoya, Japan). Samples were observed at an acceleration voltage of 3.0 kV using a scanning electron microscope (SEM) (JSM-7500F; JEOL Ltd., Tokyo, Japan).
  • SEM scanning electron microscope
  • FIG. 11 shows a transmission electron microscope image of collagen fibers formed in the presence of VN. hVN promoted the formation of collagen fibers with few gaps in which the fibers were closely adjacent to each other.
  • Example 6 Effect of hVN sugar chains on cathepsin K activity VN was purified from human and porcine plasma by two-step heparin affinity chromatography before and after urea treatment (Yatohgo et al, Cell Struct Funct, 13, 281-). 92, 1998). Heparin-Sepharose 6B columns were prepared by reductive amination (Date et al., Methods Mol Biol. 1200: 53-67, 2014).
  • VN (500 ⁇ g) in 20 mM citric acid-phosphate buffer (pH 6.0) containing 1 mM CaCl2 and 0.5 mM PMSF with N-glucosidase (3.3 units) or Neuirainidase (16.7 units) at 37 ° C. Processed for 48 hours. After glycosidase treatment of the VN, the VN was dialyzed against 20 mM Tris-HCl buffered saline (TBS, pH 7.2). De-N-glycosylation or desialylation was confirmed by a decrease in VN molecular weight by SDS-PAGE. N-gly indicates hVN treated with N-glycosidase F. Neu indicates hVN treated with Neuirainidase. Controls were incubated without glycosidases at 37 ° C. for 48 hours.
  • the cathepsin K activity was assayed in the same manner as in Example 1.
  • the reaction time was 60 min, the VN concentration was 1 ⁇ M, and the pH was 5.5.
  • FIG. 12 shows the effect of hVN sugar chains on cathepsin K activity.
  • hVN also had a cathepsin K inhibitory effect by N-type sugar chain removal (N-gly) and sialic acid removal (Neu).
  • N-gly N-type sugar chain removal
  • Neu sialic acid removal
  • Example 7 Effect of hVN sugar chain on self-activation of procathepsin K
  • a self-activation assay of proCatK was performed in the same manner as in Example 2. The measurement was carried out at a reaction time of 10 min and a VN concentration of 2 ⁇ M (pH 4.0).
  • FIG. 13 shows the effect of hVN sugar chains on the self-activation of procathepsin K.
  • Pro-cathepsin K Pro-CatK (inactive form)
  • cathepsin K CatK (active form)
  • self-activation pre-reaction lane without VN 1, post-reaction lane 2 without VN.
  • Control-hVN inhibited self-activation of procathepsin K to cathepsin K (lane 3).
  • N-gly-hVN (lane 4) and Neu-hVN (lane 5) inhibited self-activation from procathepsin K to cathepsin K, but the effect was weaker than that of Control-hVN (lane 3).
  • Example 8 Degradation of VN by cathepsin K (effect of VN sugar chain) Cathepsin K was self-activated from procatehepsin K (5 ⁇ M) by incubation at 37 ° C. for 10 minutes in 100 mM acetate buffer (pH 4.0) containing 2.5 mM EDTA / DTT. Cathepsin K was diluted to 2, 20, 60, 200 nM in ice-cold 200 mM acetate buffer (pH 5.5) containing 2.5 mM EDTA / DTT and mixed with 4 ⁇ M VN in a 1: 1 ratio. The mixed solution was incubated at 37 ° C.
  • FIG. 14 shows the degradation of VN by cathepsin K (effect of VN sugar chain).
  • Example 9 Acid dissolution promoting action of VN (effect of VN sugar chain) Similar to Example 3, an acid solubilization assay for collagen was performed. The VN concentration was measured at 0.1 mg / ml.
  • FIG. 15 shows the acid dissolution promoting action of VN (effect of VN sugar chain). There was no difference between Control-hVN, N-glycy-hVN, and Neu-hVN in the acid dissolution promoting action of VN. From this, it was shown that there is no influence of the sugar chain of VN on the acid dissolution promoting action of VN.
  • Example 10 Promotion of collagen fiber formation in VN (effect of VN sugar chain) An assay for collagen fibrosis was performed as in Example 4. The VN concentration was measured at 0.1 mg / ml.
  • FIG. 16 shows the promotion of collagen fiber formation in VN (effect of VN sugar chain). There was no difference between Control-hVN, N-gly-hVN, and Neu-hVN in the collagen fiber formation promoting action of VN. From this, it was shown that there is no influence of the sugar chain of VN on the collagen fiber formation promoting action of VN.
  • Example 11 Prediction of VN cathepsin K binding site by data analysis
  • the three-dimensional structure of VN has been partially determined, and the total length has not been elucidated. This indicates that the VN has parts that do not have a fixed three-dimensional structure (disordered regions that do not usually have a fixed structure, hereinafter also referred to as disordered regions).
  • the disordered region stabilizes from a non-structured structure to an ordered and fixed structure by binding to other molecules (Yura, K. & Hayward, S. The interwinding nature of protein-protein interfaces and its implication for protein complex formation. Bioinformatics 25, 3108-3113, doi: 10.1093 / bioinformatics / btp563 (2009).).
  • the binding site of VN to cathepsin K exists in the disordered region.
  • Disordered regions were calculated by four methods: PONDR, DisEMBL, PrDOS, and FolderIndex to determine the binding site of VN to cathepsin K.
  • PrDOS Protein DisOrder prediction System http://prdos.hgc.jp/cgi-bin/top.cgi Ishida, T. & Kinoshita, K. PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res 35, W460-464, doi: 10.1093 / nar / gkm363 (2007).
  • FoldIndex https://fold.weizmann.ac.il/ Prilusky, J. et al. FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21, 3435-3438, doi: 10.1093 / bioinformatics / bti537 (2005).
  • NCBI protein sequence EAW510821.1 SEQ ID NO: 1
  • the disordered region of hVN was amino acid SEQ ID NOs: 100 to 130 and 292 to 296 to 364 to 397 shown by the broken line in FIG.
  • the area of the broken line is drawn based on UniProt entry P04004 (UniProt Consortium, T. UniProt: the universal protein knowledgebase. Nucleic Acids Res 46, 2699, doi: 10.1093 / nar / gky092 (2016)).
  • the black line is Protein Databank (Kinjo, A. R. et al. Protein Data Bank Japan (PDBj): updated user interfaces, resource description framework, analysis tools for large structures.
  • Nucleic Acids Res 45, D282- It is a region with a three-dimensional structure known in nar / gkw962 (2017)).
  • the N-type sugar chain binding sites located at Asn86, 169, and 242 show a schematic but most abundant N-type sugar chain structure in hVN.
  • the cathepsin K binding site of VN is the amino acid sequence preservation region of hVN and pVN.
  • HVN and pVN amino acids by ClustalW (ver. 2.1) (Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948, doi: 10.1093 / bioinformatics / btm404 (2007))
  • ClustalW ver. 2.1
  • the broken line in FIG. 18 points to the same amino acid sequence as the broken line in FIG. 17, and is a calculated disordered region.
  • the amino acid sequence number of hVN shown by the broken line in FIG. 18 was also conserved in the amino acid sequence of pVN. From these results, it was shown that the disordered region corresponding to the binding site of VN to cathepsin K is amino acid SEQ ID NOs: 100 to 130 and 292 to 296 to 364 to 397 of hVN.
  • the black line in FIG. 18 points to the same amino acid sequence as the black line in FIG. 17, and is a region having a three-dimensional structure known in Protein Databank. Amino acid SEQ ID NOs: 100 to 130 and 292 to 296 to 396 to 397 of hVN are shown in SEQ ID NOs: 3 to 5, respectively.
  • the accuracy of the calculation of the disordered region is 95% or more.
  • the false positive rate of the four methods PONDR, DisEMBL, PrDOS, and FoundIndex (False positive rate. In this case, the percentage of residues that were calculated as the disturbed region but not actually the disturbed region) is as follows: It's a street. PONDR 22% DisEMBL 19% PrDOS 5% FolderIndex 10%
  • the binding site of vitronectin to cathepsin K is amino acid SEQ ID NOs: 100-130, 292-296, 364-397 of human vitronectin (hVN), which is a disordered region that does not normally have a fixed structure. It was expected to be.
  • the amino acid sequence numbers (100-130, 292-296, 364-397) of these human vitronectins (hVN) are also conserved in pig VN (pVN) and are considered to be active sites that inhibit cathepsin K. Be done.

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Abstract

The present invention addresses the problem of providing a novel cathepsin K inhibitor. According to the present invention, provided are: a cathepsin K inhibitor including vitronectin, or a fragment thereof that binds to cathepsin K; an inhibitor of procathepsin K self-activation; an agent for accelerating fibrotic collagen dissolution; and an agent for accelerating collagen fiber formation.

Description

カテプシンＫ阻害剤Cathepsin K inhibitor
　本発明は、ビトロネクチン、又はカテプシンＫと結合するその断片を含むカテプシンＫ阻害剤に関する。 The present invention relates to a cathepsin K inhibitor comprising vitronectin or a fragment thereof that binds to cathepsin K.
　高齢化に伴い骨粗鬆症の患者が増加している。骨粗鬆症は、破骨細胞による骨の分解と吸収（骨吸収）と、骨芽細胞による骨形成とのバランスが崩れ、骨吸収が過剰になり、骨がもろくなる疾患である。骨粗鬆症に対する薬物療法に際しては骨吸収阻害薬（例えば、カルシトニン製剤、エストロゲン製剤、ビタミンＫ製剤、ビスホスホネート製剤等）を用いるのが主流である。しかし、骨吸収阻害薬は、骨形成も阻害するという問題がある。そのため、骨形成を阻害せず骨吸収のみを阻害する薬の開発が求められている。 The number of patients with osteoporosis is increasing with the aging of the population. Bone osteoporosis is a disease in which the balance between bone decomposition and resorption (bone resorption) by osteoblasts and bone formation by osteoblasts is lost, and bone resorption becomes excessive and the bone becomes brittle. Bone resorption inhibitors (for example, calcitonin preparations, estrogen preparations, vitamin K preparations, bisphosphonate preparations, etc.) are mainly used for drug therapy for osteoporosis. However, bone resorption inhibitors also have the problem of inhibiting bone formation. Therefore, there is a need for the development of a drug that does not inhibit bone formation but inhibits only bone resorption.
  カテプシンＫは、システインプロテアーゼのパパインスーパーファミリーの一部であるシステインカテプシンファミリーの酵素の１つである。カテプシンＫは、破骨細胞から前駆体プロカテプシンＫとして分泌され、酸性下で自己活性化し、骨の主成分であるＩ型コラーゲンを分解するプロテアーゼである。カテプシンＫ阻害剤は、骨の分解のみを阻害し、骨形成をほとんど阻害しないため、骨粗鬆症治療薬として有望である。 Cathepsin K is one of the enzymes of the cysteine cathepsin family, which is part of the papain superfamily of cysteine proteases. Cathepsin K is a protease that is secreted from osteoclasts as a precursor procatehepsin K, self-activates under acidic conditions, and decomposes type I collagen, which is the main component of bone. Cathepsin K inhibitors are promising as therapeutic agents for osteoporosis because they inhibit only bone decomposition and hardly inhibit bone formation.
　ビトロネクチン（ＶＮ）は、主に肝臓で産生され、細胞外マトリックスや血液中に存在し、複数のリガンド結合部位をもつ多機能な接着性糖タンパク質である。ヒトＶＮは３本の複合型Ｎ－型糖鎖をもち、哺乳類間でＮ－型糖鎖結合部位は高く保存されている（非特許文献１～４）。ＶＮは、コラーゲンと強く相互作用する（非特許文献５及び６）。ＶＮの糖鎖は細胞の接着進展に影響を与えるため、骨芽細胞や破骨細胞の接着進展に影響を与えることが考えられ（非特許文献７）、ＶＮは骨粗鬆症において重要な役割を担うと考えられる。ＶＮペプチドが骨形成細胞の接着・伸展を促進し、卵巣摘出の骨粗鬆症モデルラットで有効であることなどが報告されている（非特許文献８）。しかしながら、ＶＮのカテプシンへの影響については不明である。 Vitronectin (VN) is a multifunctional adhesive glycoprotein that is mainly produced in the liver, is present in the extracellular matrix and blood, and has multiple ligand binding sites. Human VN has three complex N-type sugar chains, and the N-type sugar chain binding site is highly conserved among mammals (Non-Patent Documents 1 to 4). VN strongly interacts with collagen (Non-Patent Documents 5 and 6). Since the sugar chain of VN affects the adhesion progress of cells, it is considered that it affects the adhesion progress of osteoblasts and osteoclasts (Non-Patent Document 7), and VN plays an important role in osteoporosis. Conceivable. It has been reported that the VN peptide promotes the adhesion and extension of bone-forming cells and is effective in ovariectomized osteoporosis model rats (Non-Patent Document 8). However, the effect of VN on cathepsins is unknown.
　本発明は、新規なカテプシンＫ阻害剤を提供することを解決すべき課題とする。 The present invention is a problem to be solved to provide a novel cathepsin K inhibitor.
　本発明者らは、上記課題を解決するために鋭意検討した結果、ビトロネクチンが、カテプシンＫ活性阻害作用、プロカテプシンＫ自己活性化阻害作用、並びにコラーゲン酸溶解促進作用という、破骨細胞での骨吸収を抑制する３つの作用を有することを見出した。さらに本発明者らは、ビトロネクチンが、骨形成細胞において産生されるコラーゲンの線維形成を促進する作用を有することを見出した。さらに本発明者らは、ビトロネクチンのカテプシンＫへの結合部位は、普段定まった構造を取らないdisordered regions（ヒトビトロネクチン（ｈＶＮ）のアミノ酸配列番号１００－１３０、２９２－２９６、３６４－３９７）であることを見出した。本発明は上記の知見に基づいて完成したものである。 As a result of diligent studies to solve the above problems, the present inventors have found that vitronectin has a cathepsin K activity inhibitory action, a cathepsin K self-activation inhibitory action, and a collagen acid lysis promoting action, which are bones in osteoclasts. It has been found to have three actions of suppressing absorption. Furthermore, the present inventors have found that vitronectin has an action of promoting the fibrosis of collagen produced in bone-forming cells. Furthermore, we present that the binding site of vitronectin to cathepsin K is disordered regions (amino acid SEQ ID NOs: 100-130, 292-296, 364-397 of human vitronectin (hVN)) that do not normally have a fixed structure. I found that. The present invention has been completed based on the above findings.
　すなわち、本発明によれば、以下の発明が提供される。
＜１＞　ビトロネクチン、又はカテプシンＫと結合するその断片を含む、カテプシンＫ阻害剤。
＜２＞　ビトロネクチン、又はカテプシンＫと結合するその断片を含む、プロカプテシンＫの自己活性化阻害剤。
＜３＞　ビトロネクチン、又はカテプシンＫと結合するその断片を含む、線維化コラーゲンの溶解促進剤。
＜４＞　ビトロネクチン、又はカテプシンＫと結合するその断片を含む、コラーゲンの線維形成の促進剤。
＜５＞　破骨細胞の骨吸収を抑制するために使用する、＜１＞から＜３＞の何れか一に記載の剤。
＜６＞　ビトロネクチンがヒトビトロネクチン又はブタビトロネクチンである、＜１＞から＜５＞の何れか一に記載の剤。
＜７＞　ビトロネクチンが、Ｎ－型糖鎖を有している、＜１＞から＜６＞の何れか一に記載の剤。 That is, according to the present invention, the following invention is provided.
<1> A cathepsin K inhibitor comprising vitronectin or a fragment thereof that binds to cathepsin K.
<2> A self-activation inhibitor of procaptesin K, which comprises vitronectin or a fragment thereof that binds to cathepsin K.
<3> A lysis promoter for fibrotic collagen, which comprises vitronectin or a fragment thereof that binds to cathepsin K.
<4> An agent for promoting collagen fibrosis, which comprises a fragment thereof that binds to vitronectin or cathepsin K.
<5> The agent according to any one of <1> to <3>, which is used for suppressing bone resorption of osteoclasts.
<6> The agent according to any one of <1> to <5>, wherein the vitronectin is human vitronectin or porcine vitronectin.
<7> The agent according to any one of <1> to <6>, wherein the vitronectin has an N-type sugar chain.
　本発明によればさらに、
　ビトロネクチン、又はカテプシンＫと結合するその断片を対象に投与することを含む、カテプシンＫを阻害する方法；
　ビトロネクチン、又はカテプシンＫと結合するその断片を対象に投与することを含む、プロカプテシンＫの自己活性化を阻害する方法；
　ビトロネクチン、又はカテプシンＫと結合するその断片を対象に投与することを含む、線維化コラーゲンの溶解を促進する方法；並びに、
　ビトロネクチン、又はカテプシンＫと結合するその断片を対象に投与することを含む、コラーゲンの線維形成を促進する方法；
が提供される。 Further according to the present invention
Methods of inhibiting cathepsin K, comprising administering to the subject a fragment of vitronectin, or a fragment thereof that binds to cathepsin K;
Methods of Inhibiting Self-Activation of Procaptesin K, Containing Administration of Vitronectin, or Fragments thereof that Bind Cathepsin K, to Subjects;
Methods of promoting the lysis of fibrotic collagen, including administering to the subject a fragment of vitronectin, or a fragment thereof that binds to cathepsin K;
Methods of promoting collagen fibrosis, comprising administering to the subject a fragment of vitronectin, or a fragment thereof that binds to cathepsin K;
Is provided.
　本発明によればさらに、
　カテプシンＫを阻害する処置において使用するためのビトロネクチン、又はカテプシンＫと結合するその断片；
　プロカプテシンＫの自己活性化を阻害する処置において使用するためのビトロネクチン、又はカテプシンＫと結合するその断片；
　線維化コラーゲンの溶解を促進する処置において使用するためのビトロネクチン、又はカテプシンＫと結合するその断片；並びに
　コラーゲンの線維形成を促進する処置において使用するためのビトロネクチン、又はカテプシンＫと結合するその断片；
が提供される。 Further according to the present invention
Vitronectin for use in treatments that inhibit cathepsin K, or a fragment thereof that binds to cathepsin K;
Vitronectin, or a fragment thereof that binds to cathepsin K, for use in treatments that inhibit the self-activation of procaptesin K;
Vitronectin or a fragment thereof that binds to cathepsin K for use in a procedure that promotes lysis of fibrotic collagen; and a fragment thereof that binds to vitronectin or cathepsin K for use in a procedure that promotes fibrosis of collagen;
Is provided.
　本発明によればさらに、
　カテプシンＫ阻害剤の製造のための、ビトロネクチン、又はカテプシンＫと結合するその断片の使用；
　プロカプテシンＫの自己活性化阻害剤の製造のための、ビトロネクチン、又はカテプシンＫと結合するその断片の使用；
　線維化コラーゲンの溶解促進剤の製造のための、ビトロネクチン、又はカテプシンＫと結合するその断片の使用；並びに
　コラーゲンの線維形成の促進剤の製造のための、ビトロネクチン、又はカテプシンＫと結合するその断片の使用；
が提供される。 Further according to the present invention
Use of vitronectin, or a fragment thereof that binds to cathepsin K, for the production of cathepsin K inhibitors;
Use of vitronectin, or a fragment thereof that binds to cathepsin K, for the production of self-activation inhibitors of procaptesin K;
Use of a fragment thereof that binds to vitronectin or cathepsin K for the production of a lysis promoter of fibrotic collagen; and a fragment thereof that binds to vitronectin or cathepsin K for the production of a promoter of collagen fibrosis. Use of;
Is provided.
　本発明によれば、新規なカテプシンＫ阻害剤が提供される。本発明のカテプシンＫ阻害剤は、骨粗鬆症治療薬として有用である。 According to the present invention, a novel cathepsin K inhibitor is provided. The cathepsin K inhibitor of the present invention is useful as a therapeutic agent for osteoporosis.
図１は、様々なｐＨにおけるＶＮのカテプシンＫ活性阻害を示す。FIG. 1 shows inhibition of VN cathepsin K activity at various pHs. 図２は、ＶＮのカテプシンＫ活性阻害（ｐＨ，％）（反応時間３０ｍｉｎのデータ）を示す。FIG. 2 shows inhibition of VN cathepsin K activity (pH,%) (data with a reaction time of 30 min). 図３は、ＶＮのカテプシンＫ活性阻害（濃度依存性）（反応時間３０ｍｉｎのデータ）を示す。FIG. 3 shows inhibition of VN cathepsin K activity (concentration-dependent) (data with a reaction time of 30 min). 図４は、ｐＶＮのカテプシンＫ活性阻害（拮抗阻害）（反応時間３０ｍｉｎのデータ）を示す。FIG. 4 shows inhibition of cathepsin K activity (competitive inhibition) of pVN (data with a reaction time of 30 min). 図５は、様々なｐＨにおけるｈＶＮのプロカテプシンＫの自己活性化阻害を示す。FIG. 5 shows the inhibition of self-activation of hVN procathepsin K at various pHs. 図６は、コラーゲン酸溶解におけるＶＮの影響を示す。FIG. 6 shows the effect of VN on collagen acid dissolution. 図７は、コラーゲン酸溶解におけるｐＶＮ／ｈＶＮの影響（反応時間２０ｍｉｎのデータ）を示す。FIG. 7 shows the effect of pVN / hVN on collagen acid dissolution (data with a reaction time of 20 min). 図８は、コラーゲン酸溶解におけるｐＶＮ／ｈＶＮの影響（反応時間２０ｍｉｎのデータ）を示す。FIG. 8 shows the effect of pVN / hVN on collagen acid dissolution (data with a reaction time of 20 min). 図９は、ＶＮのコラーゲン線維形成促進（コラーゲンの種類（ブタ・ウシ）の差）を示す。FIG. 9 shows the promotion of collagen fiber formation in VN (difference in collagen type (pig / bovine)). 図１０は、ＶＮのコラーゲン線維形成促進（濃度依存性）を示す。FIG. 10 shows the promotion of collagen fiber formation (concentration dependence) of VN. 図１１は、ＶＮ存在下で形成されたコラーゲン線維の透過型電子顕微鏡画像を示す。FIG. 11 shows a transmission electron micrograph of collagen fibers formed in the presence of VN. 図１２は、カテプシンＫ活性におけるｈＶＮ糖鎖の影響を示す。FIG. 12 shows the effect of hVN sugar chains on cathepsin K activity. 図１３は、プロカテプシンＫの自己活性化におけるｈＶＮ糖鎖の影響を示す。FIG. 13 shows the effect of hVN sugar chains on the self-activation of procathepsin K. 図１４は、カテプシンＫによるＶＮの分解（ＶＮ糖鎖の影響）を示す。FIG. 14 shows the degradation of VN by cathepsin K (effect of VN sugar chain). 図１５は、ＶＮの酸溶解促進作用（ＶＮ糖鎖の影響）を示す。FIG. 15 shows the acid dissolution promoting action of VN (effect of VN sugar chain). 図１６は、ＶＮのコラーゲン線維形成促進（ＶＮ糖鎖の影響）を示す。FIG. 16 shows the promotion of collagen fiber formation in VN (effect of VN sugar chain). 図１７は、ｈＶＮと普段定まった構造を取らないdisordered regions予測の図を示す。FIG. 17 shows a diagram of disordered regions prediction that does not normally have a fixed structure with hVN. 図１８は、ｈＶＮとｐＶＮの多重整列を示す。FIG. 18 shows a multiple alignment of hVN and pVN.
　以下、本発明について更に具体的に説明する。
　本発明は、ビトロネクチン、又はカテプシンＫと結合するその断片を含む、カテプシンＫ阻害剤、プロカプテシンＫの自己活性化阻害剤、線維化コラーゲンの溶解促進剤、並びにコラーゲンの線維形成の促進剤に関する。
　本明細書の実施例に示す通り、ビトロネクチンは、カテプシンＫの阻害作用、プロカプテシンＫの自己活性化阻害作用、及び線維化コラーゲンの溶解促進作用を有することが実証され、特にカテプシンＫを選択的に阻害することができる。ビトロネクチンは、これらの作用を有することから、本発明の上記剤は、破骨細胞の骨吸収を抑制するために使用することができる。 Hereinafter, the present invention will be described in more detail.
The present invention relates to a cathepsin K inhibitor, a self-activation inhibitor of procaptesin K, a fibrotic collagen lysis promoter, and a collagen fibrosis promoter, which comprises a fragment thereof that binds to vitronectin or cathepsin K.
As shown in the examples of the present specification, vitronectin has been demonstrated to have an inhibitory effect on cathepsin K, an inhibitory effect on self-activation of procapthecin K, and a lysis promoting effect on fibrotic collagen, and particularly selectively cathepsin K. Can be inhibited. Since vitronectin has these effects, the above-mentioned agent of the present invention can be used to suppress bone resorption of osteoclasts.
　本発明で用いるビトロネクチンの由来は特に限定されないが、好ましくは哺乳動物由来のビトロネクチンを使用することができ、例えば、ヒトビトロネクチン又はブタビトロネクチンを使用することができる。ヒトビトロネクチンは４７８アミノ酸から構成され、動物種の違いにより、３８８～６５５アミノ酸から構成されている。ヒトビトロネクチンのアミノ酸配列は、NCBI Reference Sequence: NP_000629.3に登録されており、ブタビトロネクチンのアミノ酸配列は、NCBI Reference Sequence: NP_999269.1に登録されている。
　ヒト等の生体内で生産される天然ビトロネクチンは、アミノ酸の一部に糖鎖（Ｎ－型糖鎖）が結合した糖タンパク質である。本発明においては、Ｎ－型糖鎖が結合しているビトロネクチンを使用することが好ましい。さらに、末端にシアル酸が付加した複合型Ｎ－型糖鎖が複数結合しているビトロネクチンを使用することが好ましい。 The origin of the vitronectin used in the present invention is not particularly limited, but a mammalian-derived vitronectin can be preferably used, and for example, human vitronectin or porcine vitronectin can be used. Human vitronectin is composed of 478 amino acids, and is composed of 388 to 655 amino acids depending on the animal species. The amino acid sequence of human vitronectin is registered in NCBI Reference Sequence: NP_000629.3, and the amino acid sequence of porcine vitronectin is registered in NCBI Reference Sequence: NP_999269.1.
Natural vitronectin produced in a living body such as human is a glycoprotein in which a sugar chain (N-type sugar chain) is bound to a part of amino acids. In the present invention, it is preferable to use vitronectin to which an N-type sugar chain is bound. Further, it is preferable to use vitronectin to which a plurality of complex N-type sugar chains to which sialic acid is added at the end are bound.
　ビトロネクチンとは、ビトロネクチンレセプターを保有する細胞に対して接着活性を持つ４７８アミノ酸残基からなるタンパク質である。ビトロネクチンは、本発明の効果を奏する限り、部分配列を欠失したものであってもよい。ビトロネクチンは、Ｎ末端からソマトメジンＢ領域、コネクティング領域、ヘモペキシン領域から成り、本発明の効果を奏する限り、そのうちの何れか１以上の領域からなる断片、又はペプチド断片であってもよい（Yoneda A et al., Biochemistry 1998, 37, 18, 6351-6360）。具体的には、Ｎ末端側にある２０アミノ酸からなるシグナルドメインが欠失した組み換えビトロネクチン；Ｃ末端部分（３９９番目のアミノ酸～４７８番目のアミノ酸配列部分）が欠失した組み換えビトロネクチンなどでもよい。 Vitronectin is a protein consisting of 478 amino acid residues having adhesive activity to cells carrying the vitronectin receptor. Vitronectin may be a partial sequence deletion as long as it exhibits the effect of the present invention. Vitronectin is composed of a somatomedin B region, a connecting region, and a hemopexin region from the N-terminal, and may be a fragment consisting of any one or more of the regions or a peptide fragment as long as the effect of the present invention is exhibited (Yoneda A et). al., Biochemistry 1998, 37, 18, 6351-6360). Specifically, recombinant vitronectin in which the signal domain consisting of 20 amino acids on the N-terminal side is deleted; recombinant vitronectin in which the C-terminal portion (the 399th amino acid to the 478th amino acid sequence portion) is deleted may be used.
　ビトロネクチンは、天然由来のビトロネクチン、遺伝子組み換え技術で作製した組み換えビトロネクチン、又は化学合成したビトロネクチンの何れでもよい。市販品としては、例えば、ビトロネクチン，ヒト血漿由来（富士フイルム和光純薬工業（株）製）、ｓｙｎｔｈｅｍａｘ（Ｃｏｒｎｉｎｇ  Ｉｎｃｏｒｐｏｒａｔｅｄ製）、Ｖｉｔｒｏｎｅｃｔｉｎ（ＶＴＮ－Ｎ）（Ｔｈｅｒｍｏ  Ｆｉｓｈｅｒ  Ｓｃｉｅｎｔｉｆｉｃ製）などを使用することもできる。 The vitronectin may be either naturally-derived vitronectin, recombinant vitronectin produced by genetically modified technology, or chemically synthesized vitronectin. As commercially available products, for example, vitronectin, human plasma-derived (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Inc.), Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific), and the like can also be used. can.
　本発明においては、カテプシンＫと結合する、ビトロネクチンの断片を使用することもできる。カテプシンＫと結合する、ビトロネクチンの断片としては、例えば、ヒトビトロネクチン（ｈＶＮ）の場合には、ｈＶＮのアミノ酸配列番号１００－１３０（配列番号３）、２９２－２９６（配列番号４）、または３６４－３９７（配列番号５）のうちの何れか少なくとも1以上を含む断片を挙げることができる。 In the present invention, a fragment of vitronectin that binds to cathepsin K can also be used. Fragments of vitronectin that bind to cathepsin K include, for example, in the case of human vitronectin (hVN), amino acid SEQ ID NOs: 100-130 (SEQ ID NO: 3), 292-296 (SEQ ID NO: 4), or 364 of hVN. Fragments containing at least one of 397 (SEQ ID NO: 5) can be mentioned.
　本発明によるカテプシンＫ阻害剤、プロカプテシンＫの自己活性化阻害剤、線維化コラーゲンの溶解促進剤、及びコラーゲンの線維形成の促進剤（以下、本発明の薬剤という）は、例えば、ロコモティブシンドローム（運動器症候群）、骨粗鬆症、変形性骨関節症、慢性関節リウマチ、骨パジェット病、高カルシウム血症、癌の骨転移、骨痛、線維性疾患、動脈硬化症、又は皮膚筋炎症の治療又は予防のために使用することができる。 The catepsin K inhibitor, the self-activation inhibitor of procaptesin K, the lysis promoter of fibrotic collagen, and the fibrosis promoter of collagen (hereinafter referred to as the agent of the present invention) according to the present invention are, for example, locomotive syndrome (exercise). Treatment or prevention of organ syndrome), osteoporosis, osteoarthritis osteoarthritis, rheumatoid arthritis, bone Paget's disease, hypercalcemia, bone metastasis of cancer, bone pain, fibrous disease, arteriosclerosis, or cutaneous muscle inflammation Can be used for.
　本発明の薬剤は、有効成分であるビトロネクチン、又はカテプシンＫと結合するその断片と、１又は２以上の製剤学的に許容される製剤用添加物とを含む、試薬組成物又は医薬組成物の形態で提供されてもよい。 The agent of the present invention is a reagent composition or a pharmaceutical composition comprising the active ingredient vitronectin or a fragment thereof that binds to cathepsin K and one or more pharmaceutically acceptable pharmaceutical additives. It may be provided in the form.
　本発明の薬剤の製造に用いられる製剤用添加物の種類は特に限定されず、当業者が適宜選択可能である。例えば、賦形剤、崩壊剤又は崩壊補助剤、結合剤、滑沢剤、コーティング剤、基剤、溶解剤又は溶解補助剤、分散剤、懸濁剤、乳化剤、緩衝剤、抗酸化剤、防腐剤、等張化剤、ｐＨ調節剤、溶解剤、安定化剤などを用いることができ、これらの目的で使用される個々の具体的成分は当業者に周知されている。 The type of pharmaceutical additive used for producing the drug of the present invention is not particularly limited and can be appropriately selected by those skilled in the art. For example, excipients, disintegrants or disintegrant aids, binders, lubricants, coating agents, bases, solubilizers or solubilizers, dispersants, suspensions, emulsifiers, buffers, antioxidants, preservatives. Agents, tonicity agents, pH regulators, solubilizers, stabilizers and the like can be used, and the individual specific ingredients used for these purposes are well known to those skilled in the art.
  注射剤又は点滴用製剤は、ビトロネクチン、又はカテプシンＫと結合するその断片が溶剤に溶解、懸濁または乳化された液剤であってもよく、あるいは使用時に溶剤に溶解、懸濁または乳化される凍結乾燥製剤などの固形剤であってもよい、溶剤としては、注射用蒸留水、生理食塩水、植物油、プロピレングリコール、ポリエチレングリコール、エタノールのようなアルコール類等、またはこれらの組み合わせなどが例示される。 The injectable or drip-form may be a solution in which a fragment thereof that binds to vitronectin or catepsin K is dissolved, suspended or emulsified in a solvent, or is frozen in which it is dissolved, suspended or emulsified in a solvent at the time of use. Examples of the solvent, which may be a solid agent such as a dry preparation, include distilled water for injection, physiological saline, vegetable oil, alcohols such as propylene glycol, polyethylene glycol, and ethanol, or a combination thereof. ..
　本発明の薬剤は、種々の形態で投与することができるが、好適な投与形態としては、経口投与でも非経口投与（例えば、静脈内、筋肉内、皮下又は皮内等への注射、直腸内投与、経粘膜投与など）でもよいが、非経口投与が好ましい。 The agent of the present invention can be administered in various forms, and suitable administration forms include oral administration and parenteral administration (for example, intravenous, intramuscular, subcutaneous or intradermal injection, intrarectal injection, etc.). Administration, transmucosal administration, etc.) may be used, but parenteral administration is preferable.
　経口投与に適する医薬組成物としては、例えば、錠剤、顆粒剤、カプセル剤、散剤、溶液剤、懸濁剤、シロップ剤などを挙げることができ、非経口投与に適する医薬組成物としては、例えば、注射剤、点滴剤、坐剤、経皮吸収剤などを挙げることができるが、これらに限定されることはない。 Examples of the pharmaceutical composition suitable for oral administration include tablets, granules, capsules, powders, solutions, suspensions, syrups and the like, and examples of the pharmaceutical composition suitable for parenteral administration include tablets, granules, capsules, powders, solutions and syrups. , Injections, infusions, suppositories, transdermal absorbents and the like, but are not limited thereto.
  本発明の薬剤の投与対象としては、哺乳動物、例えば、ヒト、およびサル、ヒツジ、ウシ、ウマ、イヌ、ネコ、ウサギ、ラット、マウス等の非ヒト動物が挙げられる。本発明の薬剤の投与対象はヒト（好ましくは患者）である。 The administration target of the agent of the present invention includes mammals, for example, humans and non-human animals such as monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, and mice. The administration target of the agent of the present invention is a human (preferably a patient).
　本発明の薬剤の投与量は患者の年齢、性別、体重、症状、及び投与経路などの条件に応じて適宜増減されるべきであるが、一般的には、成人一日あたりの有効成分の量として１μg／ｋｇから１，０００ｍｇ／ｋｇ程度の範囲であり、好ましくは１０μｇ／ｋｇから１００ｍｇ／ｋｇ程度の範囲である。上記投与量の薬剤は一日一回に投与してもよいし、数回（例えば、２～４回程度）に分けて投与してもよい。 The dose of the agent of the present invention should be appropriately increased or decreased according to conditions such as the age, sex, weight, symptoms, and route of administration of the patient, but generally, the amount of the active ingredient per adult day. It is in the range of about 1 μg / kg to about 1,000 mg / kg, preferably in the range of about 10 μg / kg to about 100 mg / kg. The drug having the above dose may be administered once a day, or may be administered in several divided doses (for example, about 2 to 4 doses).
　以下の実施例により本発明を具体的に説明するが、本発明は実施例によって限定されることはない。 The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the examples.
　実施例における表記の意味を以下に示す。
ＣａｔＫ：カテプシンＫ
ＶＮ：ビトロネクチン
ｐＶＮ：ブタビトロネクチン（血漿からヘパリンアフィニティークロマトグラフィーによって精製したもの）
ｈＶＮ：ヒトビトロネクチン（血漿からヘパリンアフィニティークロマトグラフィーによって精製したもの） The meanings of the notations in the examples are shown below.
CatK: Cathepsin K
VN: Vitronectin pVN: Buta Vitronectin (purified from plasma by heparin affinity chromatography)
hVN: Human vitronectin (purified from plasma by heparin affinity chromatography)
実施例１：カテプシンＫ活性のアッセイ
　ＣａｔＫ活性は、９６ウエルプレート中で蛍光基質を使用して測定した（Ａｄｅｌｅｋｅ　Ｈ　ｅｔ　ａｌ。、ＰＮＡＳ、１１１（４９）２０１４、１７４７４－７９、Ｐｅｔｅｒ　Ａ．、Ｊ　Ｂｉｏｌ　Ｃｈｅｍ。、２８９（３１）２１５６２－２１５７２）。基質としてのＺ－Ｐｈｅ－Ａｒｇ－ＡＭＣ（Ｅｎｚｏ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅ）は２ｍｇ／ｍｌでＤＭＳＯに溶解し、使用前は－８０℃で保存した。プロカテプシンＫ（５μＭ）（Ｅｎｚｏ　Ｌｉｆｅ　Ｓｃｉｅｎｃｅ）は、２．５ｍＭ　ＥＤＴＡ／ＤＴＴを含む１００ｍＭ酢酸緩衝液（ｐＨ４．０）で３７℃で３０分間自己活性化してカテプシンＫにした後に、５ｍＭ　ＥＤＴＡ／ＤＴＴで４ｎＭに希釈した。反応混合物１００μｌ（２．５ｍＭ　ＥＤＴＡ／ＤＴＴを含む酢酸バッファー（ｐＨ４．０－６．０）またはＴｒｉｓ－ＨＣｌバッファー（ｐＨ７．２）中の２μＭ　ＶＮ、１０μＭ　Ｚ－Ｐｈｅ－Ａｒｇ－ＡＭＣおよび０．５ｎＭ　ＣａｔＫ）を９６ウエルアッセイプレートに添加し、３７℃で１８０分間インキュベートした。Ｃｙｔａｔｉｏｎ３（ＢｉｏＴｅｋ）を使用して３８０ｎｍで励起し、４６０ｎｍで蛍光発光測定を行った。 Example 1: Assay for Cathepsin K Activity CatK activity was measured using a fluorescent substrate in a 96-well plate (Adeleke Het al., PNAS, 111 (49) 2014, 17474-79, Peter A., J. Biol Chem. 289 (31) 21562-21572). Z-Phe-Arg-AMC (Enzo Life Science) as a substrate was dissolved in DMSO at 2 mg / ml and stored at -80 ° C before use. Procathepsin K (5 μM) (Enzo Life Science) was self-activated in 100 mM acetate buffer (pH 4.0) containing 2.5 mM EDTA / DTT at 37 ° C. for 30 minutes to cathepsin K, followed by 5 mM EDTA / DTT. Diluted to 4 nM. 2 μM VN, 10 μM Z-Phe-Arg-AMC and 0.5 nM in 100 μl of the reaction mixture (acetic acid buffer (pH 4.0-6.0) containing 2.5 mM EDTA / DTT) or Tris-HCl buffer (pH 7.2). CatK) was added to the 96-well assay plate and incubated at 37 ° C. for 180 minutes. Excitation was performed at 380 nm using Cytation3 (BioTek) and fluorescence emission measurements were performed at 460 nm.
　図１は、様々なｐＨにおけるＶＮのカテプシンＫ活性阻害を示す。ｐＶＮ（＋）はどのｐＨにおいてもカテプシンＫ活性を著しく阻害した。（－）はｐＶＮなしを示す。
　図２は、ＶＮのカテプシンＫ活性阻害（ｐＨ，％）（反応時間３０ｍｉｎのデータ）を示す。ｐＶＮ、ｈＶＮは、ｐＨ４－７．２においてカテプシンＫ活性を阻害した。
　図３は、ＶＮのカテプシンＫ活性阻害（濃度依存性）（反応時間３０ｍｉｎのデータ）を示す。ＶＮ濃度は０～２ｎＭ（ｐＨ５．５）で測定した。ｐＶＮ、ｈＶＮは、ｐＨ５．５においてカテプシンＫ活性を濃度依存的に阻害した。ＩＣ５０値がｐＶＮの方が小さいことから、ｐＶＮの方がｈＶＮよりも、カテプシンＫ阻害作用が少し高い結果となった。
　図４は、ｐＶＮのカテプシンＫ活性阻害（拮抗阻害）（反応時間３０ｍｉｎのデータ）を示す。ＶＮ濃度は１５．６３、３１．２５、６２．５、１２５、２５０ｎＭ（ｐＨ５．５）で測定した。ＳはＺ－Ｐｈｅ－Ａｒｇ－ＡＭＣ濃度（μＭ）を示す。ｐＶＮ、ｈＶＮによるｐＨ５．５におけるカテプシンＫ活性阻害は、拮抗阻害であることが示された。 FIG. 1 shows inhibition of VN cathepsin K activity at various pHs. pVN (+) markedly inhibited cathepsin K activity at any pH. (-) Indicates no pVN.
FIG. 2 shows inhibition of VN cathepsin K activity (pH,%) (data with a reaction time of 30 min). pVN and hVN inhibited cathepsin K activity at pH 4-7.2.
FIG. 3 shows inhibition of VN cathepsin K activity (concentration-dependent) (data with a reaction time of 30 min). The VN concentration was measured at 0 to 2 nM (pH 5.5). pVN and hVN inhibited cathepsin K activity at pH 5.5 in a concentration-dependent manner. Since the IC50 value was smaller in pVN, the result was that pVN had a slightly higher cathepsin K inhibitory effect than hVN.
FIG. 4 shows inhibition of cathepsin K activity (competitive inhibition) of pVN (data with a reaction time of 30 min). The VN concentration was measured at 15.63, 31.25, 62.5, 125, 250 nM (pH 5.5). S indicates the Z-Phe-Arg-AMC concentration (μM). Inhibition of cathepsin K activity at pH 5.5 by pVN and hVN was shown to be competitive inhibition.
実施例２：ｐｒｏＣａｔＫの自己活性化アッセイ
　２．５ｍＭ　ＥＤＴＡ／ＤＴＴを含む１００ｍＭ酢酸緩衝液（ｐＨ４．０－５．５）中のＰｒｏＣａｔＫ（３μＭ）を、ＶＮの存在下又は非存在下において３７℃でインキュベートした。３７℃で１０分間または３０分間のインキュベーション後、２ｘＳＤＳ－ＰＡＧＥサンプルバッファーをすぐに添加し、９８℃で１０分間加熱した。タンパク質サンプルを、２－メルカプトエタノールを含む１５％Ｔｒｉｓ－グリシンゲルを使用して電気泳動し、ＥｚＳｔａｉｎ　Ｓｉｌｖｅ（ＡＥ－１３６０、ＡＴＴＯ）で染色した。ｐｒｏＣａｔＫとＣａｔＫの両方の総バンド強度は、ＩｍａｇｅＪ（ＮＩＨ）によって定量化した。 Example 2: Self-activation assay for proCatK ProCatK (3 μM) in 100 mM acetate buffer (pH 4.0-5.5) containing 2.5 mM EDTA / DTT at 37 ° C. in the presence or absence of VN. Incubated in. After incubation at 37 ° C. for 10 or 30 minutes, 2xSDS-PAGE sample buffer was immediately added and heated at 98 ° C. for 10 minutes. Protein samples were electrophoresed using a 15% Tris-glycine gel containing 2-mercaptoethanol and stained with EzStain Silver (AE-1360, ATTO). The total band intensities of both proCatK and CatK were quantified by ImageJ (NIH).
　図５は、様々なｐＨにおけるｈＶＮのプロカテプシンＫの自己活性化阻害を示す。ｐＨ４－４．５においてプロカテプシンＫ（Ｐｒｏ－ＣａｔＫ（不活性型））はカテプシンＫ（Ｍａｔｕｒｅ（活性型））自己活性化に活性化される（－ｈＶＮのレーン）。ｈＶＮはプロカテプシンＫからカテプシンＫへの自己活性化を阻害した（＋ｈＶＮのレーン）。 FIG. 5 shows the inhibition of self-activation of hVN procathepsin K at various pH. At pH 4-4.5, cathepsin K (Pro-CatK (inactive form)) is activated by cathepsin K (Mature (active form)) self-activation (-hVN lane). hVN inhibited self-activation from procathepsin K to cathepsin K (+ hVN lane).
実施例３：コラーゲンの酸可溶化アッセイ
　Ｉｗａｋｉ９６ウエルアッセイプレート（ＡＧＣ　ＴＥＣＨＮＯ　ＧＬＡＳＳ　ｃｏ.,ｌｔｄ）において、１００μｌのＴＢＳ中に０．１ｍｇ／ｍｌ　ＶＮの存在下又は非存在下で、可溶化Ｉ型コラーゲン溶液（１ｍｇ／ｍｌ）（株式会社ニッピ）を３７℃で２時間処理し、コラーゲン線維を形成させた。１ＭのｐＨバッファー（１Ｍ酢酸バッファー（ｐＨ４．０－６．０）またはＴｒｉｓ－ＨＣｌバッファー（ｐＨ７．２）、１０μｌ）を形成させたコラーゲン線維に添加した。直ちに、各ウエルのサンプルの濁度をＣｙｔａｔｉｏｎ３（ＢｉｏＴｅｋ）によって３７℃で１分ごとに３５０ｎｍで測定した（Ｇｌｙｃｏｂｉｏｌｏｇｙ、Ｔａｔａｒａ　ｅｔ　ａｌ.,２０１７）。 Example 3: Collagen Acid Solubilization Assay In an Iwaki 96-well assay plate (AGC TECHNO GLASS co., Ltd), solubilized type I collagen in the presence or absence of 0.1 mg / ml VN in 100 μl TBS. The solution (1 mg / ml) (Nippi Co., Ltd.) was treated at 37 ° C. for 2 hours to form collagen fibers. 1 M pH buffer (1 M acetate buffer (pH 4.0-6.0) or Tris-HCl buffer (pH 7.2), 10 μl) was added to the formed collagen fibers. Immediately, the turbidity of the sample in each well was measured by Cytation 3 (BioTek) at 37 ° C. every minute at 350 nm (Glycobiology, Tatara et al., 2017).
　図６は、コラーゲン酸溶解におけるＶＮの影響を示す。線維化コラーゲンはｐＨ４－４．５で溶解した（Ｃｏｎｔｒｏｌ）。ｈＶＮは、ｐＨ４．５－５．５での線維化コラーゲンの溶解を促進した（ｈＶＮ）。
　図７は、コラーゲン酸溶解におけるｐＶＮ／ｈＶＮの影響（反応時間２０ｍｉｎのデータ）を示す。ｈＶＮ，ｐＶＮともに、ｐＨ４．５－５．５での線維化コラーゲンの溶解を促進した。
　図８は、コラーゲン酸溶解におけるｐＶＮ／ｈＶＮの影響（反応時間２０ｍｉｎのデータ）を示す。ＶＮ濃度は０－１００μｇ／ｍｌでｐＨ５．０で測定した。ｈＶＮ，ｐＶＮは濃度依存的に線維化コラーゲンの酸溶解を促進した。 FIG. 6 shows the effect of VN on collagen acid dissolution. Fibrotic collagen was lysed at pH 4-4.5 (Control). hVN promoted the dissolution of fibrotic collagen at pH 4.5-5.5 (hVN).
FIG. 7 shows the effect of pVN / hVN on collagen acid dissolution (data with a reaction time of 20 min). Both hVN and pVN promoted the dissolution of fibrotic collagen at pH 4.5-5.5.
FIG. 8 shows the effect of pVN / hVN on collagen acid dissolution (data with a reaction time of 20 min). The VN concentration was measured at 0-100 μg / ml at pH 5.0. hVN and pVN promoted acid dissolution of fibrotic collagen in a concentration-dependent manner.
実施例４：コラーゲン線維形成のアッセイ
　ＴＢＳ中のコラーゲン（２ｍｇ／ｍｌ、５０μｌ）とＶＮ（０．１ｍｇ／ｍｌ、５０μｌ）をＩｗａｋｉ９６ウエルアッセイプレートのウエルに添加した。攪拌後、ＶＮの存在下又は非存在下のコラーゲン溶液を３７℃で２時間インキュベートして、コラーゲン線維を形成させた。各ウエルの濁度を、Ｃｙｔａｔｉｏｎ３（ＢｉｏＴｅｋ）によって５分ごとに３５０ｎｍで測定した。 Example 4: Collagen Fibrosis Assay Collagen (2 mg / ml, 50 μl) and VN (0.1 mg / ml, 50 μl) in TBS were added to the wells of the Iwaki 96-well assay plate. After stirring, the collagen solution in the presence or absence of VN was incubated at 37 ° C. for 2 hours to form collagen fibers. The turbidity of each well was measured every 5 minutes at 350 nm by Cytation3 (BioTek).
　図９は、ＶＮのコラーゲン線維形成促進（コラーゲンの種類（ブタ・ウシ）の差）を示す。ｈＶＮはブタ、ウシ由来のＩ型コラーゲンの線維化を促進したが、ｐＶＮはブタ由来のＩ型コラーゲンの線維化を促進するが、ウシ由来のＩ型コラーゲンの線維化を抑制した。
　図１０は、ＶＮのコラーゲン線維形成促進（濃度依存性）を示す。ＶＮ濃度は０－１００μｇ／ｍｌとした。ｈＶＮ、ｐＶＮはともにブタ由来のＩ型コラーゲンの線維化を濃度依存的に促進した。 FIG. 9 shows the promotion of collagen fiber formation in VN (difference in collagen type (pig / bovine)). hVN promoted the fibrosis of type I collagen derived from pigs and cattle, while pVN promoted the fibrosis of type I collagen derived from pigs but suppressed the fibrosis of type I collagen derived from cattle.
FIG. 10 shows the promotion of collagen fiber formation (concentration dependence) of VN. The VN concentration was 0-100 μg / ml. Both hVN and pVN promoted the fibrosis of type I collagen derived from pigs in a concentration-dependent manner.
実施例５：コラーゲン線維の透過型電子顕微鏡画像
　ＴＢＳ中のコラーゲン（２ｍｇ／ｍｌ、５０μｌ）と０．１ｍｇ／ｍｌのＶＮ（表示濃度、５０μｌ）をＩｗａｋｉ９６ウエルアッセイプレートのウエルに添加した。攪拌後、ＶＮの存在下又は非存在下のコラーゲン溶液を３７℃で２時間インキュベートして、フィブリルを形成させた。 Example 5: Transmission electron micrograph of collagen fibers Collagen (2 mg / ml, 50 μl) in TBS and 0.1 mg / ml VN (labeled concentration, 50 μl) were added to the wells of the Iwaki 96-well assay plate. After stirring, the collagen solution in the presence or absence of VN was incubated at 37 ° C. for 2 hours to form fibril.
　コラーゲンサンプルを、０．１Ｍカコジル酸バッファー（ｐＨ７．４）中の２％パラホルムアルデヒド（ＰＦＡ）および２％グルタルアルデヒド（ＧＡ）で４℃で一晩固定した。サンプルは、０．１Ｍカコジル酸バッファー（ｐＨ７．４）中の１％タンニン酸で、４℃でさらに２時間固定した。固定後、サンプルを０．１Ｍカコジル酸緩衝液で３０分間ずつ４回洗浄し、０．１Ｍカコジル酸緩衝液中２％四酸化オスミウム（ＯｓＯ４）で４℃で３時間、後固定した。サンプルは、５０％エタノールで３０分間４℃で１回、７０％エタノールで３０分間４℃で１回、９０％エタノールで３０分間室温で１回、その後、１００％エタノールで３０分間室温で４回脱水した。これらの脱水操作後に、サンプルを１００％エタノールで室温で一晩連続して脱水した。サンプルを５０％ｔｅｒｔ－ブチルアルコール／エタノールに１時間置換し、１００％ｔｅｒｔ－ブチルアルコールに室温で１時間、３回置換した。置換後、サンプルを４℃で凍結し、真空乾燥した。オスミウムプラズマコーター（ＮＬ－ＯＰＣ８０Ａ；　Ｎｉｐｐｏｎ　Ｌａｓｅｒ＆Ｅｌｅｃｔｒｏｎｉｃｓ　Ｌａｂｏｒａｔｏｒｙ、Ｎａｇｏｙａ、Ｊａｐａｎ）を使用して、サンプルをオスミウムの薄層（３０ｎｍ）でコートした。サンプルを、走査型電子顕微鏡（ＳＥＭ））（ＪＳＭ－７５００Ｆ；　ＪＥＯＬ　Ｌｔｄ．、Ｔｏｋｙｏ、Ｊａｐａｎ）を用いて加速電圧３．０ｋＶで観察した。 Collagen samples were fixed overnight at 4 ° C. with 2% paraformaldehyde (PFA) and 2% glutaraldehyde (GA) in 0.1 M cacodylic acid buffer (pH 7.4). Samples were fixed at 4 ° C. for an additional 2 hours with 1% tannic acid in 0.1 M cacodylic acid buffer (pH 7.4). After fixation, the sample was washed 4 times with 0.1 M cacodylic acid buffer for 30 minutes each, and post-fixed with 2% osmium tetroxide (OsO4) in 0.1 M cacodylic acid buffer at 4 ° C. for 3 hours. Samples were sampled once at 4 ° C for 30 minutes with 50% ethanol, once at 4 ° C for 30 minutes with 70% ethanol, once at room temperature for 30 minutes with 90% ethanol, and then 4 times at room temperature for 30 minutes with 100% ethanol. Dehydrated. After these dehydration operations, the samples were continuously dehydrated overnight at room temperature with 100% ethanol. The sample was replaced with 50% tert-butyl alcohol / ethanol for 1 hour and 100% tert-butyl alcohol at room temperature for 1 hour 3 times. After the replacement, the sample was frozen at 4 ° C. and vacuum dried. Samples were coated with a thin layer of osmium (30 nm) using an osmium plasma coater (NL-OPC80A; Nippon Laser & Electricals Laboratory, Nagoya, Japan). Samples were observed at an acceleration voltage of 3.0 kV using a scanning electron microscope (SEM) (JSM-7500F; JEOL Ltd., Tokyo, Japan).
　図１１は、ＶＮ存在下で形成されたコラーゲン線維の透過型電子顕微鏡画像を示す。ｈＶＮは、線維同士が密に隣り合った隙間の少ないコラーゲン線維の形成を促した。 FIG. 11 shows a transmission electron microscope image of collagen fibers formed in the presence of VN. hVN promoted the formation of collagen fibers with few gaps in which the fibers were closely adjacent to each other.
実施例６：カテプシンＫ活性におけるｈＶＮ糖鎖の影響
　ＶＮは、尿素処理の前後に２段階のヘパリンアフィニティークロマトグラフィーによってヒトとブタの血漿から精製した（Ｙａｔｏｈｇｏ　ｅｔ　ａｌ、Ｃｅｌｌ　Ｓｔｒｕｃｔ　Ｆｕｎｃｔ、１３、２８１－９２、１９９８）。ヘパリン－セファロース６Ｂカラムは、還元アミノ化法によって作製した（Ｄａｔｅ　ｅｔ　ａｌ.,Ｍｅｔｈｏｄｓ　Ｍｏｌ　Ｂｉｏｌ。１２００：５３－６７、２０１４）。ＶＮ（５００μｇ）を、１ｍＭ　ＣａＣｌ２および０．５ｍＭ　ＰＭＳＦを含む２０ｍＭクエン酸－リン酸バッファー（ｐＨ６．０）において、Ｎ－グルコシダーゼ（３．３ユニット）又はＮｅｕｉｒａｉｎｉｄａｓｅ（１６．７ユニット）により３７℃で４８時間処理した。ＶＮのグリコシダーゼ処理後、ＶＮを２０　ｍＭ　Ｔｒｉｓ－ＨＣｌ緩衝生理食塩水（ＴＢＳ、ｐＨ７．２）に対して透析した。脱Ｎ－グリコシル化または脱シアリル化は、ＳＤＳ－ＰＡＧＥによるＶＮの分子量の減少により確認した。
　Ｎ－ｇｌｙは、Ｎ－グリコシダーゼＦで処理したｈＶＮを示す。
　Ｎｅｕは、Ｎｅｕｉｒａｉｎｉｄａｓｅで処理したｈＶＮを示す。
　対照は、グリコシダーゼなしで３７℃４８時間インキュベートした。 Example 6: Effect of hVN sugar chains on cathepsin K activity VN was purified from human and porcine plasma by two-step heparin affinity chromatography before and after urea treatment (Yatohgo et al, Cell Struct Funct, 13, 281-). 92, 1998). Heparin-Sepharose 6B columns were prepared by reductive amination (Date et al., Methods Mol Biol. 1200: 53-67, 2014). VN (500 μg) in 20 mM citric acid-phosphate buffer (pH 6.0) containing 1 mM CaCl2 and 0.5 mM PMSF with N-glucosidase (3.3 units) or Neuirainidase (16.7 units) at 37 ° C. Processed for 48 hours. After glycosidase treatment of the VN, the VN was dialyzed against 20 mM Tris-HCl buffered saline (TBS, pH 7.2). De-N-glycosylation or desialylation was confirmed by a decrease in VN molecular weight by SDS-PAGE.
N-gly indicates hVN treated with N-glycosidase F.
Neu indicates hVN treated with Neuirainidase.
Controls were incubated without glycosidases at 37 ° C. for 48 hours.
　実施例１と同様に、カテプシンＫ活性のアッセイを行った。反応時間は６０ｍｉｎであり、ＶＮ濃度は１μＭ、ｐＨ５．５で測定した。 The cathepsin K activity was assayed in the same manner as in Example 1. The reaction time was 60 min, the VN concentration was 1 μM, and the pH was 5.5.
　図１２は、カテプシンＫ活性におけるｈＶＮ糖鎖の影響を示す。ｈＶＮは、Ｎ－型糖鎖除去（Ｎ－ｇｌｙ）やシアル酸除去（Ｎｅｕ）によっても、カテプシンＫ阻害作用を持っていた。Ｃｏｎｔｒｏｌ－ｈＶＮに比べて、Ｎ－ｇｌｙやＮｅｕ－ｈＶＮではカテプシンＫ阻害効果が減弱した。 FIG. 12 shows the effect of hVN sugar chains on cathepsin K activity. hVN also had a cathepsin K inhibitory effect by N-type sugar chain removal (N-gly) and sialic acid removal (Neu). Compared with Control-hVN, the cathepsin K inhibitory effect was attenuated in N-glycy and Neu-hVN.
実施例７：プロカテプシンＫの自己活性化におけるｈＶＮ糖鎖の影響
　実施例２と同様にして、ｐｒｏＣａｔＫの自己活性化アッセイを行った。反応時間１０ｍｉｎ、ＶＮ濃度２μＭで測定（ｐＨ４．０）を行った。 Example 7: Effect of hVN sugar chain on self-activation of procathepsin K A self-activation assay of proCatK was performed in the same manner as in Example 2. The measurement was carried out at a reaction time of 10 min and a VN concentration of 2 μM (pH 4.0).
　図１３は、プロカテプシンＫの自己活性化におけるｈＶＮ糖鎖の影響を示す。
　プロカテプシンＫ（Ｐｒｏ－ＣａｔＫ（不活性型））はカテプシンＫ（ＣａｔＫ（活性型））自己活性化に活性化された（ＶＮなしでの反応前レーン１，ＶＮなしでの反応後レーン２）。Ｃｏｎｔｒｏｌ－ｈＶＮは、プロカテプシンＫからカテプシンＫへの自己活性化を阻害した（レーン３）。Ｎ－ｇｌｙ－ｈＶＮ（レーン４）、Ｎｅｕ－ｈＶＮ（レーン５）は、プロカテプシンＫからカテプシンＫへの自己活性化を阻害するが、その効果はＣｏｎｔｒｏｌ－ｈＶＮ（レーン３）よりも弱かった。
レーン６；Ｃｏｎｔｒｏｌ－ｈＶＮのみ（カテプシンＫなし）
レーン７；Ｎ－ｇｌｙ－ｈＶＮのみ（カテプシンＫなし）
レーン８；Ｎｅｕ－ｈＶＮのみ（カテプシンＫなし） FIG. 13 shows the effect of hVN sugar chains on the self-activation of procathepsin K.
Pro-cathepsin K (Pro-CatK (inactive form)) was activated by cathepsin K (CatK (active form)) self-activation (pre-reaction lane without VN 1, post-reaction lane 2 without VN). .. Control-hVN inhibited self-activation of procathepsin K to cathepsin K (lane 3). N-gly-hVN (lane 4) and Neu-hVN (lane 5) inhibited self-activation from procathepsin K to cathepsin K, but the effect was weaker than that of Control-hVN (lane 3).
Lane 6; Control-hVN only (without cathepsin K)
Lane 7; N-gly-hVN only (without cathepsin K)
Lane 8; Neu-hVN only (without cathepsin K)
実施例８：カテプシンＫによるＶＮの分解（ＶＮ糖鎖の影響）
　カテプシンＫは、２．５ｍＭ　ＥＤＴＡ／ＤＴＴを含む１００ｍＭ酢酸緩衝液（ｐＨ４．０）中での３７℃１０分間のインキュベーションにより、プロカテプシンＫ（５μＭ）から自己活性化した。カテプシンＫを、２．５ｍＭ　ＥＤＴＡ／ＤＴＴを含む氷冷２００ｍＭ酢酸バッファー（ｐＨ５．５）で２、２０、６０、２００ｎＭに希釈し、４μＭ　ＶＮと１：１の比率で混合した。混合溶液を３７℃で０、１０、３０、６０、１２０分間インキュベートして、カテプシンＫによりＶＮを分解した。インキュベーション後、氷冷２μＭ　Ｅ－６４（カテプシンＫを含むプロテイナーゼの阻害剤、最終濃度１μＭ）および２ｘＳＤＳ－ＰＡＧＥサンプルバッファーを混合溶液に添加した。サンプルは直ちに９８℃で１０分間加熱した。サンプルを、２－メルカプトエタノールを含む７％Ｔｒｉｓ－グリシンゲルを使用して電気泳動し、ＳｙｐｒｏＲｕｂｙ染色により染色した。タンパク質のバンドはＩｍａｇｅＱｕａｎｔ　ＬＡＳ　４０００（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）で検出した。ＩｍａｇｅＪ（ＮＩＨ）により、インキュベーション時間０分の未分解ＶＮバンドの分子量に対応するＶＮバンドの強度を定量化した。 Example 8: Degradation of VN by cathepsin K (effect of VN sugar chain)
Cathepsin K was self-activated from procatehepsin K (5 μM) by incubation at 37 ° C. for 10 minutes in 100 mM acetate buffer (pH 4.0) containing 2.5 mM EDTA / DTT. Cathepsin K was diluted to 2, 20, 60, 200 nM in ice-cold 200 mM acetate buffer (pH 5.5) containing 2.5 mM EDTA / DTT and mixed with 4 μM VN in a 1: 1 ratio. The mixed solution was incubated at 37 ° C. for 0, 10, 30, 60 and 120 minutes to degrade the VN with cathepsin K. After incubation, ice-cold 2 μM E-64 (inhibitor of proteinase containing cathepsin K, final concentration 1 μM) and 2xSDS-PAGE sample buffer were added to the mixed solution. The sample was immediately heated at 98 ° C. for 10 minutes. Samples were electrophoresed using a 7% Tris-glycine gel containing 2-mercaptoethanol and stained by SyproRuby staining. Protein bands were detected with ImageQuant LAS 4000 (GE Healthcare). ImageJ (NIH) quantified the intensity of the VN band corresponding to the molecular weight of the undegraded VN band at 0 minutes incubation time.
　図１４は、カテプシンＫによるＶＮの分解（ＶＮ糖鎖の影響）を示す。
　カテプシンＫ（最終濃度１０ｎＭ）とｈＶＮを１２０分間反応させたところ、ＶＮ分子量が低下し、カテプシンＫによりＶＮが分解されることがわかった。Ｃｏｎｔｒｏｌ－ｈＶＮは、Ｎ－ｇｌｙ－ｈＶＮやＮｅｕ－ｈＶＮ　に比べて、ＶＮの全長を示すバンドが多く残っていたことから、ｈＶＮの糖鎖はカテプシンＫに対する分解抵抗性を示すのに役立つことが示された。 FIG. 14 shows the degradation of VN by cathepsin K (effect of VN sugar chain).
When cathepsin K (final concentration 10 nM) was reacted with hVN for 120 minutes, it was found that the VN molecular weight decreased and cathepsin K decomposed VN. Since Control-hVN had more bands indicating the total length of VN than N-glycy-hVN and Neu-hVN, the sugar chain of hVN may be useful for showing degradation resistance to cathepsin K. Shown.
実施例９：ＶＮの酸溶解促進作用（ＶＮ糖鎖の影響）
　実施例３と同様に、コラーゲンの酸可溶化アッセイを行った。ＶＮ濃度は０．１ｍｇ／ｍｌで測定した。
　図１５は、ＶＮの酸溶解促進作用（ＶＮ糖鎖の影響）を示す。ＶＮの酸溶解促進作用において、Ｃｏｎｔｒｏｌ－ｈＶＮ、Ｎ－ｇｌｙ－ｈＶＮ、Ｎｅｕ－ｈＶＮは差がなかった。このことから、ＶＮの酸溶解促進作用において、ＶＮの糖鎖の影響はないことが示された。 Example 9: Acid dissolution promoting action of VN (effect of VN sugar chain)
Similar to Example 3, an acid solubilization assay for collagen was performed. The VN concentration was measured at 0.1 mg / ml.
FIG. 15 shows the acid dissolution promoting action of VN (effect of VN sugar chain). There was no difference between Control-hVN, N-glycy-hVN, and Neu-hVN in the acid dissolution promoting action of VN. From this, it was shown that there is no influence of the sugar chain of VN on the acid dissolution promoting action of VN.
実施例１０：ＶＮのコラーゲン線維形成促進（ＶＮ糖鎖の影響）
　実施例４と同様に、コラーゲン線維形成のアッセイを行った。ＶＮ濃度は０．１ｍｇ／ｍｌで測定した。
　図１６は、ＶＮのコラーゲン線維形成促進（ＶＮ糖鎖の影響）を示す。ＶＮのコラーゲン線維形成促進作用において、Ｃｏｎｔｒｏｌ－ｈＶＮ、Ｎ－ｇｌｙ－ｈＶＮ、Ｎｅｕ－ｈＶＮは差がなかった。このことから、ＶＮのコラーゲン線維形成促進作用において、ＶＮの糖鎖の影響はないことが示された。 Example 10: Promotion of collagen fiber formation in VN (effect of VN sugar chain)
An assay for collagen fibrosis was performed as in Example 4. The VN concentration was measured at 0.1 mg / ml.
FIG. 16 shows the promotion of collagen fiber formation in VN (effect of VN sugar chain). There was no difference between Control-hVN, N-gly-hVN, and Neu-hVN in the collagen fiber formation promoting action of VN. From this, it was shown that there is no influence of the sugar chain of VN on the collagen fiber formation promoting action of VN.
実施例１１：データ解析によるVNのカテプシンＫ結合部位の予測
　ＶＮの三次元構造は部分的にしか決定されておらず、全長は解明されていない。これは、ＶＮに一定の立体構造を取らない部分（普段定まった構造を取らないdisordered regions、以下ｄｉｓｏｒｄｅｒｅｄ領域とも呼ぶ）が存在することを示している。ｄｉｓｏｒｄｅｒｅｄ領域は、他の分子と結合することで、まった構造を取らない構造から秩序のある定まった構造に安定化する（Yura, K. & Hayward, S. The interwinding nature of protein-protein interfaces and its implication for protein complex formation. Bioinformatics 25, 3108-3113, doi:10.1093/bioinformatics/btp563 (2009).）。つまり、ＶＮのカテプシンＫへの結合部位は、ｄｉｓｏｒｄｅｒｅｄ領域に存在する。ＶＮのカテプシンＫへの結合部位を決定するために、ＰＯＮＤＲ、ＤｉｓＥＭＢＬ、ＰｒＤＯＳ、およびＦｏｌｄＩｎｄｅｘの４つの方法によりｄｉｓｏｒｄｅｒｅｄ領域を算出した。 Example 11: Prediction of VN cathepsin K binding site by data analysis The three-dimensional structure of VN has been partially determined, and the total length has not been elucidated. This indicates that the VN has parts that do not have a fixed three-dimensional structure (disordered regions that do not usually have a fixed structure, hereinafter also referred to as disordered regions). The disordered region stabilizes from a non-structured structure to an ordered and fixed structure by binding to other molecules (Yura, K. & Hayward, S. The interwinding nature of protein-protein interfaces and its implication for protein complex formation. Bioinformatics 25, 3108-3113, doi: 10.1093 / bioinformatics / btp563 (2009).). That is, the binding site of VN to cathepsin K exists in the disordered region. Disordered regions were calculated by four methods: PONDR, DisEMBL, PrDOS, and FolderIndex to determine the binding site of VN to cathepsin K.
PONDR：
Predictors of Natural Disordered Regions　http://www.pondr.com/pondr-tut.html
Romero, P. et al. Sequence complexity of disordered protein. Proteins 42, 38-48, doi:10.1002/1097-0134(20010101)42:1<38::aid-prot50>3.0.co;2-3 (2001)、 PONDR:
Predictors of Natural Disordered Regions http://www.pondr.com/pondr-tut.html
Romero, P. et al. Sequence complexity of disordered protein. Proteins 42, 38-48, doi: 10.1002 / 1097-0134 (20010101) 42: 1 <38 :: aid-prot50>3.0.co; 2-3 (2001) ),
DisEMBL：
Intrinsic Protein Disorder Prediction 1.5　http://dis.embl.de/
Linding, R. et al. Protein disorder prediction: implications for structural proteomics. Structure 11, 1453-1459, doi:10.1016/j.str.2003.10.002 (2003). DisEMBL:
Intrinsic Protein Disorder Prediction 1.5 http://dis.embl.de/
Linding, R. et al. Protein disorder prediction: implications for structural proteomics. Structure 11, 1453-1459, doi: 10.1016 / j.str. 2003.10.002 (2003).
PrDOS：
Protein DisOrder prediction System　http://prdos.hgc.jp/cgi-bin/top.cgi
Ishida, T. & Kinoshita, K. PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res 35, W460-464, doi:10.1093/nar/gkm363 (2007). PrDOS:
Protein DisOrder prediction System http://prdos.hgc.jp/cgi-bin/top.cgi
Ishida, T. & Kinoshita, K. PrDOS: prediction of disordered protein regions from amino acid sequence. Nucleic Acids Res 35, W460-464, doi: 10.1093 / nar / gkm363 (2007).
FoldIndex：
https://fold.weizmann.ac.il/
Prilusky, J. et al. FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21, 3435-3438, doi:10.1093/bioinformatics/bti537 (2005). FoldIndex:
https://fold.weizmann.ac.il/
Prilusky, J. et al. FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 21, 3435-3438, doi: 10.1093 / bioinformatics / bti537 (2005).
　ｈＶＮのアミノ酸配列は、ＮＣＢＩ　ｐｒｏｔｅｉｎ　ｓｅｑｕｅｎｃｅ　ＥＡＷ５１０８２１．１（配列番号１）を使用した。結果を図１７に示す。 As the amino acid sequence of hVN, NCBI protein sequence EAW510821.1 (SEQ ID NO: 1) was used. The results are shown in FIG.
　ｈＶＮのｄｉｓｏｒｄｅｒｅｄ領域は、図１７の破線で示したアミノ酸配列番号１００から１３０、２９２から２９６、３６４から３９７であった。破線の領域はＵｎｉｐｒｏｔエントリＰ０４００４（UniProt Consortium, T. UniProt: the universal protein knowledgebase. Nucleic Acids Res 46, 2699, doi:10.1093/nar/gky092 (2018)）に基づいて描いた。黒線はＰｒｏｔｅｉｎ　Ｄａｔａｂａｎｋ(Kinjo, A. R. et al. Protein Data Bank Japan (PDBj): updated user interfaces, resource description framework, analysis tools for large structures. Nucleic Acids Res 45, D282-D288, doi:10.1093/nar/gkw962 (2017))で既知の３次元構造を持つ領域である。Ａｓｎ８６、１６９、および２４２に位置するＮ－型糖鎖結合部位は、概略的であるがｈＶＮに最も多く存在するＮ－型糖鎖構造が示されている。 The disordered region of hVN was amino acid SEQ ID NOs: 100 to 130 and 292 to 296 to 364 to 397 shown by the broken line in FIG. The area of the broken line is drawn based on UniProt entry P04004 (UniProt Consortium, T. UniProt: the universal protein knowledgebase. Nucleic Acids Res 46, 2699, doi: 10.1093 / nar / gky092 (2018)). The black line is Protein Databank (Kinjo, A. R. et al. Protein Data Bank Japan (PDBj): updated user interfaces, resource description framework, analysis tools for large structures. Nucleic Acids Res 45, D282- It is a region with a three-dimensional structure known in nar / gkw962 (2017)). The N-type sugar chain binding sites located at Asn86, 169, and 242 show a schematic but most abundant N-type sugar chain structure in hVN.
　ｈＶＮとｐＶＮはカテプシンＫ活性を阻害することから、ＶＮのカテプシンＫ結合部位は、ｈＶＮとｐＶＮとのアミノ酸配列保存領域である。ＣｌｕｓｔａｌＷ（ｖｅｒ．２．１）（Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948, doi:10.1093/bioinformatics/btm404 (2007)）によってｈＶＮとｐＶＮのアミノ酸配列の多重整列を行った。ｈＶＮとｐＶＮのアミノ酸配列は、それぞれＮＣＢＩ　ｐｒｏｔｅｉｎ　ｓｅｑｕｅｎｃｅ　ＥＡＷ５１０８２１．１（配列番号１）とＢＡＡ０９６１６．１（配列番号２）を使用した。結果を図１８に示す。 Since hVN and pVN inhibit cathepsin K activity, the cathepsin K binding site of VN is the amino acid sequence preservation region of hVN and pVN. HVN and pVN amino acids by ClustalW (ver. 2.1) (Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948, doi: 10.1093 / bioinformatics / btm404 (2007)) Multiple alignment of the sequence was performed. For the amino acid sequences of hVN and pVN, NCBI protein sequence EAW510821.1 (SEQ ID NO: 1) and BAA09616.1 (SEQ ID NO: 2) were used, respectively. The results are shown in FIG.
＜配列番号１＞

EAW51082.1 vitronectin [Homo sapiens]
MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFRGQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRAMWLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL <SEQ ID NO: 1>

EAW51082.1 vitronectin [Homo sapiens]
MAPLRPLLILALLAWVALADQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDVFTMPEDEYTVYDDGEEKNNATVHEQVGGPSLTSDLQAQSKGNPEQTPVLKPEEEAPAPEVGASKPEGIDSRPETLHPGRPQPPAEEELCSGKPFDAFTDLKNGSLFAFRGQYCYELDEKAVRPGYPKLIRDVWGIEGPIDAAFTRINCQGKTYLFKGSQYWRFEDGVLDPDYPRNISDGFDGIPDNVDAALALPAHSYSGRERVYFFKGKQYWEYQFQHQPSQEECEGSSLSAVFEHFAMMQRDSWEDIFELLFWGRTSAGTRQPQFISRDWHGVPGQVDAAMAGRIYISGMAPRPSLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPSRAMWLSLFSSEESNLGANNYDDYRMDWLVPATCEPIQSVFFFSGDKYYRVNLRTRRVDTVDPPYPRSIAQYWLGCPAPGHL
＜配列番号２＞

BAA09616.1 vitronectin [Sus scrofa]
MAPLRPLLMLALLAWVALADQESCKGRCTDGFIAERKCQCDELCSYYQSCCTDYVAECKPQVTRGDVFLQPDDEYRAYDYHEETRHNTSVQEEQRIPVLLAKTEETPVLKPEEEAPPPGPQTDDLGVPEEELCSGKPFDAFTNLKNGSVFAFRGLYCYELDEKAVRPGYPKLIQDVWGIKGPIDAAFTRINCQGKTYLFKGSQYWRFDDGVLDPNYPREISEGFKGIPDDVDAALALPAHSYSGRERVYFFKGKQYWEYVFQQQPSREECEGSSPSDVFAHFALMQRDSWEDIFRLLFWSHSFGGAIEPRVISQDWLGLPEQVDAAMAGQIYISGSALKPSQPKMTKSARRSGKRYRSRRGRGRGRGHKPQPEVPPAVSFNLAALVLQ <SEQ ID NO: 2>

BAA09616.1 vitronectin [Sus scrofa]
MAPLRPLLMLALLAWVALADQESCKGRCTDGFIAERKCQCDELCSYYQSCCTDYVAECKPQVTRGDVFLQPDDEYRAYDYHEETRHNTSVQEEQRIPVLLAKTEETPVLKPEEEAPPPGPQTDDLGVPEEELCSGKPFDAFTNLKNGSVFAFRGLYCYELDEKAVRPGYPKLIQDVWGIKGPIDAAFTRINCQGKTYLFKGSQYWRFDDGVLDPNYPREISEGFKGIPDDVDAALALPAHSYSGRERVYFFKGKQYWEYVFQQQPSREECEGSSPSDVFAHFALMQRDSWEDIFRLLFWSHSFGGAIEPRVISQDWLGLPEQVDAAMAGQIYISGSALKPSQPKMTKSARRSGKRYRSRRGRGRGRGHKPQPEVPPAVSFNLAALVLQ
　図１８の破線は、図１７の破線と同じアミノ酸配列を指しており、算出されたｄｉｓｏｒｄｅｒｅｄ領域である。図１８の破線で示したｈＶＮのアミノ酸配列番号はｐＶＮのアミノ酸配列でも保存されていた。これらの結果から、ＶＮのカテプシンＫへの結合部位に相当するｄｉｓｏｒｄｅｒｅｄ領域は、ｈＶＮのアミノ酸配列番号１００から１３０、２９２から２９６、３６４から３９７であることが示された。なお、図１８の黒線は、図１７の黒線と同じアミノ酸配列を指しており、Ｐｒｏｔｅｉｎ　Ｄａｔａｂａｎｋで既知の３次元構造を持つ領域である。ｈＶＮのアミノ酸配列番号１００から１３０、２９２から２９６、３６４から３９７をそれぞれ配列番号３から５に示す。 The broken line in FIG. 18 points to the same amino acid sequence as the broken line in FIG. 17, and is a calculated disordered region. The amino acid sequence number of hVN shown by the broken line in FIG. 18 was also conserved in the amino acid sequence of pVN. From these results, it was shown that the disordered region corresponding to the binding site of VN to cathepsin K is amino acid SEQ ID NOs: 100 to 130 and 292 to 296 to 364 to 397 of hVN. The black line in FIG. 18 points to the same amino acid sequence as the black line in FIG. 17, and is a region having a three-dimensional structure known in Protein Databank. Amino acid SEQ ID NOs: 100 to 130 and 292 to 296 to 396 to 397 of hVN are shown in SEQ ID NOs: 3 to 5, respectively.
＜配列番号３＞
SDLQAQSKGNPEQTPVLKPEEEAPAPEVGAS
＜配列番号４＞
ECEGS
＜配列番号５＞
SLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPS <SEQ ID NO: 3>
SDLQAQSKGNPEQTPVLKPEEEAPAPEVGAS
<SEQ ID NO: 4>
ECEGS
<SEQ ID NO: 5>
SLAKKQRFRHRNRKGYRSQRGHSRGRNQNSRRPS
　前記ｄｉｓｏｒｄｅｒｅｄ領域の算出の精度は、９５％以上である。ＰＯＮＤＲ、ＤｉｓＥＭＢＬ、ＰｒＤＯＳ、およびＦｏｌｄＩｎｄｅｘの４つの方法の偽正算出率（Ｆａｌｓｅ ｐｏｓｉｔｉｖｅ ｒａｔｅ。この場合、ｄｉｓｏｒｄｅｒｅｄ領域と算出したが、実際にはｄｉｓｏｒｄｅｒｅｄ領域ではない残基の割合。）は、一般に次の通りである。
ＰＯＮＤＲ　２２％
ＤｉｓＥＭＢＬ　１９％
ＰｒＤＯＳ　５％
ＦｏｌｄＩｎｄｅｘ　１０％ The accuracy of the calculation of the disordered region is 95% or more. The false positive rate of the four methods PONDR, DisEMBL, PrDOS, and FoundIndex (False positive rate. In this case, the percentage of residues that were calculated as the disturbed region but not actually the disturbed region) is as follows: It's a street.
PONDR 22%
DisEMBL 19%
PrDOS 5%
FolderIndex 10%
　今回の算出はこれらのコンセンサスに基づくため、一番精度が高い予測に寄ることになる。よって偽正算出率は５％となり、算出の精度は９５％になる。または、すべての方法で間違って算出する確率は０．０２％（０．２２×０．１９×０．０５×０．１０＝０．０００２）であり、完全コンセンサス予測で間違える可能性は０．０２％である。したがって、本法においては極めて高確率でｄｉｓｏｒｄｅｒｅｄ領域の算出ができている。 Since this calculation is based on these consensus, it depends on the most accurate prediction. Therefore, the false correct calculation rate is 5%, and the accuracy of the calculation is 95%. Alternatively, the probability of incorrect calculation by all methods is 0.02% (0.22 x 0.19 x 0.05 x 0.10 = 0.0002), and the possibility of making a mistake in the complete consensus prediction is 0. It is 02%. Therefore, in this method, the disordered region can be calculated with an extremely high probability.
　上記の通り、ビトロネクチンのカテプシンＫへの結合部位は、普段定まった構造を取らないｄｉｓｏｒｄｅｒｅｄ領域（disordered regions）であるヒトビトロネクチン（ｈＶＮ）のアミノ酸配列番号１００－１３０、２９２－２９６、３６４－３９７であると予想された。また、これらのヒトビトロネクチン（ｈＶＮ）のアミノ酸配列番号（１００－１３０、２９２－２９６、３６４－３９７）は、ブタＶＮ（ｐＶＮ）でも保存されており、カテプシンＫを阻害する活性部位であると考えられる。 As described above, the binding site of vitronectin to cathepsin K is amino acid SEQ ID NOs: 100-130, 292-296, 364-397 of human vitronectin (hVN), which is a disordered region that does not normally have a fixed structure. It was expected to be. In addition, the amino acid sequence numbers (100-130, 292-296, 364-397) of these human vitronectins (hVN) are also conserved in pig VN (pVN) and are considered to be active sites that inhibit cathepsin K. Be done.

Claims (7)

  1. ビトロネクチン、又はカテプシンＫと結合するその断片を含む、カテプシンＫ阻害剤。 A cathepsin K inhibitor comprising vitronectin, or a fragment thereof that binds to cathepsin K.
  2. ビトロネクチン、又はカテプシンＫと結合するその断片を含む、プロカプテシンＫの自己活性化阻害剤。 A self-activation inhibitor of procaptesin K, comprising a fragment thereof that binds to vitronectin, or cathepsin K.
  3. ビトロネクチン、又はカテプシンＫと結合するその断片を含む、線維化コラーゲンの溶解促進剤。 A lysis promoter for fibrotic collagen, comprising a fragment thereof that binds to vitronectin, or cathepsin K.
  4. ビトロネクチン、又はカテプシンＫと結合するその断片を含む、コラーゲンの線維形成の促進剤。 A promoter of collagen fibrosis, comprising a fragment thereof that binds to vitronectin, or cathepsin K.
  5. 破骨細胞の骨吸収を抑制するために使用する、請求項１から３の何れか一項に記載の剤。 The agent according to any one of claims 1 to 3, which is used for suppressing bone resorption of osteoclasts.
  6. ビトロネクチンがヒトビトロネクチン又はブタビトロネクチンである、請求項１から５の何れか一項に記載の剤。 The agent according to any one of claims 1 to 5, wherein the vitronectin is human vitronectin or porcine vitronectin.
  7. ビトロネクチンが、Ｎ－型糖鎖を有している、請求項１から６の何れか一項に記載の剤。 The agent according to any one of claims 1 to 6, wherein the vitronectin has an N-type sugar chain.
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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATE, KIMIE ET AL.: "Discovery of cathepsin K inhibitory activity by glycoprotein vitronectin and role of glycosylation of VN", ABSTRACTS OF THE 39TH ANNUAL MEETING OF THE JAPANESE SOCIETY OF CARBOHYDRATE RESEARCH, 21 November 2020 (2020-11-21), pages 182 *
KIMIE, SAKAGAMI: "Inhibitory effect of extracellular matrix glycoprotein vitronectin on collagen degradation", THE 93RD ANNUAL MEETING OF THE JAPANESE BIOCHEMICAL SOCIETY (WEB, SUMMARIES, September 2020 (2020-09-01) *
MIN, S. K. ET AL.: "A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation", CELL DEATH AND DIFFERENTIATION, vol. 25, 2018, pages 268 - 281, XP055895654 *
NOVINEC, M. ET AL.: "Cathepsin K exists in two functionally distinct conformations at neutral pH.", FEBS JOURNAL, vol. 276, 2009, pages 379 - 380 *
UEMURA, T. ET AL.: "mRNA expression of MT1-MMP, MMP-9, cathepsin K, and TRAP in highly enriched osteoclasts cultured on several matrix proteins and ivory surfaces", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 64, no. 8, 2000, pages 1771 - 1773, XP055895660 *

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