WO2021256200A1 - Inhibiteur de cathepsine k - Google Patents

Inhibiteur de cathepsine k Download PDF

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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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cathepsin
vitronectin
hvn
collagen
fragment
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PCT/JP2021/019927
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Japanese (ja)
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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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    • 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

La présente invention aborde le problème de la fourniture d'un nouvel inhibiteur de cathepsine K. La présente invention concerne : un Inhibiteur de cathepsine K comprenant de la vitronectine, ou un fragment de celle-ci qui se lie à la cathepsine K ; un inhibiteur d'auto-activation de procathepsine K ; un agent pour accélérer la dissolution de collagène fibreux ; et un agent pour accélérer la formation de fibres de collagène.
PCT/JP2021/019927 2020-06-19 2021-05-26 Inhibiteur de cathepsine k WO2021256200A1 (fr)

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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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