WO2005054460A1 - Cristal d'un domaine de reconnaissance de ldl oxydee du recepteur de ldl oxydee lox-1, sa stereostructure et son utilisation - Google Patents

Cristal d'un domaine de reconnaissance de ldl oxydee du recepteur de ldl oxydee lox-1, sa stereostructure et son utilisation Download PDF

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WO2005054460A1
WO2005054460A1 PCT/JP2004/017890 JP2004017890W WO2005054460A1 WO 2005054460 A1 WO2005054460 A1 WO 2005054460A1 JP 2004017890 W JP2004017890 W JP 2004017890W WO 2005054460 A1 WO2005054460 A1 WO 2005054460A1
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lox
binding fragment
ligand
atomic coordinates
ligand binding
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PCT/JP2004/017890
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English (en)
Japanese (ja)
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Izuru Ohki
Tomoko Ishigaki
Takuji Oyama
Kosuke Morikawa
Shin-Ichi Tate
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to JP2005515952A priority Critical patent/JPWO2005054460A1/ja
Publication of WO2005054460A1 publication Critical patent/WO2005054460A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present invention relates to the fields of pharmaceuticals and three-dimensional structures. More particularly, the present invention relates to a novel crystal of an oxidized LDL (Low Density Lipoprotein) receptor LOX-1 and its use in drug search.
  • LDL Low Density Lipoprotein
  • the present invention relates to a crystal of an oxidized LDL recognition domain of an oxidized LDL receptor protein useful for the development of a medicament for preventing or treating arteriosclerosis, and an X-ray crystal structure analysis using the crystal.
  • the three-dimensional structure of the resulting oxidized LDL recognition domain is a crystal of an oxidized LDL recognition domain of an oxidized LDL receptor protein useful for the development of a medicament for preventing or treating arteriosclerosis.
  • Arteriosclerosis is a vascular disease that causes angina, myocardial infarction, obstructive arteriosclerosis, cerebrovascular disorder, renal failure and is life-threatening. Atherosclerotic changes in blood vessels are referred to as aging of the blood vessels, which progressively progresses with age, and is a culprit that threatens the quality of life of older people because it causes the various diseases described above. Prevention of sclerosis * Development of treatments is an important issue in today's aging society. Oxidized LDL (low density lipoprotein) in the blood is a major causative factor in the development of atherosclerosis.
  • Oxidized LDL low density lipoprotein
  • Non-patent Document 4 Suzuki, T. et al., Clinical Biochemistry 35, 347-353 (2002)
  • Oxidized LDL in blood binds to LOX-1 which is an oxidized LDL receptor on vascular endothelial cells.
  • ROS reactive oxygen species
  • Non-Patent Document 6 Am J Physiol Cell Physiol. 280, C719-C741 (2001)) Because of this, the connection between vascular endothelial cells is weakened and a gap is created in the vascular wall.
  • the Shiroi LDL has the function of dissociating monocytes that have entered the vascular endothelial tissue into macrophages, and at the same time, further proliferating the macrophages (Non-Patent Document 8: Martens, JS et al. J. Biol. Chem. 274, 10903-10910 (1999)).
  • Non-Patent Document 8 Martens, JS et al. J. Biol. Chem. 274, 10903-10910 (1999)
  • scavenger receptors CD36, LOX-1, etc.
  • Non-Patent Document 9 Krieger, MJ Biol. Chem. 268, 4569-4572 (1993)).
  • LOX-1 on vascular endothelial cells also increases LOX-1 expression on vascular endothelial cells through a similar oxidative stress response by binding to oxidized LDL. It is thought that the above-mentioned vascular endothelial cell response starting from binding to 1 may progress autonomously.
  • LOX-1 Since LOX-1 is also known to have cell adhesion activity, the presentation of a large amount of LOX-1 on vascular endothelial cells promotes the retention of monocytes in the blood on the blood vessel wall. It also promotes the invasion of monocytes into the vascular endothelial tissue. In this respect, LOX-1 can be said to be more effective in promoting the onset of arteriosclerosis.
  • LOX-1 is present not only in large amounts on vascular endothelial cells and macrophages, but also on vascular smooth muscle cells as described above, and oxidized LDL generated in vascular endothelial tissue is Apoptosis against vascular smooth muscle cells by binding to LOX-1 It has been shown to induce monocis (Non-Patent Document 10: Kataoka, H. et al, Arteriosclear Thromb Vase Biol. 21, 955-960 (2001)). In other words, LOX-1 is involved in the entire onset of arteriosclerosis, including the induction of arteriosclerosis, from macrophage foaming to smooth muscle cell apoptosis induction.
  • a recombinant expression method of a protein is well known as a method for preparing a large amount of a desired protein and producing crystals. Recombinant expression has made it possible to prepare large quantities of proteins that are naturally available and difficult to prepare in large quantities.
  • a receptor present on a cell surface is a protein that specifically binds to a ligand corresponding to the receptor and transmits various signals into a cell, and has a low expression level per cell. It is difficult to prepare large quantities from natural sources. Large quantities can be prepared using recombinant expression methods.
  • Receptors present on the cell surface are diverse, and their corresponding ligands are also different. Therefore, in order to detect and Z or quantify a specific ligand, a receptor that specifically binds to that ligand must be used. It is useful to prepare and use in large quantities. By immobilizing the receptor or receptor fragment prepared in large quantities on a solid phase and preparing a receptor chip using a diagnostic marker for abnormal cells or disease as a ligand, the presence of abnormal cells in the cell population It is expected that a useful tool for the detection of or the diagnosis of a disease can be provided.
  • the existence of a plurality of receptors capable of recognizing and binding denatured LDL, abnormal cells such as apoptotic cells and senescent erythrocytes, and bacteria invading the living body has been discovered. Many of these receptors are expected to have a region required for recognition of a target (ligand) to be recognized. These receptors themselves, or If only the regions required for recognition are used, it may be possible to easily detect abnormal cells such as denatured LDL, apoptotic cells, and bacteria, which are ligands.
  • the binding between the receptor and its corresponding ligand is specific, a material that removes the ligand by immobilizing the receptor or the ligand-binding fragment thereof expressed in large amounts on a solid phase. It is possible to provide. For example, by using a receptor for degenerated LDL to remove degenerated LDL in the blood, it is possible to treat diseases such as arteriosclerosis and hyperlipidemia caused by abnormal LDL dynamics.
  • Host cells for recombinant expression include bacterial cells, animal cells, plant cells, fungal cells, and the like.
  • bacterial cells such as Escherichia coli are widely used as host cells because of their rapid growth rate, relatively simple operation, and low preparation cost, and are particularly suitable for production on an industrial scale.
  • the product protein is often accumulated as an insoluble substance in the cell as an inclusion body. This inclusion body is insoluble, and the three-dimensional structure of the inclusion body protein is different from the three-dimensional structure of the protein in a natural state, and thus requires soluble and refolding.
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2002-306163
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2002-306163
  • the purity used in the soluble and refolding steps is reduced due to contamination of the refolded protein with substances to be used, and the presence of incompletely refolded protein is reduced. Is a problem.
  • the decrease in purity and the heterogeneity can be detected as a low specific activity of the refolded protein as compared with the natural protein.
  • Non-patent Document 13 Daugherty, D ⁇ . Et al. (1998) J. Biol. Chem. 273). , 3396 ⁇ 33971; Sundari.CS et al. (1999) FEBS, Lett., 443, 215-219; ⁇ 135).
  • a conventional refolding method can be used to obtain a highly purified and homogeneous refolded protein. Lack of preparation results in reduced sensitivity in qualitative and quantitative detection. Also, when the ligand-binding fragment of the receptor is refolded and used as a material for removing ligands present in blood, for example, when the purity and homogeneity of the refolded protein are low, In this case, substances other than the target ligand are removed by interaction with other factors in the blood.
  • Patent Document 1 JP 2003-169693
  • Patent document 2 JP-A-2002-306163
  • Non-patent document l Daugherty, D ⁇ . Et al. (1998) J. Biol. Chem. 273, 3396-33971
  • Non-patent document 2 Sundari, C.S. et al. (1999) FEBS, Lett., 443, 215-219
  • Non-Patent Document 3 Machida, S. et al. (2000) FEBS, Lett., 486, 131-135
  • Non-patent document 4 Suzuki, T. et al, Clinical Biochemistry 35, 347-353 (2002)
  • Non-patent document 5 Cominacini, et al "J. Biol. Chem. 275, 12633-12638 (2000)
  • Non-patent document 6 Am J Physiol Cell Physiol. 280, C719-C741 (2001)
  • Non-Patent Document 7 Khan BV et al., J Clin Invest, 95, 1262-1270 (1995)
  • Non-Patent Document 8 Martens, JS et al. J. Biol. Chem. 274, 10903—10910 (1999)
  • Non-Patent Document 9 Krieger, MJ Biol. Chem. 268, 4569-4572 (1993)
  • Non-Patent Document 10 Kataoka, H. et al, Arteriosclear Thromb Vase Biol. 21, 955-960
  • An object of the present invention is to elucidate the three-dimensional structure of the oxidized LDL-binding domain of LOX-1 in order to elucidate the details of the binding mode of the oxidized LDL receptor LOX-1 to oxidized LDL.
  • elucidation of the three-dimensional structure of the CTLD-binding domain as a dimeric structure that maintains the same morphology as existing on the cell surface is the ultimate task.
  • CTLD C-type lectin-like domain
  • LOX-1 oxidizing LDL-binding domain existing as a dimer and the NECK domain (transmembrane region
  • the protein comprising the protein disclosed in the present specification (particularly the step of adding a buffer having a buffer region at an acidic pH to the solution, and adding calcium or calcium to the solution).
  • the method includes a step of reducing zinc ions.
  • the method includes a step of adding a buffer having a buffer region at a neutral pH to the dimer LOX-1.
  • the problem is solved by obtaining a crystal obtained by using a sample obtained by unwinding a protein expressed in large amounts in Escherichia coli according to /, and
  • An object of the present invention is to elucidate the details of the binding mode of the LOX-1 LDL-1 receptor to LDL-1 by elucidating the three-dimensional structure of the oxidized LDL-binding domain of LOX-1. To do that.
  • the application of X-ray crystal structure analysis method is indispensable. For this purpose it is necessary to create crystals that give high resolution diffraction images of the LOX-1 oxidized LDL binding domain.
  • the present inventors have prepared a large amount of LOX-1 LDL-binding domain, crystallized the LOX-1 LDL-binding domain of LOX-1 having a crystal form that gives high-resolution X-ray diffraction images, LOX-1 oxidation
  • the purpose of this study is to clarify the three-dimensional structure of the LDL-binding domain.
  • Still another object of the present invention is to provide a method for screening a compound capable of binding to LOX-1 and inhibiting or activating its activity. Means for solving the problem
  • the oxidized LDL-binding domain (CTLD: C Type Lectin like Domain) of LOX-1 used for crystallization is disclosed in Japanese Patent Application No. 2003-342645 of the present inventors and disclosed in the present specification.
  • a P2 222 orthorhombic LOX-1 oxidized LDL-binding domain has been reported as an example of the purity of LOX-1 CTLD obtained by this protein unwinding technique, and was obtained from different crystallization conditions.
  • the present invention provides a crystal of LOX-1 CTLD consisting of natural amino acids and its three-dimensional structure.
  • the above-mentioned problem is solved by providing a crystal and a structure of a ligand binding domain of LOX-1 which retains a dimer structure by disulfide bond in the same form as that existing on the cell surface.
  • the LOX-1 crystal structure that retains the natural dimer structure has a cavity structure at the dimer interface, and amino acid substitutions near the cavity have a dramatic effect on LOX-1 modified LDL. Lost his binding ability. This suggests that the cavities present at the dimer interface may be target sites for LOX-1 specific inhibitors.
  • the present invention provides the following.
  • a method comprising:
  • a data array comprising the atomic coordinates of a LOX-1 ligand binding fragment or a homolog or variant thereof, wherein the data array represents a three-dimensional structure by using a three-dimensional molecular modeling algorithm. Data array.
  • the atomic coordinates include the atomic coordinates of acetylated LDL, and the atomic coordinates of acetylated LDL-complexed LOX-1 ligand binding fragment shown in FIG. 7 or homologs or variants thereof.
  • LOX A computer-readable recording medium that encodes the atomic coordinates of a ligand-binding fragment or a homolog or variant thereof.
  • the atomic coordinates include the atomic coordinates of acetylated LDL, and are the acetylated LDL-complexed LOX-1 ligand-binding fragment atomic coordinates shown in FIG. 7 or homologs or variants thereof.
  • the atomic coordinates include the atomic coordinates of the acetylated LDL, and are the atomic coordinates of the acetylated LDL-complexed LOX-1 ligand-binding fragment shown in FIG. 7 or a homolog or a variant thereof.
  • the protein according to claim 14 which comprises an activity of a LOX-1 ligand-binding fragment, or a homologue or variant thereof.
  • a protein comprising an active pocket of a LOX-1 ligand-binding fragment or a homolog or a variant thereof.
  • the active pocket is defined by the atomic coordinates of the amino acid residue in SEQ ID NO: 18 or the corresponding amino acid residue corresponding to the change before and after the presence or absence of acetiluidani LDL in FIG. 8.
  • the active pocket is defined by the atomic coordinates of positions 208 to 231 of SEQ ID NO: 4 or the corresponding amino acid residue.
  • a method for obtaining a homologue of a LOX-1 ligand-binding fragment comprising comparing the atomic coordinates of a candidate compound with the atomic coordinates of a LOX-1 ligand-binding fragment.
  • a method for obtaining a homologue of a LOX-1 ligand binding fragment comprising the following steps:
  • a method comprising:
  • a method for identifying a compound capable of binding to a LOX-1 ligand binding fragment or a homolog or a variant thereof, comprising the following steps:
  • the spatial coordinates of the set of candidate conjugates are electronically screened against the spatial coordinates of the active site pocket of the LOX-1 ligand binding fragment or homolog or variant thereof, and (1) a step of identifying a compound capable of binding to a ligand binding fragment or a homolog or a variant thereof,
  • a method comprising:
  • the spatial coordinates determined in the step a) are defined by the atomic coordinates of the amino acid residue shown in SEQ ID NO: 18 or the corresponding amino acid residue. The method described in 1.
  • the two structures are compared by comparing the distance between the heteroatom forming a hydrogen bond in the candidate conjugate and the heteroatom forming the active site pocket in the three-dimensional molecular model. Identifying the candidate compound species that theoretically forms a stable complex with the active site pocket of the three-dimensional molecular model of the LOX-1 ligand binding fragment, based on the optimal hydrogen bond between
  • a method comprising:
  • the candidate compound inhibits LDL-binding activity of a LOX-1 ligand-binding fragment 58.
  • the method of claim 57 having the activity of:
  • a pharmaceutical composition comprising a compound identified by the method according to claim 57 as an active ingredient.
  • a pharmaceutical composition for treating or preventing a disease or disorder associated with a LOX1 ligand-binding fragment comprising a compound identified by the method according to claim 57 as an active ingredient.
  • a recording medium including a database, encoding data including a name and a structure of a compound identified by the method according to claim 57.
  • a transmission medium including a database, encoding data including a name and a structure of a compound identified by the method according to claim 57.
  • a method comprising:
  • the drug candidate molecule according to claim 73 which has an activity of inhibiting the LDL-binding activity of a LOX-1 ligand-binding fragment.
  • a pharmaceutical composition comprising the compound according to claim 73 or 74.
  • a method for preparing a pharmaceutical composition for treating or preventing a disease or disorder related to LOX-1 comprising:
  • a method comprising:
  • the method according to claim 76 further comprising: (81) synthesizing the compound species to produce the compound species.
  • a method comprising:
  • a recording medium comprising:
  • a computer for performing a method for obtaining a homolog of a LOX-1 ligand binding fragment comprising: A) means for comparing the atomic coordinates of the candidate compound with the atomic coordinates of the LOX-1 ligand binding fragment;
  • a computer-readable recording medium on which a program for causing a computer to execute a method for obtaining a homolog of a LOX-1 ligand binding fragment is recorded, the method comprising:
  • a recording medium comprising:
  • a computer for performing a method for obtaining a homolog of a LOX-1 ligand binding fragment comprising:
  • a program for causing a computer to execute a method for obtaining a variant of a LOX-1 ligand-binding fragment comprising: A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the LOX-1 ligand binding fragment to obtain a three-dimensional molecular model; and
  • a computer-readable recording medium having recorded thereon a program for causing a computer to execute a method for obtaining a modified LOX-1 ligand-binding fragment, the method comprising:
  • a computer for performing a method for obtaining a variant of a LOX-1 ligand binding fragment comprising:
  • a computer-readable recording medium recording a program for causing a computer to execute a method for identifying a compound capable of binding to a LOX-1 ligand-binding fragment or a homolog or a variant thereof, the method comprising: ,
  • the spatial coordinates of the set of candidate conjugates are electronically screened against the spatial coordinates of the active site pocket of the LOX-1 ligand binding fragment or homolog or variant thereof, and (1) a step of identifying a compound capable of binding to a ligand binding fragment or a homolog or a variant thereof,
  • a recording medium comprising:
  • the spatial coordinates of the set of candidate conjugates are electronically screened against the spatial coordinates of the active site pocket of the LOX-1 ligand binding fragment or homolog or variant thereof, and (1) a step of identifying a compound capable of binding to a ligand binding fragment or a homolog or a variant thereof,
  • the two structures are compared by comparing the distance between the heteroatom forming a hydrogen bond in the candidate conjugate and the heteroatom forming the active site pocket in the three-dimensional molecular model. Identifying the candidate compound species that theoretically forms a stable complex with the active site pocket of the three-dimensional molecular model of the LOX-1 ligand binding fragment, based on the optimal hydrogen bond between
  • a computer-readable recording medium recording a program for causing a computer to execute a method for identifying a compound capable of binding to a LOX-1 ligand-binding fragment or a homolog or a variant thereof, the method comprising: Is
  • the two structures are compared by comparing the distance between the heteroatom forming a hydrogen bond in the candidate conjugate and the heteroatom forming the active site pocket in the three-dimensional molecular model. Identifying the candidate compound species that theoretically forms a stable complex with the active site pocket of the three-dimensional molecular model of the LOX-1 ligand binding fragment, based on the optimal hydrogen bond between
  • a recording medium comprising:
  • a computer for executing a method for identifying a compound capable of binding to a LOX-1 ligand-binding fragment or a homolog or a variant thereof, the method comprising: A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the LOX-1 ligand binding fragment or the atomic coordinates of a homolog or variant thereof to obtain a three-dimensional molecular model;
  • the two structures are compared by comparing the distance between the heteroatom forming a hydrogen bond in the candidate conjugate and the heteroatom forming the active site pocket in the three-dimensional molecular model. Identifying the candidate compound species that theoretically forms a stable complex with the active site pocket of the three-dimensional molecular model of the LOX-1 ligand binding fragment, based on the optimal hydrogen bond between
  • a method comprising:
  • a data array comprising the atomic coordinates of a LOX-1 ligand binding fragment or homolog or variant thereof in dimeric form, wherein said data array is obtained by using a three-dimensional molecular modeling algorithm.
  • a computer readable recording medium that encodes the atomic coordinates of a LOX-1 ligand binding fragment or a homolog or variant thereof in a dimeric form.
  • LOX-1 A protein comprising a cavity structure existing at the dimer interface of a ligand-binding fragment or a homolog or a variant thereof.
  • a method for obtaining a homolog of a dimer of a LOX-1 ligand binding fragment wherein the above-mentioned method comprises the atomic coordinates of a candidate compound and the atomic coordinates of a dimer interface of a LOX-1 ligand binding fragment.
  • a method comprising: comparing with
  • a method for obtaining a dimeric homolog of a LOX-1 ligand binding fragment comprising: The notation method is as follows:
  • a method comprising:
  • a method for obtaining a dimeric variant of a LOX-1 ligand binding fragment comprising the following steps:
  • a method for identifying a compound capable of binding to a LOX-1 ligand binding fragment or a homolog or a variant thereof comprises the following steps: A) Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the dimer of the LOX-1 ligand-binding fragment or its homologs or variants to form a dimer of the LOX-1 ligand-binding fragment Determining the spatial coordinates of the dimer interface to be performed; and
  • polypeptide forming the dimer of the LOX-1 ligand-binding fragment comprises the sequence shown in SEQ ID NO: 36.
  • polypeptide which forms the dimer of the LOX-1 ligand-binding fragment is the sequence shown in SEQ ID NO: 4, in which the cavity-forming portion containing tributophan (W) at position 150 is conserved. The method described in.
  • a method comprising:
  • a pharmaceutical composition for treating or preventing a disease associated with LOX-1 comprising a conjugate identified by the method according to item 125 or 131 as an active ingredient.
  • a recording medium including a database, which codes data including the name and structure of the compound identified by the method described in Item 125 or 131.
  • a transmission medium including a database, which codes data including the name and structure of the compound identified by the method described in Item 125 or 131.
  • a method comprising:
  • a pharmaceutical thread for treating or preventing a disease or disorder related to LOX-1 A method of preparing a composition, comprising:
  • a method comprising:
  • a method comprising:
  • a compound capable of binding to LOX1 is identified by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of a dimer-forming LOX-1 ligand-binding fragment or a homolog thereof.
  • a computer-readable recording medium on which a program for causing a computer to execute the method is recorded.
  • high-resolution LOX-1 crystal information can be obtained by using a specific refolding method, and the information can be applied to drug development to treat and treat cardiovascular diseases.
  • the effect of prevention can be achieved.
  • FIG. 1 shows that the protein obtained by the refolding method of the present invention has a narrower range of molecular weight than the conventional refolding method using cyclic carbohydrate cycloamylose, and thus is purified with higher purity. Show that you have been.
  • Fig. 2 shows that the protein obtained by the refolding method of the present invention is purified to a higher purity by the conventional cyclic saccharide cycloamylose with fewer contaminants than the refolding method. .
  • Fig. 3 shows that the protein obtained by the refolding method of the present invention is refolded as a single molecular species, whereas the protein obtained by the conventional method is incomplete refolding. This indicates that the mixed proteins are present.
  • FIG. 4 shows that proteins obtained by the refolding method of the present invention are free of incomplete refolding proteins.
  • FIG. 5 shows that LOX-1 CTLD refolded using the refolding method of the present invention has the same level of affinity for oxidized LDL and acetylated LDL as natural LOX-1. Show that you have.
  • FIG. 6 shows the protein strength obtained as a result of refolding the entire extracellular domain of LOX-1 by the refolding method of the present invention. Indicates a body.
  • 8 Me (+) indicates that
  • ⁇ Me (1) indicates that ⁇ -mercaptoethanol was not added
  • Fig. 7 shows the atomic coordinates of LOX-1.
  • Figure 7 shows the atomic coordinates of LOX-1 (continued) ⁇
  • FIG. 7 shows the atomic coordinates of LOX- -I (continued) ⁇
  • Figure 7 shows the atomic coordinates of LOX- -I (continued) ⁇
  • Figure 7 shows the atomic coordinates of LOX- -I (continued) ⁇
  • Figure 7 shows the atomic coordinates of LOX- -I (continued) ⁇
  • FIG. 7 shows the atomic coordinates of LOX- -I (continued) ⁇
  • Figure 7 shows the atomic coordinates of LOX- -I (continued) ⁇
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Fig. 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • FIG. 7-12 Fig. 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • Figure 7 shows the atomic coordinates of LOX-1 (continued).
  • FIG. 8 shows an NMR chart of LOX-1 with and without acetylated LDL.
  • FIG. 9 shows an example of crystallization optimization.
  • FIG. 10 shows the results obtained as a result of crystallization.
  • FIG. 11 shows the LDL-binding domain of LOX-1.
  • FIG. 12 is a system configuration example of the present invention.
  • FIG. 13 is an example of a recording medium of the present invention.
  • FIG. 14 is an example of an optical recording medium of the present invention.
  • FIG. 15 shows the alignment of LOX-1 from various sources.
  • FIG. 16 is a model diagram in a case where CTLD-NECK forming a dimer of the present invention is used. At the dimer interface, small cavities are present, which appear to play a critical role in dimer binding and activation. Therefore, it became clear that this site was used as a conventional site of a drug interaction site.
  • LO LO
  • CTLD Indicates the presence of cavities observed at the interface of the dimer structure of NECK. Variants of W150, the major amino acid residue surrounding this cavity, are prominent in LOX
  • the conjugate which selectively binds and disturbs the structure of the dimer interface is considered to be a LOX-1-selective oxidized LDL binding inhibitor.
  • the image is shown below. The mechanism of the binding inhibitory action at this time is that the structure of the dimer interface is disturbed, and the basic spine structure (basic spine) (right Figure) is disturbed and prevents sufficient binding of LOX-1 to oxidized LDL.
  • Fig. 17-1 shows the atomic coordinates of LOX-1 in pdb format when CTLD-NECK was used.
  • Figure 17-_2 is a continuation of Figure 17--1.
  • FIG. 17-_3 is a continuation of FIG. 17--2.
  • FIG. 17-_4 is a continuation of FIG. 17--3.
  • FIG. 17-_5 is a continuation of FIG. 17--4.
  • FIG. 17-_6 is a continuation of FIG. 17--5.
  • FIG. 17-_7 is a continuation of FIG. 17--6.
  • FIG. 17-_8 is a continuation of FIG. 17--7.
  • FIG. 17-_9 is a continuation of FIG. 17--8.
  • Figure 17--10 is a continuation of Figure 17--9.
  • Fig. 17--11 is a continuation of Fig. 17--10.
  • Fig. 17--12 is a continuation of Fig. 17--11.
  • Figure 17--13 is a continuation of Figure 17--12.
  • Figure 17--14 is a continuation of Figure 17--13.
  • Figure 17--15 is a continuation of Figure 17--14.
  • Figure 17--16 is a continuation of Figure 17--15.
  • Fig. 17--17 is a continuation of Fig. 17--16.
  • Figure 17--18 is a continuation of Figure 17--17.
  • Figure 17--19 is a continuation of Figure 17--18.
  • Figure 17--20 is a continuation of Figure 17--19.
  • Fig. 17--21 is a continuation of Fig. 17--20.
  • Fig. 17--22 is a continuation of Fig. 17--21.
  • Figure 17--23 is a continuation of Figure 17--22.
  • Figure 17--24 is a continuation of Figure 17--23.
  • Figure 17--25 is a continuation of Figure 17--24.
  • FIG. 17- -26; Fig. 17- -26 is a continuation of Fig. 17--25.
  • FIG. 17- -27; Fig. 17- -27 is a continuation of Fig. 17- -26.
  • FIG. 17- -28; Fig. 17- -28 is a continuation of Fig. 17- -27.
  • FIG. 17- -29 is a continuation of Fig. 17- -28.
  • FIG. 17--30; Fig. 17--30 is a continuation of Fig. 17- -29.
  • FIG. 17- -31; Fig. 17- -31 is a continuation of Fig. 17--30.
  • FIG. 17- -32; Fig. 17- -32 is a continuation of Fig. 17- -31.
  • FIG. 17- -33; Fig. 17- -33 is a continuation of Fig. 17- -32.
  • FIG. 17- -34; Fig. 17- -34 is a continuation of Fig. 17- -33.
  • FIG. 17- -35; Fig. 17- -35 is a continuation of Fig. 17- -34.
  • FIG. 17- -36; Fig. 17- -36 is a continuation of Fig. 17- -35.
  • FIG. 17- -37; Fig. 17- -37 is a continuation of Fig. 17- -36.
  • FIG. 17- -38; Fig. 17- -38 is a continuation of Fig. 17- -37.
  • FIG. 17- -41; Fig. 17- -41 is a continuation of Fig. 17--40.
  • FIG. 17- -42; Fig. 17- -42 is a continuation of Fig. 17- -41.
  • FIG. 17- -43; Fig. 17- -43 is a continuation of Fig. 17- -42.
  • SEQ ID NO: 1 shows the nucleic acid sequence of a human LOX-1 ligand binding fragment (CTLD).
  • SEQ ID NO: 2 shows the amino acid sequence of a human LOX-1 ligand binding fragment.
  • SEQ ID NO: 3 shows the nucleic acid sequence of human LOX-1.
  • SEQ ID NO: 4 shows the amino acid sequence of human LOX-1.
  • SEQ ID NO: 5 shows the nucleic acid sequence of the extracellular region of human LOX-1.
  • SEQ ID NO: 6 shows the amino acid sequence of the extracellular region of human LOX-1.
  • SEQ ID NO: 7 shows the amino acid sequence of Pyotin-dani motif.
  • SEQ ID NO: 8 is another example of the amino acid sequence of Pyotin-dani motif.
  • SEQ ID NO: 9 is another example of the amino acid sequence of Pyotin-dani motif.
  • SEQ ID NO: 10 is another example of the amino acid sequence of Pyotin-dani motif.
  • SEQ ID NO: 1111 is an example of an amino acid sequence of a Factor Xa recognition sequence.
  • SEQ ID NO: 12 is an example of the amino acid sequence of the enterokinase recognition sequence.
  • SEQ ID NO: 13 is an example of a cellulose binding domain tag.
  • SEQ ID NO: 14 is an example of a calmodulin-binding peptide tag.
  • SEQ ID NO: 15 is an example of an S protein binding peptide tag.
  • SEQ ID NO: 16 is an example of a T7 tag.
  • SEQ ID NO: 17 shows the nucleic acid sequence of the active pocket of hLOX-1.
  • SEQ ID NO: 18 shows the amino acid sequence of the active pocket of hLOX-1 (positions 191 to 240 of SEQ ID NO: 4).
  • SEQ ID NO: 19 shows the nucleic acid sequence of a ligand-binding fragment of LOX-1 of Egret (rabbit).
  • SEQ ID NO: 20 shows the amino acid sequence of a ligand binding fragment of LOX-1 of Egret (rabbit).
  • SEQ ID NO: 21 shows the nucleic acid sequence of LOX-1 of Egret (rabbit).
  • SEQ ID NO: 22 shows the amino acid sequence of LOX-1 of Egret (rabbit).
  • SEQ ID NO: 23 shows the nucleic acid sequence of a porcine LOX-1 ligand binding fragment.
  • SEQ ID NO: 24 shows the amino acid sequence of a porcine LOX-1 ligand binding fragment.
  • SEQ ID NO: 25 shows the nucleic acid sequence of pig LOX-1.
  • SEQ ID NO: 26 shows the amino acid sequence of porcine LOX-1.
  • SEQ ID NO: 27 shows the nucleic acid sequence of a mouse LOX-1 ligand binding fragment.
  • SEQ ID NO: 28 shows the amino acid sequence of a mouse LOX-1 ligand binding fragment.
  • SEQ ID NO: 29 shows the nucleic acid sequence of mouse LOX-1.
  • SEQ ID NO: 30 shows the amino acid sequence of mouse LOX-1.
  • SEQ ID NO: 31 shows the nucleic acid sequence of a rat LOX-1 ligand binding fragment.
  • SEQ ID NO: 32 shows the amino acid sequence of a rat LOX-1 ligand binding fragment.
  • SEQ ID NO: 33 shows the nucleic acid sequence of rat LOX-1.
  • SEQ ID NO: 34 shows the amino acid sequence of rat LOX-1.
  • SEQ ID NO: 35 shows the nucleic acid sequence of CTLD-NECK.
  • SEQ ID NO: 36 shows the amino acid sequence of CTLD-NECK (corresponding to amino acid numbers 129 to 273 of SEQ ID NO: 4).
  • LOX-1 is an abbreviation for lectin-like LDL receptor 1, and refers to a receptor of the LDL receptor-related protein family, which is a type of scavenger receptor.
  • LOX-1 is a very inducible gene, and its expression is induced under conditions such as hypertension, hyperlipidemia, and diabetes that promote arteriosclerosis.
  • oxidized LDL acts on vascular endothelial cells via LOX-1, it causes the production of active oxygen and the accompanying decrease in NO activity.
  • the expression of a cell adhesion molecule, chemokine is also induced, and a state of so-called endothelial dysfunction is caused by bow I.
  • LDL undergoes oxidative degeneration in vascular endothelial cells and vascular smooth muscle cells. Oxidized LDL is taken up and degraded by lectin-like oxidized LDL receptor LOX-1 in vascular endothelial cells. In addition, it is taken up by macrophages by the oxidized LDL decavenger receptor and foams the macrophages.
  • Representative LOX-1 sequences are shown in SEQ ID NOs: 3 and 4 (nucleic acid and amino acid sequences, respectively; from human aortic endothelial cells).
  • Representative other animal origins of LOX-1 include, for example, SEQ ID NOS: 21 and 22 (perhaps rabbit (rabbit) nucleic acid and amino acid sequences, respectively), SEQ ID NOs: 25 and 26 (porcine nucleic acid and And amino acid sequences), SEQ ID NOs: 29 and 30 (mouse nucleic acid and amino acid sequences, respectively), and SEQ ID NOs: 33 and 34 (rat nucleic acid and amino acid sequences, respectively).
  • the term "dimer” refers to a molecule formed by the same species or similar molecules interacting to form a complex. Examples of such an interaction include a covalent bond, a hydrogen bond, and the like.
  • the LOX-1 of the present invention is preferably a covalent bond via a disulfide bond. LOX-1 is said to exhibit a dimer structure through disulfide bonds when naturally occurring or when present on the cell surface, and exhibit the above activity. Therefore, analyzing the dimer plays an important role in inhibiting or activating LOX-1.
  • the dimer of LOX-1 preferably contains 14 residues (129 to 142 of SEQ ID NO: 4) of the NECK region (amino acids 60 to 142 of SEQ ID NO: 4).
  • 14 residues 129 to 142 of SEQ ID NO: 4
  • amino acids 60 to 142 of SEQ ID NO: 4 amino acids 60 to 142 of SEQ ID NO: 4.
  • dimer interface refers to a region that interacts with the other monomer when LOX-1 forms a dimer.
  • a typical example is the NECK domain, but is not limited thereto.
  • NECK region refers to a region of SEQ ID NO: 4 (amino acid sequence of human LOX-1) which is in a disulfide bond necessary for dimer formation, Typically, it refers to the region of amino acid number 60-142. In the present invention, it is particularly preferable to include 14 residues (129 to 142 of SEQ ID NO: 4). In addition to this, tryptophan of amino acid number 150 of SEQ ID NO: 4 and position 144 of CTLD region It is desirable that the 155th cysteine is preserved.
  • the LOX-1 crystal structure which retains the natural dimer structure, has a cavity structure present at the dimer interface and an amino acid substitution present near the cavity dramatically alters the LOX-1 modified LDL. It was found that the binding ability was lost. Therefore, it became clear that the cavities existing at the dimer interface of LOX-1 can be a target site for LOX-1 specific inhibitors.
  • LDL low-density lipoprotein that promotes arteriosclerosis.
  • a disease or disorder associated with LOX-1 refers to a disease or disorder caused by abnormal levels or properties of LDL due to LOX-1 or another abnormal state.
  • diseases or disorders include general diseases of the circulatory system, for example, circulatory diseases caused by arteriosclerosis such as arteriosclerosis, angina, myocardial infarction, and cerebral infarction. It is not limited to. Therefore, in the present invention, even if the activity of LOX-1 itself is normal, abnormal levels of LDL are within the range of diseases or disorders associated with LOX-1.
  • the term "regulates the activity of LOX-1" means that when used for a certain LOX-1, usually the activity of the protein is changed to an activity that does not normally have the activity of the protein. . Thus, regulation includes an increase (increase) or decrease in LOX-1 activity (eg, the ability to bind LDL).
  • prevention refers to treatment of a disease or disorder before such condition is caused so that the condition does not occur.
  • treatment refers to the prevention of a disease or disorder from occurring when such a disease or disorder occurs, preferably the status quo is maintained. More preferably, it means reducing, and more preferably reducing.
  • candidate compound refers to a candidate compound that can be used for treating a disease or disorder of interest.
  • a compound may be referred to as a candidate compound if it is expected to be effective for the disease or disorder of interest.
  • compound species refers to one compound having desired properties, such as having a specific target activity, for a set of compounds. For example, if a compound that modulates LOX-1 activity is identified in a collection of compounds that modulate LOX1 activity, such compound may be referred to as a compound species. In this specification, it is simply referred to as a compound.
  • the term "library” refers to a compound such as a compound for screening.
  • a fixed set The library 1 may be a set of compounds having similar properties or a set of random compounds.
  • use is made of, but not limited to, a collection of compounds that are expected to have similar properties.
  • interaction refers to an action between a certain molecule and another molecule when two or more molecules exist. Such interactions include, but are not limited to, hydrogen bonding, van der Derska, ionic interactions, non-ionic interactions, receptor ligand interactions, electrostatic interactions and host-guest interactions. In the screening of the present invention, in particular, hydrogen bonds may be used.
  • evaluation refers to, when used in screening, determining whether or not a candidate compound has the ability to satisfy the requirements of an index (eg, activity as a medicine) for such an index. Say. Such an evaluation can be performed using a method known in the art.For example, in the case of LOX-1, the LOX-1 activity was changed by using its ligand, such as Ojiride LDL. You can do it by looking at the power.
  • stereostructure (computer) model of a protein refers to a model of the stereostructure of a certain compound expressed using a combi- ter.
  • Such models can be displayed using computer programs known in the art, such as programs supported by CCP4, DENZO (HK L2000), MolScript (Avatar Software AB) , Raster3D, PyMOL (DeLano Scientific), TURBO—FRODO (AFMB—CNRS), O (A. Jones ⁇ Uppsala U niversitet, Sweden), ImageMagic (John Chrysty), RasMol (University of Massachusetts, Amherst MA USA), etc. But not limited to them.
  • Such a program can generate a model using, for example, data of atomic coordinates as shown in FIG.
  • data array refers to a series of data such as a sequence indicating atomic coordinates.
  • a "recording medium” can be any medium as long as data can be recorded.
  • Such media include, for example, hard disks, MO, CD-R ⁇ CD-RW, CD-ROM, DVD-RAM, DVD-R, DVD-RW ⁇ DVD + RW, Examples include, but are not limited to, DVD-ROMs and memory cards.
  • the "transmission medium” can be any medium as long as data can be transmitted.
  • Such media include, but are not limited to, the Internet, intranets, LANs, WANs, and the like.
  • the "application” refers to a program for using a computer according to each purpose of use.
  • the term "pharmacophore” refers to a combination of atoms and functional groups (and their three-dimensional positions). Enables interaction with proteins and demonstrates their pharmacological activity. A three-dimensional “functional form” formed by the steric (physical) and electric fields of a drug molecule, which results in the pharmacological activity of the molecule.
  • a variety of approaches have been developed to study the medicinal lead conjugates, and the measurable activity of the lid conjugates against specific targets, and a series of structure-activity relationships pharmacophores can be designed.
  • biological test refers to determining whether or not a compound has a certain activity using an actual biological reaction. Biological tests include in vitro and in vivo tests. Therefore, biological testing is an opposite concept to in silico.
  • Antagonist refers to a substance that acts antagonistically on the binding of a biologically active substance (eg, an agonist) to a receptor and manifests itself as a physiological action via that receptor. Refers to substances that do not pass. Antagonists fall into the category of inhibitors.
  • inhibitor refers to a molecule that inhibits the action of a certain biologically active substance.
  • agonist refers to a substance that binds to a receptor of a certain biologically active substance and exhibits the same or similar action as that of the substance.
  • binding pocket refers to a region of a molecule or molecular complex that associates with another chemical or compound as a result of its shape.
  • active pocket refers to a molecule or a molecular complex containing a site capable of interacting with a substrate or a coenzyme when a protein is referred to. Refers to the area of the body.
  • the "acetylated LDL-complexed LOX-1 active site binding pocket” has an acetylated LDL-complexed LOX- A part of a molecule or molecular complex similar to all or any part of one active site binding pocket.
  • the commonality of this shape is, for example, less than 3.OA, preferably less than 3.OA from the skeleton atom of the amino acid constituting the binding pocket in the acetylated LDL-conjugated LOX-1. Is defined by a root mean square deviation of less than 2.5A. The method of obtaining this calculation is as described elsewhere herein.
  • the "active site binding pocket" or "active site” of acetylated LDL-conjugated LOX-1 refers to the region of LOX-1 that is responsible for the binding region of acetylated LDL.
  • the data shown in FIG. 8 can also identify amino acid residues at the binding site. Each of these amino acids can be defined by a set of structural coordinates as shown in FIG.
  • structural coordinates refer to orthogonal coordinates derived from a mathematical expression relating to a pattern obtained on the diffraction of a monochromatic X-ray beam by atoms (scattering centers) of LOX-1 or a complex thereof.
  • the diffraction data is used to calculate an electron density map of the repeating unit of the crystal.
  • the electron density map is then used to determine the position of individual atoms in LOX-1.
  • the set of structural coordinates for an enzyme or enzyme complex such as LOX-1 or a portion thereof is a set of relative points that define a shape in three dimensions. To understand. Thus, a whole different set of coordinates can define similar or identical shapes, and furthermore, slight changes in individual coordinates have little effect on the overall shape. With respect to the binding pocket, these changes are not expected to significantly alter the nature of the ligands that can associate with these pockets.
  • association refers to the condition of proximity between a chemical substance or compound, or a part thereof, and a LOX-1 molecule or a part thereof.
  • the association can be non-covalent (where the point of attachment is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions), or it can be covalent.
  • the above change in coordinates is based on the mathematical formula of acetylated LDL complexed LOX-1 structural coordinates. Can be generated by manual operations.
  • the structural coordinates shown in FIG. 7 can be manipulated by crystallographic substitution of structural coordinates, division of structural coordinates, integer addition or subtraction to a set of structural coordinates, or any combination of the above.
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein, and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotides” or “derivative polynucleotides.” “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide having an unusual force or a bond between nucleotides containing a derivative of a nucleotide, and is used interchangeably.
  • Such an oligonutrient Specifically, for example, 2,1-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, a phosphodiester bond in an oligonucleotide, Is converted to N3, -P5, phosphoramidate bond, derivative oligonucleotide in which ribose and phosphoric acid diester bond are converted to peptide nucleic acid bond, and peracyl in oligonucleotide is C5.
  • oligonucleotide substituted with propynylperacyl derivative oligonucleotide in which peracyl in oligonucleotide is substituted with C-5 thiazole peracyl, derivative oligonucleotide in which cytosine in oligonucleotide is substituted with C5 propyl-cytosine
  • oligonucleotides in which cytosines in oligonucleotides are substituted with phenoxazine-modified cytosine derivatives in which ribose in DNA is substituted with 2,1O-propylribose
  • oligonucleotides Derivative oligonucleotides in which ribose is substituted with 2'-methoxyethoxy ribose.
  • the oligonucleotide may be single-stranded or double-stranded, but preferably used is single-stranded, but the present invention is not limited to single-stranded. Accordingly, in the present specification, the “derivative” of an oligonucleotide refers to an oligonucleotide containing a derivative of a nucleotide as described above. Unless otherwise indicated, a particular nucleic acid sequence may also have conservatively modified variants (e.g., degenerate codon substitutions) and It is contemplated to include complement sequences.
  • degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and a Z or deoxyinosine residue.
  • nucleic acid as used herein also encompasses the concepts of gene, cDNA, mRNA. Certain nucleic acid sequences also include “splice variants.” Similarly, a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of the nucleic acid. As the name suggests, "splice variants" are the products of alternative splicing of a gene. After transfer, The initial nucleic acid transcript can be spliced such that different nucleic acid splice products encode different polypeptides. The mechanism of production of splice variants varies, but involves alternative splicing of exons. Another polypeptide derived from the same nucleic acid by read-through transcription is also included in this definition. Any product of a splicing reaction, including recombinant forms of the splice product, is included in this definition.
  • the terms "protein,” “polypeptide,” “oligopeptide,” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. .
  • the polymer may be linear or branched or cyclic.
  • the amino acids may be naturally occurring or non-naturally occurring or modified amino acids.
  • the term can also be assembled into a complex of multiple polypeptide chains.
  • the term also encompasses naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component).
  • This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptide-like conjugates (eg, ⁇ butoid), and those known in the art. Other modifications are included.
  • polypeptides containing one or more analogs of an amino acid eg, including unnatural amino acids, etc.
  • peptide-like conjugates eg, ⁇ butoid
  • the term "gene” refers to a factor that defines a genetic trait. Usually, they are arranged in a certain order on a chromosome. Structural genes that define the primary structure of a protein, ⁇ , and regulatory genes that control its expression are called. As used herein, in certain circumstances, “gene” refers to "polynucleotide”, “oligonucleotide” and “nucleic acid” and Z or "protein”, “polypeptide”, “oligopeptide” and “peptide”. May be referred to.
  • homology of a gene refers to the degree of identity between two or more gene sequences.
  • the higher the homology between two genes the higher the identity or similarity between their sequences.
  • the ability of the two genes to have homology can be determined by direct sequence comparison or, in the case of nucleic acids, the hybridization method under stringent conditions.
  • the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably Genes are homologous if they are at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical.
  • the comparison of sequence identity, homology and similarity is calculated using BLAST, a sequence analysis tool, with default parameters.
  • the identity search can be performed, for example, using NCBI's BLAST 2.2.9 (issued on May 12, 2004).
  • the value of identity usually refers to a value obtained when the above-mentioned BLAST is used and aligned under default conditions. However, if a higher value appears due to a parameter change, the highest value shall be the value of identity. If the identity is evaluated in multiple areas, the highest value among them is the identity value.
  • polypeptide or nucleic acid of the present invention also has the same identity as the sequence (for example, SEQ ID NO: 3 or 4) in which a wild-type amino acid sequence or nucleic acid sequence is specifically described herein. Not homologous ones can also be used.
  • Such a polypeptide or nucleic acid molecule of the present invention having homology to a wild-type polypeptide or nucleic acid includes nucleic acids, for example, when compared using BLAST default parameters, At least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, A nucleic acid molecule comprising a nucleic acid sequence having about 85%, about 90%, about 95%, about 99% identity or similarity, or at least about 30%, about 35%, about 40%, about 40% for polypeptides 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% identity Or a polypeptide having an amino acid sequence having similarity, but is not limited thereto.
  • expression of a gene, polynucleotide, polypeptide or the like means that the gene or the like undergoes a certain action in vivo and takes on another form.
  • it means that the gene, polynucleotide or the like is transcribed and translated into a polypeptide, but transcription and production of mRNA may also be a form of expression. More preferably, such polypeptide forms may be post-translationally processed.
  • amino acid may be natural or non-natural.
  • “Derivatives” “Amino acid” or “amino acid analog” refers to one which is different from a naturally occurring amino acid but has the same function as the original amino acid. Such derivative amino acids and amino acid analogs are well known in the art.
  • natural amino acid refers to the L isomer of a natural amino acid. Natural amino acids include glycine, alanine, palin, leucine, isoleucine, serine, methionine, threonine, fenylalanine, tyrosine, tryptophan, cystine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, ⁇ -carboxyglutamic acid, and arginine. , Ortin, and lysine. Unless otherwise indicated, all amino acids referred to in the present specification are in the form of L-form amino acids, and the forms using the amino acids are also within the scope of the present invention.
  • unnatural amino acid refers to an amino acid that is not normally found in proteins.
  • unnatural amino acids include D- or L-forms of norleucine, para-tropheninoleanine, homopheninoleanine, para-funoleolopheninoleanine, 3-amino-2-benzylpropionic acid, and homoarginine. And D-phenylalanine.
  • amino acid analog refers to a molecule that is not an amino acid but is similar to the physical properties and amino acids or functions of an amino acid.
  • Amino acid analogs include, for example, etionine, napanin, 2-methylglutamine and the like.
  • Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • nucleotide may be a natural or non-natural.
  • “Derivative nucleotides” or “nucleotide analogs” are those that are different from naturally occurring nucleotides, but that have the same function as the original nucleotides. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-dimethyl ribonucleatide, peptide nucleic acids ( ⁇ ).
  • Amino acids are represented by their commonly known three-letter symbols or by IUPAC-IUB Biochemical. It may be referred to herein by any of the single letter symbols recommended by the Nomenclature Commission. Such abbreviations are as follows:
  • X Xaa Unknown or other amino acid.
  • Nucleotides may also be referred to by the generally accepted one letter code.
  • the "corresponding" amino acid or nucleic acid refers to a predetermined amino acid or a domain thereof in a polypeptide or polynucleotide as a reference for comparison with a certain polypeptide molecule or polynucleotide molecule, respectively. It has an amino acid or nucleic acid or a domain thereof which is predicted to have the same action as that of the enzyme. Particularly, in the case of an enzyme molecule, it is present at the same position in the active site and has the same catalytic activity. Refers to contributing amino acids or their domains or nucleic acids encoding them.
  • nucleic acid and amino acid sequences corresponding to the human LOX-1 ligand binding fragment sequences shown in SEQ ID NOs: 1 and 2 will be apparent to those skilled in the art.
  • corresponding amino acids can also be determined by analyzing the three-dimensional structure. Techniques for such three-dimensional structure analysis are well known to those skilled in the art.
  • corresponding amino acids can also be identified by analyzing complexes with substances that interact with the polypeptide of interest, such as substrates. Such identification methods are also well known to those skilled in the art, and examples of such methods are given in Laube, H. et al. (1992) Biochemistry 31,2735-2748.
  • a "corresponding" gene for example, LOX-1 gene
  • LOX-1 gene has, in a certain species, an activity similar to that of a predetermined gene in a species serving as a reference for comparison, or It refers to a gene that is predicted to have, and when there are a plurality of genes having such an effect, it refers to those having the same evolutionary origin.
  • the corresponding gene of a gene may be an ortholog or species homolog of that gene. Therefore, genes corresponding to human LOX-1 and the like can be found in other animals. Like that The corresponding gene can be identified using techniques well known in the art.
  • a corresponding gene in an animal can be obtained by using the sequence of a gene (eg, human LOX-1) as a reference for the corresponding gene as a query sequence in the animal (eg, mouse, rat, dog, cat). By searching a sequence database.
  • a gene eg, human LOX-1
  • a query sequence in the animal eg, mouse, rat, dog, cat
  • searching a sequence database e.g, a sequence obtained by such a search can also be used.
  • fragment refers to a polypeptide or a polypeptide having a sequence length of up to 11-1 with respect to a full-length polypeptide or polynucleotide (length is n), respectively. Polynucleotide.
  • the length of the fragment can be appropriately changed depending on the purpose.For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 for a polypeptide. , 15, 20, 25, 30, 40, 50 and more amino acids, and the length represented by an integer not specifically listed herein (eg, 11, etc.) is also a lower limit. Can be appropriate.
  • nucleotide 5 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides can be mentioned, and specifically listed here.
  • Non-integer lengths eg, 11, etc.
  • the lengths of polypeptides and polynucleotides can be represented by the numbers of amino acids or nucleic acids, respectively, as described above, but the above-mentioned numbers are not absolute, as long as they have the same function.
  • the above number as an upper limit or adjustment is intended to include a few above and below (or, for example, 10% above and below). In order to express such an intention, in this specification, "about” may be used before a number.
  • a receptor “fragment” specifically binds to a ligand to which a full-length receptor can specifically bind.
  • a preferred fragment of the lectin-like oxidized LDL receptor is a fragment containing a C-type lectin-like region (CTLD).
  • biological activity refers to a factor (for example, a polypeptide or protein).
  • Protein activity refers to the activity that can be possessed in a living body, and includes activities that exert various functions. For example, if a factor is a receptor (eg, LOX-1), its biological activity includes its ligand binding activity (eg, LOX-1 activity). In another example, where an agent is a ligand, the ligand involves binding to the corresponding receptor. Such a biological activity can be measured by techniques well known in the art. Also, when the LOX-1 activity is a biological activity, such activity can be measured as described herein.
  • a method for producing the polypeptide of the present invention for example, a bacterium, which is a prokaryote that produces the polypeptide, is cultured, and the recombinant receptor protein is accumulated in the bacterium as an inclusion body, and the host There is a method for obtaining the polypeptide by destroying bacteria.
  • TEINAPTDGKVEKVLVKERDAVQGGQGLIKIGDLELJ (SEQ ID NO: 7).
  • the amino acid sequence “GLNDIFEAQKIEWHE” (SEQ ID NO: 8) is also available as a biotinylation motif. Even if a mutation is introduced into these sequences other than the K (lysine) residue that is actually subjected to the biotinylation, the sequence in which a substitution other than the lysine residue is substituted also has no significant effect on the biotinylation activity.
  • the recognition sequence "IEGR” (SEQ ID NO: 11) for FactorXa, which is an endoproteinase
  • the recognition sequence "DDDDK” (SEQ ID NO: 12) for enterokinase are used between the biotinidromochi and the foreign protein to be expressed.
  • exogenous protein can be purified by cleavage with FactorXa or enterokinase.
  • the amino acid sequence “IEGR” is inserted between these biotinylated motifs and CTLD. It is also possible to purify only CTLD.
  • transformant refers to all or a part of an organism such as a cell produced by transforming a host cell. Transformants include prokaryotic cells. A transformant is also referred to as a transformed cell, a transformed tissue, a transformed host, or the like, depending on the subject, and in the present specification, refers to a specific form in a specific context. obtain.
  • the host bacterial cell for obtaining the transformant is not particularly limited as long as it expresses a polypeptide having a physiological activity, and various types of host cells conventionally used in genetic engineering are used. Bacterial cells can be used. Prokaryotic cells include prokaryotic cells belonging to the genus Escherichia, Serratia, Batinoles, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia coli XL1-Blue, Escherichia coll XL 2— Blue ⁇ Escherichia coll DHl, Escherichia con M C1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.
  • a certain amino acid in a certain protein is used in the present invention! /
  • the desired modification e.g., substitution in the interaction region with acetylated LDL
  • the desired modification without significant loss or loss of interaction binding capacity, e.g., the cationic region or epitope
  • it can be substituted with another amino acid. It is the protein's ability to interact and its properties that define the biological function of a protein.
  • certain amino acid substitutions may be made in the amino acid sequence, or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after the substitution.
  • various modifications can be made in the peptide disclosed herein or the corresponding DNA encoding the peptide without apparent loss of biological utility.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydrophobic amino acid index in conferring interactive biological functions on proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-132, 1982).
  • the hydrophobic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of that protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
  • Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge properties.
  • one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still yield a protein having a similar biological function (eg, a protein equivalent in enzymatic activity)
  • the hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.
  • hydrophilicity index can also be taken into account when making modifications. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: alginine (+3.0); lysine (+3.0); Aspartic acid (+ 3.0 ⁇ 1); Glutamic acid (+ 3.0 ⁇ 1) Serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (10.4); proline (10.5 ⁇ 1); Alanine (10.5); histidine (10.5); cysteine (-1.0); methionine (1-1.3); valine (1-1.5); leucine (1-1.8); isoloisin ( I-1.8); Tyrosine (I-2.3); Hue-alanan (I-2.5); and Tryptophan (I-3.4).
  • an amino acid can be substituted for another that has a similar hydrophilicity index and still provide a bioisostere.
  • the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • conservative substitution refers to an amino acid substitution, in which the hydrophilicity index or Z and hydrophobicity index of the original amino acid and the amino acid to be substituted are similar to those described above. And permutation. Examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; And the like, but are not limited thereto.
  • variant refers to a substance, such as an original polypeptide or polynucleotide, that has been partially modified or its tertiary structure data (or atomic coordinates).
  • variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like.
  • Alleles refer to genetic variants that belong to the same locus and are distinct from each other. Therefore, an “allelic variant” refers to a variant that has an allelic relationship to a certain gene.
  • “Species homolog or homolog” refers to homology (preferably 60% or more homology, more preferably 80% or more) of a certain gene at the amino acid or nucleotide level with a certain gene. %, 85% or more, 90% or more, 95% or more homology).
  • a method for obtaining such a species homolog is apparent from the description of the present specification, and a database known in the art such as DDBJ or the like can also obtain information.
  • human LOX-1 is specifically disclosed in the present specification, the present invention is not limited to such a sequence, and it is not limited to such a sequence. Such receptors are also within the scope of the present invention.
  • substitution, addition or deletion of a polypeptide or polynucleotide means an amino acid or a substitute thereof, or a nucleotide, with respect to an original polypeptide or polynucleotide, respectively.
  • substitute power means to be replaced, added or removed.
  • Techniques for such substitution, addition or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis techniques.
  • the number of substitutions, additions, or deletions may be any number as long as it is one or more.Such numbers indicate the desired function (for example, a cancer marker, Markers) can be increased. For example, such a number can be one or several, and preferably is no more than 20%, no more than 10% of the total length, or no more than 100, no more than 50, no more than 25, etc. obtain.
  • ortholog is also called an orthologous gene and refers to a gene derived from speciation from a common ancestor of two genes.
  • the human a-hemoglobin gene and the mouse a-hemoglobin gene are orthologs.
  • the human ⁇ -hemoglobin gene and the 13-hemoglobin gene ⁇ -hemoglobin gene (Gene generated by gene duplication). Since orthologs are useful for estimating molecular phylogenetic trees, orthologs of human LOX-1 of the present invention may also be useful in the present invention.
  • wild type of LOX-1 refers to the most widely occurring LOX-1 naturally occurring species among the species of origin. Usually, LOX-1 first identified in some species is said to be wild-type. Wild type is also referred to as "natural standard type”. Such a wild-type LOX-1 has the sequence shown in SEQ ID NOs: 3 and 4.
  • modification of interest refers to, for example, a site or amino acid residue in the LOX-1 amino acid sequence that interacts with a LOX-1 ligand or substrate (eg, LDL). Or a modification that alters the site of such interaction.
  • at least one amino acid sequence attributable to the change in the figure shown in FIG. 8 of LOX-1 shown in SEQ ID NO: 2 is selected from the group consisting of at least one amino acid.
  • the amino acids other than the amino acids at the respective positions shown in SEQ ID NO: 2 are included.
  • the domain responsible for the interaction with a substrate or a coenzyme can also be analyzed by X-ray analysis using a well-known X-ray crystal structure analysis technique (for example, BW7b beamline (DESYZEMBL, Hamburg, Germany)).
  • a well-known X-ray crystal structure analysis technique for example, BW7b beamline (DESYZEMBL, Hamburg, Germany)
  • MarReseach Imaging Plate Detector V data measurement, data processing using programs such as DENZO and SCLAEPACK), and computer modeling techniques (eg, CNS program, XPL02D program, program 0, PROCHECK, WHATCHECK, WHATIF , Etc.). Therefore, any LOX-1 can introduce the modification of the present invention by introducing a modification in an interaction domain with a substrate or a coenzyme identified using the above-described method.
  • Such modifications preferably involve substituting amino acids in the wild type for other amino acids.
  • the LOX-1 of the present invention can enhance enzymatic activity and reduce side reactions by modifying amino acid residues in the interaction domain with the ligand. Since such an interaction domain was not previously known, it can be said that the present invention has an effect that cannot be predicted by the conventional technology.
  • nucleic acid sequences As used herein, the term "conservative (modified) variants" applies to both amino acid sequences and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, and essentially the same if the nucleic acid does not encode an amino acid sequence. Sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
  • nucleic acid variations can be attributed to one species of conservatively modified mutations: Modification (mutation) ".
  • Every nucleic acid sequence herein which encodes a polypeptide also includes every possible silent variation of the nucleic acid.
  • the silent modification includes "silent substitution” in which the encoding nucleic acid is not changed, and includes the case where the nucleic acid does not encode an amino acid in the first place.
  • silent alteration is synonymous with silent substitution.
  • siren substitution refers to a substitution of a nucleic acid sequence encoding one amino acid with another nucleic acid sequence encoding the same amino acid in a nucleic acid sequence. Based on the phenomenon of degeneracy in the genetic code, when there are a plurality of nucleic acid sequences encoding a certain amino acid (for example, glycine, etc.), such a siren can be substituted. Thus, a polypeptide having an amino acid sequence encoded by a nucleic acid sequence generated by a silent substitution has the same amino acid sequence as the original polypeptide.
  • the amino acid residues that interact with the modification (the LOX-1 polypeptide substrate (eg, LDL)) other than the wild-type Substitution with an amino acid residue (for example, in the residue of the wild-type LOX-1 polypeptide corresponding to at least one residue of the ligand binding site of LOX-1 shown in SEQ ID NO: 2; It is also possible to include silent substitutions at the nucleic acid sequence level by having an amino acid other than the amino acid at each position).
  • each codon in a nucleic acid (except AUG, which is usually the only codon encoding methionine, and TGG, which is usually the only codon encoding tryptophan), is required to produce a functionally identical molecule. It is understood that it can be modified. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of a polypeptide.
  • amino acid addition in addition to the amino acid substitution, amino acid addition, Deletions or modifications can also be made.
  • Amino acid substitution refers to substitution of one or more, for example, 110, preferably 115, and more preferably 113 amino acids of the original peptide.
  • the addition of amino acids refers to adding one or more, for example, 110, preferably 115, and more preferably 113 amino acids to the original peptide chain.
  • Amino acid deletion refers to deletion of one or more, for example, 110, preferably 115, and more preferably 113 amino acids from the original peptide.
  • Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), and the like.
  • the amino acid to be substituted or added may be a natural amino acid or an unnatural amino acid, or an amino acid analog. U, prefer natural amino acids.
  • peptide analog or “polypeptide analog” are used interchangeably and refer to a peptide or polypeptide that is a compound different from a peptide or polypeptide. Functional or biological function is equivalent.
  • peptide analogs include those in which one or more amino acid analogs have been added or substituted for the original peptide.
  • Peptide analogs have a function similar to that of the original peptide (e.g., similar pKa values, similar functional groups, similar binding modes to other molecules, Such additions or substitutions have been made to be substantially similar to those of similar nature.
  • Such peptide analogs can be made using techniques well known in the art.
  • a peptide analog can be a polymer that includes an amino acid analog.
  • polypeptide encompasses this peptide analog unless otherwise specified.
  • a "vector” refers to one that can transfer a desired polynucleotide sequence into a desired cell.
  • a vector includes a promoter capable of autonomous replication in a host cell such as an animal individual, or a thread capable of incorporation into a chromosome, and a promoter suitable for transcription of the polynucleotide of the present invention.
  • a vector can be a plasmid.
  • expression vector refers to a structural gene and a promoter that regulates its expression, and various regulatory elements operably linked in a host cell. Nucleic acid sequence. The regulatory elements may preferably include a terminator, a selectable marker such as a drug resistance gene, and an enhancer. Biological (eg, animal) It is well known to those skilled in the art that the type of expression vector and the type of regulatory element used, S, may vary depending on the host cell.
  • the term "recombinant vector” refers to a vector capable of transferring a target polynucleotide sequence into a target cell.
  • a vector is capable of autonomous replication in a host cell such as an animal individual or can be integrated into a chromosome, and contains a promoter at a position suitable for transcription of the polynucleotide of the present invention. Things are illustrated.
  • pBTrp2 As the "recombinant vector" for prokaryotic cells, pBTrp2, pBTacl, pBTac2 (all sold from Roche Molecular Biochemicals), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega ), PQE-8 (QIAGEN), pKYPIO (JP-A-58-110600), pKYP200 (Agric. Biol. Chem., 48, 669 (1984)), pL SAl (Agric. Biol. Chem., 53, 277 ( 1989)), pGELl (Proc. Natl. Acad. Sci.
  • terminal 1 is located downstream of the region encoding the protein of the gene, and is a sequence involved in terminating transcription when DNA is transcribed into mRNA and adding a poly A sequence. It is. Terminators are known to be involved in mRNA stability and affect gene expression levels.
  • promoter refers to a region on DNA that determines the start site of transcription of a gene and that directly regulates the frequency of transcription.
  • the promoter region usually contains a putative protein. In many cases (but not limited to) the region within about 2 kbp upstream of the first exon of the protein coding region, if the protein coding region in the genomic nucleotide sequence is predicted using DNA analysis software, the promoter region Can be estimated.
  • the putative promoter region varies for each structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is within about 2 kbp upstream of the first exon translation start site.
  • enhancers can be used to enhance the expression efficiency of a target gene.
  • enhancers are well known in the art.
  • a plurality of enhancers may be used, but one may or may not be used.
  • operably linked refers to a transcription / translation regulatory sequence (eg, promoter, heterozygous gene, or the like) or a translational regulator having expression (operation) of a desired sequence. It means to be placed under the control of the sequence.
  • the promoter In order for a promoter to be operably linked to a gene, the promoter is usually, but not necessarily, positioned immediately upstream of the gene.
  • any technique for introducing a nucleic acid molecule into cells may be used.
  • transformation, transduction, transfection, and the like can be mentioned.
  • Techniques for introducing such nucleic acid molecules are well known and commonly used in the art, and are described, for example, in Ausubel FA et al. (1988), Current Protocols in Molecular Biology, Wiley, New York; NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Separate experimental medicine "Gene transfer & expression analysis experiment method" Yodosha, 1997, etc. It is described in. Introduction of the gene can be confirmed using the methods described herein, such as Northern plot, Western blot analysis, or other well-known conventional techniques.
  • the protein identified in the present invention can be produced using the method as described above.
  • a polyclonal antibody can be prepared by administering a purified full-length or partial fragment of the obtained polypeptide or a peptide having a partial amino acid sequence of the protein of the present invention as an antigen and administering it to an animal. it can.
  • animals such as rabbits, goats, rats, mice, and hamsters can be used as animals to be administered.
  • the dosage of the antigen is preferably 50-100 ⁇ g per animal.
  • a peptide it is desirable to use, as an antigen, a peptide obtained by covalently bonding the peptide to a carrier protein such as keyhole limpet haemocyanin or thyroglobulin.
  • the peptide serving as the antigen can be synthesized by a peptide synthesizer.
  • the administration of the antigen is performed 3 to 10 times every 1 to 2 weeks after the first administration.
  • hybridoma that specifically reacts with the partial fragment polypeptide of the polypeptide of the present invention is selected by enzyme immunoassay or the like.
  • the thus-obtained hybridoma-produced monoclonal antibody can be used for various purposes.
  • Such an antibody can be used, for example, for the immunological detection method of the polypeptide of the present invention.
  • ELISA using a titer plate 'fluorescent antibody method, western blot method, Epidemiological tissue staining and the like can be mentioned.
  • the polypeptide of the present invention can also be used for an immunological quantification method.
  • the polypeptide is a sandwich ELISA method using two monoclonal antibodies with different Epitopu of antibodies reactive with the polypeptide of the present invention in the liquid phase, labeled with a radioisotope such as 1 26 1 Radioimmunoassay method using the protein of the present invention and an antibody that recognizes the protein of the present invention.
  • the expression level of the DNA encoding the polypeptide of the present invention is quantified at the mRNA level by the Northern hybridization method or the PCR method.
  • Such techniques are well-known in the art and are also described in the references listed herein.
  • the interaction between the model produced in the present invention and a library of candidate conjugates can be screened using array technology.
  • array technology The following describes an array technique that can be used.
  • solid phase refers to a support to which a molecule such as an antibody can be immobilized.
  • the shape of the solid phase may be planar, spherical, or other shapes. Further, the solid phase of the present invention may be in a gel state.
  • the solid phase is preferably a glass substrate base material having a metal thin film containing gold, silver or aluminum on one surface.
  • a frequency conversion element for example, a crystal oscillator or a surface acoustic wave element
  • One side of the quartz plate is covered with silicone, and the other side is coated with gold electrodes and used as a solid phase.
  • the term "substrate” refers to a material (preferably a solid) which is a planar solid phase and on which the chip or array of the present invention is constructed. Thus, the substrate is included in the concept of a solid phase.
  • the material of the substrate may be a force having the property of binding to the biomolecule used in the present invention, either covalently or non-covalently, or such a force. Any solid material that can be derivatized to have properties is included.
  • any material that can form a solid surface can be used.
  • examples thereof include glass, silica, silicone, ceramic, silicon dioxide, plastic, Metals (including alloys), natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon) include, but are not limited to:
  • the substrate may be formed from a plurality of layers of different materials.
  • inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
  • a membrane used for blotting such as a nylon membrane, a nitrocellulose membrane, or a PVDF membrane
  • a material having hardness such as glass
  • a preferable material for the substrate varies depending on various parameters such as a measuring instrument, and those skilled in the art can appropriately select an appropriate material having the above-described various material strengths.
  • a "chip” or “microchip” is used interchangeably, has a variety of functions, and refers to a microminiature integrated circuit that becomes a part of a system.
  • a solid phase on which a biotin receptor is immobilized is referred to as a receptor chip and Z or a receptor microchip.
  • array refers to a pattern or a substrate (for example, a chip) having a pattern in which one or more (for example, 1000 or more) receptors are arranged and arranged.
  • Arrays that are patterned on a small substrate eg, 10 ⁇ 10 mm, etc.
  • microarrays and arrays are used interchangeably herein. Therefore, even if the substrate is patterned on a substrate larger than the substrate described above, Sometimes called an array.
  • an array consists of a set of desired receptors that are themselves immobilized on a solid surface or membrane.
  • Array preferably comprises at least 10 two identical or different receptors, more preferably at least 10 3, and more preferably least 10 4, even more preferably at least 10 5 a. These receptors are preferably located on a surface of 125 ⁇ 80 mm, more preferably 10 ⁇ 10 mm.
  • a size of a microtiter plate such as a 96-well microtiter plate or a 384-well microtiter plate, to a size of a slide glass is contemplated.
  • One or more kinds of receptors may be immobilized. The number of such types can be any number up to the number of individual spots. For example, about 10, about 100, about 500, about 1000 receptors can be immobilized.
  • the solid phase surface or film such as a substrate, any number of biomolecules, as described above (e.g., receptors) may be provided, usually, per one substrate 1, 10 8 biomolecules In other embodiments, up to 10 7 biomolecules, up to 10 6 biomolecules, up to 10 5 biomolecules, up to 10 4 biomolecules, up to 10 3 biomolecules, or up to 10 3 biomolecules in other embodiments. number of biomolecules up to two biological molecules can be arranged, but biological molecules for over 108 biological molecules may be placed. In these cases, the size of the substrate is preferably smaller. In particular, the size of a biomolecule receptor spot can be as small as the size of a single biomolecule (which can be on the order of 1-2 nm). The minimum substrate area is in some cases determined by the number of biomolecules on the substrate. In the present invention, the factor that specifically binds to cells is usually immobilized by covalent bond or physical interaction in a spot shape of 0.01 mm to 10 mm.
  • spots of biomolecules may be arranged.
  • spot refers to a certain set of biomolecules.
  • spotting refers to producing a spot of a certain biomolecule on a certain substrate or solid phase. Spotting can be performed in any manner, for example, by pipetting or the like, or can be performed by an automated device, and such methods are well known in the art.
  • a biomolecule is a receptor, a fragment of a receptor, or a variant of a receptor.
  • the term "address” refers to a unique location on a substrate that may be distinguishable from other unique locations.
  • the address is appropriate for association with the spot with that address, and takes any shape so that the entity at every each address can also identify the entity force at the other address (eg, optically). obtain.
  • the shape defining the address can be, for example, a force that can be circular, oval, square, rectangular, or an irregular shape. Therefore, “address” indicates an abstract concept, and “spot” may be used to indicate a specific concept. However, when there is no need to distinguish between the two, in this specification, “address” is used. And “spot” can be used interchangeably.
  • the size defining each address is, inter alia, the size of the substrate, the number of addresses on a particular substrate, the amount of analyte and Z or available reagents, the size of the microparticles and the array thereof.
  • the magnitude can be, for example, 1-2 nm force, which can also be in the range of a few cm, any size consistent with the application of the array.
  • the spatial arrangement and shape defining the address are designed to suit the particular application for which the microarray is used.
  • the addresses can be densely arranged and widely dispersed, or subgrouped into a desired pattern appropriate to the particular type of analyte.
  • Microarrays are described in Shujunsha eds., Cell Engineering Separate Volume, "DNA Microarrays and the Latest PCR Method", MF Templin, et al., Protein microarray technology, Drug Discovery Today, 7 (15), 815-822. (2002) [This is widely outlined!
  • Microarray power Because the amount of data obtained is enormous, data analysis software for managing the correspondence between clones and spots and performing data analysis is important. As such software, software attached to various detection systems can be used (Ermolaeva O et al. (1998) Nat. Genet. 20: 19-23).
  • the format of the database is, for example, a format called GATC (genetic analysis technology consortium) proposed by Affymetrix.
  • a method using a micropatterned surface of an alkanethiol monomolecular film is used. is there.
  • a monomolecular film of alkanethiol having a hydrophobic functional group such as a methyl group or a fluoromethyl group is formed on a glass substrate having a gold thin film deposited on one surface.
  • a photomask on which a number of light-transmitting spots having a diameter of several z / m to 1 mm is arranged is superimposed on the monomolecular film, and irradiated with ultraviolet rays.
  • the alkanethiol in the irradiated part can be decomposed and removed in the form of spots.
  • a reactive functional group introduced into the spot immobilize proteins that specifically bind to biotin, such as streptavidin and avidin, or immobilize factors that specifically bind to tags.
  • biotin such as streptavidin and avidin
  • factors that specifically bind to tags such as streptavidin and avidin.
  • the receptor protein can maintain its orientation under mild conditions without chemical treatment. Is completed.
  • a carboxyl group-containing spot a carboxyl group is converted into an active ester using N-hydroxysuccinimide, avidin or streptavidin is immobilized, and then a trace amount of a biomolecule-containing solution is applied to each spot. By dropping the liquid on the surface, the fixing can be performed.
  • the hydrophobic monolayer formed around the spot is effective for suppressing the diffusion of the solution.
  • block with an inactive protein such as serum albumin or a hydrophilic polymer such as polyethylene glycol.
  • microarrays can perform high-throughput analysis on many samples on a single substrate. It is a very effective analytical tool.
  • the present invention relates to such a microphone. Apply the concept of low-array to quickly measure the interaction between many types of biomolecules and cells. In this case, integration of microarrays is important in order to simultaneously analyze an extremely large number of specimens and to minimize the amount of biological molecules and cells required for the analysis.
  • integration of microarrays is important in order to simultaneously analyze an extremely large number of specimens and to minimize the amount of biological molecules and cells required for the analysis.
  • each spot constituting the microarray should be at least tens or thousands of cells capable of interacting with each other.
  • Its diameter is around a few / zm to 1 mmfe degree
  • microcontact printing method for the production of the microarray, various methods such as a microcontact printing method and an optical lithography method can be used.
  • a method using a micropatterned surface of an alkanethiol monomolecular film is used. is there.
  • biomolecule refers to a molecule associated with a living organism.
  • organ refers to a biological organism, including, but not limited to, animals, plants, fungi, viruses, and the like.
  • a biomolecule includes, but is not limited to, a molecule to be extracted by biopower, and is included in the definition of a biomolecule as long as it is a molecule that can affect a living body.
  • biomolecules include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, cDNA, DNA such as genomic DNA, RNA such as mRNA), Examples include, but are not limited to, polysaccharides, oligosaccharides, lipids, small molecules (eg, hormones, ligands, signal transducers, small organic molecules, combinatorial library conjugates, etc.), and composite molecules thereof.
  • Preferred biomolecules herein are receptors and receptor fragments and their ligands.
  • the term "receptor” refers to a biological structure comprising one or more binding domains that reversibly and specifically complex with one or more ligands. Wherein the complexation has a biological structure.
  • Receptors can be completely extracellular (extracellular receptors), in the cell membrane (but not in the extracellular environment and cells). (To the cytosol) or completely within the cell (an intracellular receptor). They can also function independently of cells. Receptors in the cell membrane allow the cell to communicate (e.g., signal) with space outside its borders, and to function in the transport of molecules and ions into and out of the cell .
  • the receptor may be the full length of the receptor or a fragment of the receptor.
  • a site related to ligand recognition of a receptor protein is used.
  • the site involved in ligand recognition of the receptor protein can be identified as follows.
  • homology search or domain search the ligand recognition region can be estimated from the structure of proteins having high homology and similarity in function. For example, when amino acid sequences of different receptor molecules that specifically bind to the same ligand are calculated using the default parameters of BLAST, 50% or more, preferably 55% or more, more preferably 60% or more, More preferably, the region has a homology of 65% or more and is estimated as a ligand recognition region.
  • those skilled in the art can easily make a gene encoding a mutant receptor into which a deletion mutation or amino acid substitution is introduced transiently expressed in an animal cell or the like and determine a region essential for its function.
  • ligand is a binding partner for a specific receptor or family of receptors.
  • the ligand can be an endogenous ligand for the receptor, or alternatively, a synthetic ligand for the receptor, such as a drug, drug candidate, or pharmacological tool.
  • factor that specifically binds to biotin refers to any factor that can specifically bind to biotin.
  • the binding between biotin and a factor that specifically binds to biotin may be reversible or irreversible.
  • Factors that specifically bind to biotin include, but are not limited to, avidin and streptavidin, and variants thereof.
  • factor that specifically binds to a tag refers to any factor that can specifically bind to a tag. Binding between the tag and an agent that specifically binds to the tag may be reversible or irreversible. Factors that specifically bind to tags And is well known in the art.
  • a factor that specifically binds to a tag is, for example, daltathione;
  • a factor that specifically binds to the tag is, for example, a nickel chelate column;
  • the factor that specifically binds to the tag is, for example, cellulose;
  • the factor that specifically binds to the tag is, for example, when an S protein-binding peptide tag such as SEQ ID NO: 15 is used, the factor that specifically binds to the tag is, for example, S-tag.
  • SPR Surface plasmon resonance
  • a method of arranging a prism composed of a high refractive index medium Kretschmann arrangement
  • injecting laser light and LED light is used.
  • the wave number of the plasmon wave changes due to a change in the dielectric constant of the medium in contact with the metal surface on the opposite side of the prism. That is, when a substance approaches the metal surface, the incident angle of light that gives surface plasmon resonance shifts. By utilizing this fact, it is possible to sense the coating of the metal surface with the substance.
  • This measurement method has excellent resolution in the vertical direction of the surface (on the order of 0.1 nm), and enables real-time observation of the amount of substances present on the surface in the order of ng-pgZcm 2 .
  • the ability to measure in aqueous media is also a great advantage in studying the behavior of biomolecules such as proteins.
  • a measuring device utilizing this has been developed as a device for measuring the interaction between biomolecules, and has been applied to the analysis of interactions between proteins and DNA.
  • the crystal resonator microbalance is a pair of chemically bonded pairs on the electrode of the frequency conversion element. Is fixed, the frequency conversion element is immersed in water, and the frequency change of the frequency conversion element accompanying the mass change caused by the specific binding between the binding pair and the corresponding binding pair is measured. Is detected (for example, Japanese Patent Application Laid-Open No. 6-94591).
  • the frequency conversion element include a crystal oscillator, a surface acoustic wave element (SAW), and the like.
  • the receptor chip of the present invention can also be used as a mass spectrometry chip for a mass spectrometer.
  • analysis by mass spectrometry involves the vaporization and ionization of a small sample using a high energy source such as a laser, including a laser beam.
  • a high energy source such as a laser, including a laser beam.
  • the material is vaporized by the laser beam into a gas or gaseous phase at the tip of the mass spectrometer chip, and during this process, some of the individual molecules take in protons and become ionized.
  • the molecules ionized to these positive charges are then accelerated by a short high-voltage electric field, guided (drift) into a high-vacuum chamber, and then strike the surface of a sensitive detector.
  • drift guided
  • time of flight is a function of the mass of the ionized molecule
  • the time that elapses between ionization and collision can be used to determine the mass of that molecule, which can then be used to determine the mass of that particular mass. It can be used to determine if a known molecule is present (Time of Flight Mass Spectroscopy (TOF)).
  • TOF Time of Flight Mass Spectroscopy
  • the voltage of the DC component and the high-frequency AC component is applied to specify Using a mass filter that passes only ions having the mass Z charge number (mZZ) (if necessary, to generate fragment ions), the mass Z charge number (m / Z Z) can also be detected (tandem mass spectrometry).
  • a desorption Z-ionization method in which the impact force of particles on a sample is obtained.
  • This method includes fast atom bombardment (FAB—neutral bombards a sample suspended in a volatile matrix) and secondary ion mass spectrometry (SIMS—keV—bombardment of the surface with To generate secondary ions), liquid SIMS (similar to FAB except that LSIMS is the primary species), plasma desorption mass spectrometry (MeV—except using secondary ions, Mass impact bombardment (MCI—large, similar to SIMS using cluster primary ions), laser desorption Z ionization (LDI— The surface force is also used to desorb the species z ionization), matrix-assisted laser desorption z ionization method (MALDI-excluding matrix ion species that can assist in the desorption and ionization events) LDI).
  • Typical mass spectrometry methods include laser desorption Z ionization and time-of-f
  • a measuring method using a mass spectrometer chip to which a molecule that performs affinity binding such as a receptor is bound in a mass spectrometer is disclosed in, for example, JP-A-9-501489 as follows. Exposing the surface of the mass spectrometric chip on which the receptor is immobilized to a source of the analyte molecules (for example, a mixture containing a ligand) so that the analyte molecules are bound; A step of placing the tip of the mass spectrometry chip at which one of the target molecules is bound at one end of the time-of-flight mass spectrometer and applying a vacuum and an electric field to create an accelerating potential in the spectrometer; In order to desorb the ions of the analyte molecules from the tip, at least a portion of the analyte bound to the tip of the induced mass spectrometry chip in the spectrometer is subjected to one or more laser pulses.
  • a source of the analyte molecules
  • Use the to hit In the mass spectrometer, comprising the steps of detecting the mass of the ions by flight time;-up and consisting of the step of displaying the good is urchin detected mass method.
  • this method it is possible to detect the mass of ions of a molecule (for example, a ligand that specifically binds to a receptor) bound to the mass analysis chip.
  • the mass of the analyte molecule can be measured by laser desorption Z ionization and time-of-flight mass spectrometry.
  • desorption of the analyte is performed.
  • an energy-absorbing substance for example, sinapinic acid, cinnamamide, cinnamyl bromide, 2,5-dihydroxybenzoic acid, and a-cyano 4-hydroxycainic acid
  • an energy-absorbing substance for example, sinapinic acid, cinnamamide, cinnamyl bromide, 2,5-dihydroxybenzoic acid, and a-cyano 4-hydroxycainic acid
  • a further measurement method using a mass spectrometry chip in which a molecule that performs affinity binding such as a receptor is immobilized in a mass spectrometer is disclosed in JP-T-11-512518.
  • an affinity binding molecule such as a receptor
  • a chip typically on a support having a hydrogel, and more particularly, a hydrogel of a polysaccharide such as carboxymethylated dextran, and the analyte thereof is analyzed.
  • a ligand in contact with its support After that, the presence or absence of the analyte bound to the affinity binding molecule and its mass are analyzed.
  • the receptors used in the present specification include scavenger receptors including lectin-like oxidized LDL receptor (LOX-1), receptors belonging to the insulin receptor family, receptors belonging to the EGF receptor family, PDGF Receptors belonging to the receptor family, receptors belonging to the VEGF receptor family, receptors belonging to the FGF receptor family, growth factor receptors such as the NGF receptor family, and TGF-
  • a preferred receptor is LOX-1, which belongs to the receptor of the LDL receptor-related protein family.
  • hLOX-1 human LOX-1
  • SEQ ID NOs: 5 and 6 The nucleotide sequence and amino acid sequence of the extracellular region of human LOX-1 (hLOX-1) are shown in SEQ ID NOs: 5 and 6 in the Sequence Listing, and the nucleotide sequence and amino acid sequence of hLOX-1 CTLD are shown in SEQ ID NOs: 1 and 2 in the Sequence Listing. 2 respectively.
  • SEQ ID NOs: 3 and 4 show the full length sequence of hLOX-1.
  • refolding of a receptor protein expressed as an inclusion body is performed by dropping a denatured protein into a refolding buffer solution containing arginine, reduced daltathione, and oxidized daltathione. Done.
  • a denatured protein nor the refolding buffer contains a surfactant.
  • the term "denaturing agent” refers to an agent that causes protein denaturation. Modifiers include, but are not limited to, guanidine hydrochloride, urea, dioxane, alcohol, ethylene glycol, and the like.
  • concentration using guanidine hydrochloride is preferably about 418M, more preferably about 6M.
  • refolding refers to obtaining a protein that has recovered its original tertiary structure, biological activity, and the like from a denatured protein.
  • the biological activity is, for example, a ligand binding activity.
  • inclusion body refers to a protein that is produced when large amounts of a foreign gene are recombinantly expressed in a bacterium such as Escherichia coli, and the protein is accumulated in the cell as an insoluble substance. Structure.
  • solubilizing an inclusion body refers to denaturing an insoluble inclusion body with a denaturing agent to generate a form dissolved in an aqueous solution.
  • SH protection reagent refers to a reagent that prevents a thiol group (SH group) in a protein from reacting with a substance reactive with the SH group.
  • SH protection reagents include dithiothreitol, j8-mercaptoethanol, and daltathione, but are not limited to these.
  • refolding of a receptor protein expressed as an inclusion body is performed by dropping the denatured protein into a refolding buffer containing arginine, reduced daltathione, and oxidized daltathione.
  • the dropping rate is preferably a rate of 10 to 300 1Z, more preferably a rate of 20-30 ⁇ 1 / min.
  • the pH of the refolding buffer is preferably in the range of 7.5-9.5, more preferably in the range of 8.0-8.5, but depending on the nature of the protein to be refolded. Changing the appropriate pH accordingly is well known in the art.
  • the refolding buffer contains Tris-HCl, and preferably, the concentration of Tris-HCl is 5-20 mM.
  • the concentration of arginine contained in the refolding buffer is preferably 100 mM to 600 mM, and more preferably 300 mM to 400 mM.
  • the ratio of reduced daltathione to oxidized daltathione is 1: 8 to 1:12, more preferably 1: 9 to 1:11, Most preferably, it is 1:10. More preferably, the concentration of reduced daltathione is 8-10 mM and the concentration of oxidized daltathione is 0.3-1. OmM.
  • the ratio of the volume of the denatured protein solution and the volume of the refolding buffer to be dropped is preferably 1:50 to 1: 100. Also preferred The concentration of the denatured protein dropped is 5-80 / z gZmL, more preferably 10-80 ⁇ g / mL, and still more preferably 20-80 ⁇ g / mL.
  • the denatured protein used in the refolding method of the present invention is a protein denatured by a denaturing agent, heat, or any of ultraviolet irradiation and radiation irradiation, and is preferably a denaturing agent. Is a protein denatured by
  • the denatured protein used in the refolding method of the present invention is preferably prepared by dissolving an inclusion body protein expressed in bacteria by using a soluble buffer containing a SH protective reagent and a denaturant. It is a denatured protein obtained by shading.
  • the solubilization to obtain the denatured protein is performed using a protein having a concentration of 115 mgZmL. More preferably, solubilization to obtain denatured protein is performed using a protein concentration of 2.0-3. OmgZmL.
  • the pH of the solubilizing buffer is 7.0-9.0, and in one aspect, the solubilizing buffer is 100 mM Tris-HCl buffer.
  • the SH protecting reagent is dithiothreitol, more preferably, the concentration of dithiothreitol is 5 to 200 mM, and still more preferably, the concentration of dithiothreitol is 50 to 100 mM. It is.
  • a step of stirring the solution is performed after the denatured protein solution is dropped into the refolding buffer. More preferably, the stirring step is performed for 10-48 hours, and even more preferably, the stirring step is performed for 10-14 hours. This stirring is performed at 4 ° C. at room temperature, preferably at 4 ° C.
  • the refolding method of the present invention it is possible to obtain a protein with higher purity and homogeneity than a protein prepared using a conventional refolding method. For example, by using the refolding method of the present invention, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, A protein with a purity of 99% or more is obtained. Further, when the protein obtained by using the refolding method of the present invention is analyzed by mass spectrometry, the width of mZz by MS spectrum is 70 or less, 60 or less, 50 or less, 40 or less, 30 or less. A protein with a purity of 20 or less can be obtained.
  • the present invention provides a method for preparing a ligand-binding fragment of a receptor protein that specifically binds to a desired ligand in a highly pure and homogeneous state, and binding the fragment to a solid phase to obtain the desired compound.
  • a receptor chip for detecting a ligand with high sensitivity.
  • the present invention also provides a method for detecting the desired ligand using such a receptor chip.
  • the present invention provides a method for preparing a ligand-binding fragment of a receptor protein which specifically binds to a desired ligand in a highly pure and homogeneous state, and binding the fragment to a solid phase to obtain the desired ligand.
  • a ligand removing material for specifically removing a ligand.
  • the present invention also provides a method for removing the desired ligand from the blood, comprising the following steps:
  • the blood processed by the above method is returned to the subject.
  • the present invention provides a method for removing bacteria and pathogens such as Z or virus in a sample, the method comprising the following steps:
  • the sample treated by the above method is injected into a subject.
  • the present invention further provides a modified LDL, abnormal cell, or bacterium by an intermolecular interaction analysis method, which comprises using a recombinant protein obtained by expressing a region related to ligand recognition of a receptor in a cell. To provide a detection method.
  • the present invention provides that a region related to ligand recognition of a receptor is expressed in a cell as a biotinylated protein, and then the expressed biotinylated protein is maintained in its orientation via avidin or streptavidin.
  • a method for detecting a ligand by an intermolecular interaction analysis method comprising immobilizing a protein on a solid phase and using the immobilized protein.
  • the present invention also provides a method for expressing, in a cell, a region related to ligand recognition of a receptor as a tagged protein, and then allowing the expressed tagged protein to specifically bind to the tag.
  • the present invention provides a method for detecting a ligand by an intermolecular interaction analysis method, characterized in that the protein is immobilized on a solid phase while maintaining its orientation, and the immobilized protein is used.
  • the present invention further relates to the use of a reconstituted protein obtained by refolding and reconstituting the extracellular region or ligand recognition region of a receptor accumulated in Escherichia coli into a correct three-dimensional structure.
  • a method for detecting denatured LDL, abnormal cells, or bacteria by a characteristic intermolecular interaction analysis method is provided.
  • the present invention corrects the extracellular region of the receptor or the biotinylated ligand recognition region of the receptor accumulated in Escherichia coli, refolds it into a three-dimensional structure, reconstitutes it, and then reconstitutes the reconstituted biotinidani.
  • a protein is immobilized on a solid phase while maintaining its orientation via avidin or streptavidin, and denatured LDL, abnormal cells, or bacteria by an intermolecular interaction analysis method using the immobilized protein. To provide a detection method.
  • the present invention also relates to a tagidari protein, which is obtained by refolding a tagidari extracellular region or a tagged ligand recognition region of a receptor accumulated in Escherichia coli into a positive and three-dimensional structure and then reconstituting the same. Is immobilized on a solid phase while maintaining its orientation through a factor that specifically binds to a tag, and denatured LDL and abnormal cells are analyzed by an intermolecular interaction analysis method using the immobilized protein. Alternatively, a method for detecting bacteria is provided.
  • the present invention also provides a denatured LDL containing a protein obtained by unraveling the structure of the extracellular region or the ligand recognition region of a receptor accumulated as an aggregate in Escherichia coli with a denaturing agent and then refolding it into a correct three-dimensional structure.
  • Provide kits for detecting abnormal cells or bacteria The
  • NMR nuclear magnetic resonance
  • nuclear magnetic resonance The phenomenon where resonance absorption is observed when an electromagnetic wave having a frequency corresponding to this level interval is irradiated is called nuclear magnetic resonance.
  • two-dimensional NMR refers to a method of expanding a nuclear magnetic resonance spectrum on two frequency axes. For this measurement, multiple pulses are used, and Fourier transform is performed using the time interval and the time after the observation pulse as two time axes. Signal intensity is usually represented by contour lines.
  • Typical examples of two-dimensional NMR include TROSY (transverse relaxation-optimized spectroscopy), COSY (two-dimensional shift correlation NMR), S ECSY (two-dimensional spin echo correlation spectroscopy), and FOCSY (two-dimensional alias correction spectroscopy).
  • TROSY is a method developed by Wuthrich et al. Suitable for ultra-high field NMR (Proc. Natl. Acad. Sci. USA vol. 94, 12366-12371 (1997)). In NMR of molecules with large molecular weight, nuclear magnetic relaxation is governed by (1) magnetic dipole interaction and (2) chemical shift anisotropy.
  • TROSY is a method of increasing the relaxation time and narrowing the line width by offsetting these two interactions. TROSY has enabled us to obtain two-dimensional NMR spectra of high molecular weight proteins (eg, 900 kDa or more).
  • nuclides of stable isotopes used for labeling a protein include 15 N, 13 C, 2 H or a combination thereof. Not done. Preferred 1 5 N single label as the type of stable isotope nuclei used in the labeling, 13 C single-labeled, 15 NZ 2 H double labeling, 13 CZ 2 H double labeling, 15 NZ 13 CZ 2 H three It is a heavy label.
  • the amount of change in the NMR signal in the axial direction can be used.
  • Such a change is defined as a molecular orientation-dependent change in the axial direction with respect to the frequency axis of the two-dimensional NMR developed for the stable isotope element.
  • the change in the axial direction The amount of change depends on the molecular orientation.
  • the amount of change depending on the molecular orientation in the 13 C-axis direction can be cited.
  • a protein sample used for high-order structure analysis by NMR is usually subjected to structural analysis by stable isotope labeling by biosynthesis.
  • a target protein expression system is established using genetically engineered Escherichia coli, etc., and a stable isotope-labeled carbon source or nitrogen source (glucose with all 13 C-labeled 13 C, and nitrogen with 15 N-labeled salt) Expression in a medium supplemented with an ammmodium, etc.) makes it possible to obtain stable isotope-labeled proteins of all carbon and nitrogen.
  • the obtained labeled protein can be subjected to NMR measurement by purifying by chromatography or the like and then concentrating by ultrafiltration or the like. At this time, it is desirable to pay attention to the following conditions.
  • the container to be used is attached to the glass wall surface and, in order to suppress dispersion, the equipment used, including the NMR sample tube, is made of silicon or the like. Preferably, a coating treatment is performed. It is desirable to use a Pasteur pipette for handling samples.
  • an aqueous solution is usually used.
  • proteins have many exchangeable amide protons, and if they are made into heavy water solution, a lot of information will be lost due to deuterium substitution of amide protons, which are key in spectral analysis.
  • a sample is prepared with a light aqueous solution, and then about 10% heavy water is added for NMR lock. If necessary, a surfactant, a reducing agent, etc. may be added.
  • the sample concentration is usually about ImM (for example, 0.4mM to 2mM). If the amount of sample is sufficient and the solubility is high, keep in mind that a high force concentration that can be measured even at 5 mM to 10 mM may cause association in the solution. To prevent unexpected results from association, confirm the presence or absence of association by comparing the linearity of low-concentration and high-concentration samples. Preferably.
  • the pH used is usually preferably about 5-7. This is because, by lowering the pH, the exchange rate of exchangeable protons such as amidose is reduced. However, if the pH is low, there is a concern that the higher-order structure may be affected. Therefore, it is preferable to confirm the pH with a circular dichroism (CD) spectrum or the like.
  • CD circular dichroism
  • a buffer that does not have a proton in order to prevent interference with spectrum analysis.
  • a buffer that does not have a proton in order to prevent interference with spectrum analysis.
  • Such include, but are not limited to, phosphate buffers, deuterated acetate buffers, and the like.
  • the temperature to be used is preferably about 30 to 40 ° C in order to approach the temperature in a living body.
  • the relaxation time is shortened due to paramagnetism relaxation, resulting in a loss of sensitivity, which may hinder detection of NOE correlation. It is preferable to perform degassing to prevent dissolved oxygen.
  • the degassing operation includes degassing under reduced pressure, publishing of an inert gas, and the like. Preferably, degassing under reduced pressure is used. It is also desirable not to form bubbles.
  • NMR shim adjustment is performed.
  • the pulse width is measured.
  • the pulse width may vary depending on the sample due to differences in salt strength. Therefore, it is necessary to measure the pulse width for each sample in principle.
  • the pulse sequence used in protein solution NMR a large number of RF pulses are arranged, and it is considered that the deviation in pulse width has a considerable effect on the whole. Therefore, it is desirable to measure the pulse width with the signal of the target protein before performing the multidimensional NMR measurement, and to perform the multidimensional NMR measurement using the value.
  • the sample is confirmed by NMR. It is desirable to confirm by NMR when there is a possibility that the protein has deteriorated, such as after storage for a while after sample preparation.
  • NMR ⁇ vector of a protein since thousands of signals derived from 1H appear in an overlapping manner, it is often impossible to use it for confirmation of a measurement sample, not to mention structural analysis.
  • the 1H chemical shift of a protein is roughly limited by its environment. That is, a signal derived from an amide proton (around 8 ppm), a signal derived from a side chain aromatic ring (around 7 ppm), a signal derived from an ⁇ -position proton (around 4 ppm), and a signal derived from a j8-position proton (around 2-3 ppm) ) And a signal derived from a methyl group (around lppm) are observed.
  • 13 C has a similar correlation to chemical shifts, 13 to selectively excite C in each region, a position or j8 position carbon, as another species even force per a carbonyl carbon By using this method, it can be used in various measurement methods for attribution.
  • spin coupling between different nuclei should be considered.
  • NOE correlation carboxyalkyl and is that force the amide proton and ⁇ -position proton using conformation analysis - 1 H homonuclear NOE across the Le group, the two protons, adjacent via an amide bond It indicates that it is derived from an amino acid residue, and is used for assigning the amino acid sequence in homogenous nuclear experiments.
  • This method together with the amino acid type information determined based on the spin bond and the dagger shift, is called a sequence-specific chain assignment method.
  • the higher-order structure of a protein includes an a-helix structure, which forms the amino acid sequence with steric regularity, a secondary structure such as a ⁇ -sheet structure, and a tertiary structure in which some secondary structures are spatially arranged. There is a quaternary structure in which domains composed of structures and tertiary structures are spatially arranged.Clarification of these is important in structural analysis in protein solution NMR measurement, and it is performed variously according to the purpose .
  • the primary sequence is a known amino acid sequence
  • a structure optimization calculation algorithm called a distance geometry method or a constrained molecular dynamics method is used.
  • the NOE signal as the distance information obtained from NMR is collected and collected, and given to the calculation program as a parameter to derive a higher-order structure.
  • the "distance geometry method” refers to a method in which distance information and a set of bond angles (dihedral angles) are viewed as spatial position information, and a structure is derived based on the positional information. .
  • the bond length and bond angle of the covalent bond are fixed to the standard values of general proteins, and structural calculations are performed using the dihedral angle as a variable.
  • Some structures that converge in a direction that satisfies the constraint on the distance from the NOE are extracted, and the structure that seems to be the most appropriate is the final structure.
  • This method has a small number of variables and is fast to calculate, and the load on the computer is small.
  • structural changes are limited by contact of atoms (covalent bonds cannot pass through each other), and converge to a true structure. Difficult It is used to find the initial structure such as the constrained molecular dynamics method described below.
  • the "constrained molecular dynamics method” refers to a molecular dynamics method (virtually a molecular dynamics method) known as a method generally used to simulate molecular motion. This is one of the methods to optimize the structure by vibrating and optimizing the structure), and to add the constraint of the distance from NOE as potential to optimize the structure.
  • This method uses Cartesian (Cartesian) coordinates as position information, and requires a large amount of calculation due to a large number of variables, which imposes a heavy load on the computer. Due to great progress, the calculation time has been shortened and it is frequently used.
  • the "simulated 'annealing method' is a method of molecular dynamics calculation, and unlike general molecular dynamics calculation, contributes to covalent bonds in the initial stage of calculation.
  • a method of setting the parameters weakly and intensifying the molecular motion by rapidly increasing the temperature of the system, and then gradually lowering the temperature of the system to slow down the molecular motion and strengthening each parameter to converge the structure.
  • Macromolecular structures can be described for various levels of organization. For a general discussion of this construction, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994), and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). See "Primary structure” refers to the amino acid sequence of a particular peptide. "Secondary structure” refers to a locally arranged three-dimensional structure within a polypeptide. These structures are commonly known as domains. Domains form the compact unit of a polypeptide, and are portions of that polypeptide that are typically 50-350 amino acids in length.
  • a typical domain is made of parts, such as a stretch of 13 sheets (13 strands, etc.) and a helix.
  • “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer.
  • Quaternary structure refers to a three-dimensional structure formed by the noncovalent association of independent tertiary units. Terms related to anisotropy are used in the same way as those known in the energy field.
  • crystal structure analysis refers to analysis of a crystal structure utilizing diffraction of X-ray, electron beam, or neutron beam by a crystal. While applying a nearly monochromatic parallel beam to a single crystal, the orientation of the crystal is systematically changed to increase the Bragg reflection intensity from many crystal network planes. Weissenberg. There is a method of recording the intensity on a film or imaging plate with a camera or a precession camera and measuring the intensity (rotating crystal method), or a method of systematically measuring the reflection intensity with a 4-axis diffractometer equipped with a counter tube. From the reflection extinction law obtained in this way, the space group to which it belongs can be determined.
  • the presence or absence of the center of symmetry can also be determined from the statistics of the integrated reflection intensity.
  • the number of chemical units (atoms or molecules) contained in the unit cell can be determined from the molecular formula of the crystal and the size of the unit cell.
  • the position of the heavy atom if any is determined using the Patterson function, and the contribution is used as a clue for analysis. Even when a heavy atom is not included, the structure can be solved by the isomorphous substitution method using the exogenous heavy atom as a clue.
  • complex protein molecules, etc. by putting heavy atoms such as mercury in one position, and in particular, putting another kind of heavy atom in another position, the phase of reflection can be determined by the number of forces. Analyzes the structure based on important information.
  • crystal structure analysis of such a protein can be performed using a method well known in the art. Such methods are described in, for example, X-ray crystal structure analysis of proteins (Springer's Fehrerck Tokyo Co., Ltd.), Introduction to Crystal Analysis for Life Sciences (Maruzen), and the like. Can be used arbitrarily.
  • the LOX-1 native crystal (complexed with acetylated LDL as a coenzyme if necessary) and the frozen crystal of the heavy atom derivative can be crystallized without adding a freeze-stabilizing ligating agent. It can be prepared by removing droplets or crystals in the immersion solution, immersing them directly in liquid nitrogen and freezing instantly. Frozen crystals are also freeze stable It can also be prepared by performing the above operation of instantly freezing the crystals immersed in the storage solution to which the dangling agent has been added.
  • the heavy atom isomorphous substitution method (Methods in ENZYMOLOGY Vol. 115, Diffractipn Metnoas for Biological Macromolecules, Part B, edited by H. W. Wyckoff, CHW Hirs, and SN Timasheff, and Methods in ENZYMOLOGY Vol. 276, Macromolecular Crystallography, Part A, edited by CW Carter, Jr. and RM Sweet, or multi-wavelength anomalous dispersion method (edited by SN T imasheff, and Methods in ENZYMOLOGY, Vol. 276, Macromolecular Crystallography, Part A, CW Carter, Jr.
  • the three-dimensional structure is obtained by obtaining the initial phase for calculating the electron density from the diffraction intensity difference between the diffraction data of the native crystal and the heavy atom derivative crystal, or the diffraction intensity difference between the diffraction data measured at different wavelengths. Can decide.
  • a heavy atom derivative crystal containing, for example, mercury, gold, platinum, uranium, or selenium as a heavy metal atom may be used.
  • a heavy atom derivative crystal obtained by an immersion method using a mercury conjugate EMTS or potassium dicyano gold (I) is used.
  • the diffraction data of the native crystal and the heavy atom derivative crystal are, for example, R-AXIS lie
  • Diffraction data measurement at multiple wavelengths for applying the multi-wavelength anomalous dispersion method can be performed using, for example, a SPring-8 beamline for protein crystal structure analysis.
  • the measured diffraction image data is processed into reflection intensity data using, for example, a data processing program or program DENZO (Mac Science) attached to R-AXIS lie or a similar image processing program (or software for single crystal analysis). .
  • the position of the heavy metal atom bonded to the protein in the crystal in the crystal was determined using the difference Patterson diagram from the reflection intensity data obtained by measuring the wavelength.
  • the program PHASES W. Furey ⁇ University of Pennsylvania or CCP4
  • the initial phase is determined by refining the heavy atom location parameters using a ritish Biotechnology & Biological Science Research Counsil (SERC) or similar diffraction data analysis program.
  • the determined initial phase can be determined, for example, according to the solvent smoothing method and histogram matching method using the program DM (CCP4 package) or a similar phase improvement program (electron density improvement program).
  • the phase can be improved to a highly reliable phase by gradually performing the phase expansion calculation from low resolution to high resolution.
  • Protein crystals are occupied by solvent molecules other than proteins (mainly water molecules) in 30-60% of their volume.
  • solvent region the volume occupied by the solvent molecules in the solution.
  • the phase reliability is further improved by performing electron density averaging called non-crystallographic symmetry (NCS) averaging. It is possible.
  • NCS non-crystallographic symmetry
  • the crystal of the specific LOX-1 ligand-binding fragment it is observed that the density of the crystal also includes two molecules in the asymmetric unit.
  • a non-crystallographic symmetric matrix including translation and rotation is calculated.
  • the electron density map obtained by the solvent smoothing method it is possible to identify a region called a mask where protein molecules are present.
  • NCS NCS averaging calculation using a non-crystallographic symmetric matrix and mask using the program DM, etc., the phase can be improved to a highly reliable one, and an electron density diagram used to construct a three-dimensional structure model can be obtained.
  • the three-dimensional model of LOX-1 or LOX-1 ligand-binding fragment can be obtained, for example, from an electron density diagram displayed on a three-dimensional graphic by Program 0 (A) (A. Jones, Uppsala Universitet, Sweden). It can be constructed by the following procedure. First, a plurality of regions having a characteristic amino acid sequence (for example, a partial sequence containing a tributophan residue) are searched for on an electron density map. Next, referring to the amino acid sequence with the found region as a starting point, a partial structure of amino acid residues suitable for the electron density is constructed on the three-dimensional graphics using the program O.
  • A Program 0
  • the native crystal three-dimensional structure of LOX-1 or LOX-1 ligand-binding fragment for example, LOX-1 ligand-binding fragment containing the amino acid sequence shown in SEQ ID NO: 1
  • a modified LOX-1 ligand-binding fragment for example, A variant of the LOX-1 ligand binding fragment having the sequence shown in SEQ ID NO: 1
  • the three-dimensional structure of the native crystal was determined by the molecular replacement method using the three-dimensional structure of the obtained EMTS derivative crystal, and the initial phase was determined. By following the procedures for electron density improvement, model construction, and structure refinement, the three-dimensional structure of each can be determined.
  • the determination of the three-dimensional structure of the LOX-1 ligand binding fragment of the present invention is completed.
  • the determined three-dimensional structure of LOX-1 is registered in the Protein Data Bank (PDB), which is the public data bank for the three-dimensional structure of proteins, from the perspective of topology and other functions. (Including fragments of similar proteins).
  • PDB Protein Data Bank
  • topology refers to the arrangement or spatial arrangement of secondary structural units of a protein in this specification.
  • (X helix) is one of the secondary structures of a protein or polypeptide, and is a helical structure having a pitch of 5.4A in which amino acids rotate once every 3.6 residues.
  • One of the most energetically stable structures having the following: a) Amino acids that easily form a helix include glutamic acid, lysine, alanine, leucine, etc. Conversely, amino acids that are less likely to form an OL helix Examples include palin, isoleucine, proline, and daricin, etc.
  • j8 sheet is one of the secondary structures of a protein or polypeptide, and a zigzag conformation Two or more polypeptide chains having an omension are arranged in parallel, and the amide group and the carboxyl group of the peptide are adjacent to each other.
  • parallel j8 sheet refers to a structure in which a hydrogen bond is formed between a carboyl group and an amide group to form an energetically stable sheet.
  • Anti-parallel j8 sheet refers to a ⁇ -sheet in which the sequence of amino acids in adjacent polypeptide chains is in the opposite direction.
  • ⁇ strand refers to a single peptide chain having a zigzag conformation that forms a ⁇ sheet.
  • Three-dimensional structure data or "atomic coordinate data” of a protein refers to data relating to the three-dimensional structure of the protein.
  • the three-dimensional structure data of a protein typically includes atomic coordinate data, topology, and molecular force field constant.
  • the atomic coordinate data is typically data obtained from X-ray crystal structure analysis or NMR structural analysis. Such atomic coordinate data is obtained by newly performing X-ray crystal structure analysis or NMR structural analysis. It can be obtained from power sources or from known databases (eg, Protein Data 'Bank (PDB)). Atomic coordinate data can also be data created by modeling or calculation.
  • the topology can be calculated by using a sales or freeware tool program, a self-made program may be used. Further, a molecular topology calculation program attached to a commercially available molecular force field calculation program (for example, a prepar program attached to PRE STO, Protein Engineering Laboratories, Inc.) can be used.
  • the molecular force field constant (or molecular field potential) may also use force-made data that can be calculated using a commercial or freeware tool program. Further, molecular force field constant data attached to a commercially available molecular force field calculation program (for example, AMBER, Oxford Molecular) can be used.
  • the active site of the LOX-1 ligand binding fragment includes, for example, amino acid residues 191 to 240 of the LOX-1 ligand binding fragment shown in SEQ ID NO: 4.
  • Arginine 208, 209, histidine 226, arginine 229, and arginine 231 of SEQ ID NO: 4 are known to interact closely with ligands, and therefore, in one embodiment, the enzyme
  • the modification intended for the present invention is that the residue corresponding to the arginine at position 208, arginine at 209, histidine at position 226, arginine at position 229, and arginine at position 231 is replaced with another amino acid. It is preferred that it be modified
  • a modified design can be performed for the purpose of improving the stability of LOX-1 or improving the receptor activity.
  • thermalostability refers to the activity of a protein (e.g., enzyme activity, receptor activity, etc.) even after denaturing the protein at a temperature higher than the normal biological environment (e.g., 60 ° C). Etc.) remain at least 10%, preferably at least 50%, most preferably at least 90% as compared to before heat denaturation.
  • the improvement in stability can be measured, for example, by ⁇ (difference in denaturation temperature).
  • the term “receptor activity” is used synonymously with “LOX-l activity” unless otherwise specified, and the activity is measured by a His-tag or LOX-1 introduced at the N-terminal of LOX-1.
  • the biotin-tagged LOX-1 sample obtained by biotinizing the biotin-ligated sequence introduced at the end of LOX-1 has His-tag on the SPR sensor chip! / Can be measured by detecting the changing power of the SPR sensorgram using a sensor fixed and aligned in the direction via a bio tin-tag and detecting the changing power of the SPR sensorgram.
  • the variant molecule is designed by analyzing the amino acid sequence and the three-dimensional structure of the protein or polypeptide molecule before mutation (for example, a wild-type molecule) to determine how each amino acid is determined. Predicts that a specific property (e.g., catalytic activity, interaction with another molecule, etc.) is responsible for the desired property modification (e.g., enhanced catalytic activity, increased protein stability, etc.) The calculation is performed by calculating various amino acid mutations. design Is preferably performed using a computer.
  • a specific property e.g., catalytic activity, interaction with another molecule, etc.
  • desired property modification e.g., enhanced catalytic activity, increased protein stability, etc.
  • Examples of computer programs used in such a design method include, as mentioned herein, the following: As a program for analyzing a structure, a program for processing X-ray diffraction data, DENZO ( Mac Science); P HASES (Univ. Of Pennsylvania, PA, USA) as a processing program to determine the phase; Program DM (CCP4 package, SERC) as a program to improve the initial phase; 3D graphics Program O (Uppsala Universitet, Uppsala, Sweden); three-dimensional structure refinement program; XPLOR (Yale University, CT, USA); and Swiss- PDBViewer (see above).
  • the present invention also contemplates providing a drug (eg, inhibitor, activator, etc.) by computer modeling based on the disclosure of the present invention.
  • a drug eg, inhibitor, activator, etc.
  • mass spectrometry can be used to measure a bond such as a covalent bond.
  • mass spectrometry refers to any method for analyzing ions of atoms and molecules based on differences in mass by using electromagnetic interaction. Typically, mass spectrometry is roughly classified into two methods. The first is a method in which ions are orbitally analyzed at the same time, and the apparatus is called a mass spectrograph. The second is a method for measuring the difference in mass due to the temporal difference in force ion motion or the difference in resonance conditions whose trajectory is the same, and the device is called a mass spectrometer.
  • Mass spectrometry methods are well known in the art, and include, but are not limited to, for example, MALDI-TOF. Those skilled in the art can appropriately modify such methods and use them in the present invention.
  • the binding can be calculated using information on a quantitative change in the structure of a substance such as a protein, which can be obtained by mass spectrometry.
  • Information on the quantitative change in the structure of a substance such as a protein can be displayed as information on the presence or absence of the sugar chain itself, or the entire molecule (for example, protein) containing the sugar chain is displayed as information You can also.
  • Quantitative change information also provides information on the It can be expressed as a ratio of ratios, but is not limited thereto.
  • the unit indicating the amount (or level) of each sugar chain or a molecule containing a sugar chain that constitutes such quantitative change information is, for example, ng, ⁇ mol, signal intensity in mass spectrometry, signal in spectroscopic analysis.
  • Intensity or mgZ sample g, mmolZ sample g, or relative fluorescence intensity, but is not limited thereto.
  • MALDI-TOF MALDI-TOF
  • MS Matrix Assisted Laser Desorption Ionization-Time-of- Flight
  • MALDI is a technique discovered by Tanaka et al. And developed by Hillenkamp et al. (Karas M., Hillenkamp, F., Anal. Chem. 1988, 60, 2299-230 D o. After the solution is mixed in a molar ratio of (10-2-5 x 10-4): 1, the mixed solution is dried on the target to form a crystalline state. Given above, ions from the sample such as (M + H) + and (M + Na) + are desorbed from the matrix, etc.
  • MALDI—TOF uses MALDI to measure mass based on time of flight.
  • V The mass is m
  • v the velocity of the ion
  • z the charge number of the ion
  • e the amount of element
  • t the flight time of the ion
  • KOMPA CT MALDI ⁇ from Shimadzu ZKratos can be used.
  • a pamphlet created by the manufacturer can be referred to.
  • MALDI The irradiation energy of laser irradiation used for TOF measurement can be used as the dissociation energy and! /, 1 /, and the dissociation energy can be used to analyze covalent bonds.
  • Such a mass spectrometer can be used with reference to a manual or the like provided by a manufacturer. Specifically, a sample is dropped on a target probe attached to a mass spectrometer together with a matrix (for example, solid or liquid), dried, and the mass is measured by an analyzer.
  • the matrix can be used as provided by the manufacturer, for example, disulanol, HABA, DHBA, IAA, and the like, but are not limited thereto.
  • the present invention enables, for the first time, the identification, selection, and design of chemicals (including activators and inhibitory compounds) that can bind to LOX-1 (eg, a binding pocket) by using molecular design techniques. I do.
  • the present invention also provides, in one aspect, a polypeptide comprising the binding pocket determined in the present invention.
  • the design of a compound that binds to or inhibits LOX-1 or LOX-1 ligand binding fragment (eg, a binding pocket) generally takes into account two requirements. Accompanied by First, the substance must be able to physically and structurally associate with some or all of LOX-1. Important non-covalent molecular interactions for this association include hydrogen bonding, Van der Waals interactions, hydrophobic interactions, and electrostatic interactions.
  • the material must be capable of conformation that allows it to associate directly with LOX-1 or LOX-1 ligand binding fragments. Although certain parts of the substance do not directly participate in these associations, these parts of the substance can still affect the overall conformation of the molecule. This can then have a significant effect on efficacy. Such conformational requirements may involve the entire 3D structure and orientation of the chemical associated with all or part of the binding pocket, or directly interact with LOX-1 or LOX-1 ligand binding fragments or homologs thereof. Includes the spatial arrangement between functional groups of the substance, including some chemicals.
  • the potential inhibitory or binding effect of a chemical on LOX-1 or LOX-1 ligand binding fragment can be analyzed by use of computer modeling techniques prior to its actual synthesis and testing. If the theoretical structure of a given substance indicates poor interaction and association between the substance and LOX-1 or LOX-1 ligand binding fragment, testing of the substance is excluded. However, if computer modeling shows a strong interaction, the molecule can then be synthesized and tested for its ability to bind to LOX-1 or LOX-1 ligand binding fragments.
  • Candidate conjugates of LOX-1 modulators are computationally evaluated by screening their chemicals or fragments for their ability to associate with LOX-1 and selecting positive factors. Can be done.
  • One skilled in the art may use one of several methods for screening chemicals or fragments for their ability to associate with LOX-1 or a LOX-1 ligand binding fragment.
  • This process can be performed on a computer screen based on, for example, the structural coordinates of the LOX-1 ligand binding fragment of FIG. LOX—can be initiated by visual inspection of the 1-like binding pocket. Then, the selected flag The fragments or chemicals can be arranged or docked in various orientations within the binding pocket as defined above. Docking can be achieved using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanical force fields such as CHARMM and AMBER.
  • Computer programs for performing computer modeling can also be used in the process of selecting fragments or chemicals. Such programs include:
  • GRID PJ Goodford, "A Computational Procedure for Determining Energetically Favoraole Binding Sites on Biologically Important Macromolecules J, J. Med. Chem., 28, 849—857 (1985)). GRID is Oxford University. Available from Oxford, UK.
  • MCSS Multiple Copy Simultaneous Search Method J Proteins: Structure, Function and Genetics, 11, 29-34 (1991)).
  • MCSS is available from Molecular Simulations, San Diego, CA.
  • AUTODOCK (DS Goodsell et al., "Automated Docking of Substrates to Proteins by Simulated AnnealingJ, Proteins: Structure, Function, and Genetics, 8, 195-202 (1990)).
  • AUTODOCK is a Scripps Research Institute. Available from La Jolla, CA.
  • DOCK (ID Kuntz et al., "A Geometric Approach to Macromolecule-Ligand Interactions J, J. Mol. Biol., 161, 269—page 288 (1982)). DOCK is from University of California, San Francisco, CA. Power is available.
  • CAVEAT PA Bartlett et al., "CAVEAT: A Program to Facilitate th e Structure— Derived Design of Biologically Active Molecules J (Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc., 78, 182-196 (1989)); G, Lauri and PA Barlett, "CAVEAT: a Program to Facilitate the Design of Organic Mol eculesj, J. Comput. Aided Mol. Des., 8, 51-66 (1994). CAVEAT is available from the University of California, Berkeley, CA.
  • HOOK (MB Eisen et al., "HOOK: A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site", Proteins: Struct., Funct., Genet., 19, 199-221 (1994)). HOOK is available from Molecular Simulations, San Diego, CA.
  • an inhibitory or other LOX-1 or LOX-1 ligand may be used.
  • the binding conjugate of the binding fragment is designed in whole or de novo, either with the use of empty binding sites or with the inclusion of several known inhibitor moieties as needed. Can be done.
  • LUDI H.-J. Bohm, "The Computer Program LUDI: A New Met hod for the De Novo Design of Enzyme Inhibitors J, J. Comp. Aid. Molec. Design, 661-78 (1992) LUDI is available from Molecular Simulations Incorporated, San Diego, CA. [0256] 2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, 8985 (1991)). LEGEN D is available from Molecular Simulations Incorporated, San Diego, CA.
  • SPROUT V. Gillet et al., “SPROUT: A Program for Structure Generation”, J. Comput. Aided Mol. Design, 7, 127-153 (1993)). SPROUT is available from the University of Leeds, UK.
  • an effective LOX-1 binding pocket inhibitor should preferably exhibit a relatively small energy difference between its bound and free states (ie, a small deformation energy of binding). Therefore, the most efficient LO X-1 inhibitors should preferably be designed with deformation energies of about lOkcalZ moles or less (more preferably, 7 kcalZ moles or less). LOX-1 inhibitors can interact with the binding pocket in more than one conformation at all binding energies.
  • the deformation energy of binding is considered to be the difference between the energy of the free substance and the average energy of these conformations observed when the inhibitor binds to the protein.
  • the substance designed or selected to bind to LOX-1 or LOX-1 ligand binding fragment preferably has no electrostatic repulsion interaction with the target enzyme and surrounding water molecules in its bound state As such, it can be further optimized by calculation.
  • Such non-complementary electrostatic interactions include charge-charge repulsion interactions, dipole-dipole repulsion interactions, and charge-dipole repulsion interactions.
  • Another approach possible with the present invention is the computational screening of small molecule databases for chemicals or compounds that can bind wholly or partially to LOX-1.
  • the quality of the fit of such a material to the binding site can be determined by either shape complementarity or the estimated interaction energy [E. C. Meng et al., J. Comp. Chem., 16, 505-524 (1992)].
  • the compound library used in the present invention can be prepared or obtained by any means including, but not limited to, combinatorial chemistry technology, fermentation methods, plant and cell extraction procedures, and the like. be able to. Methods for creating combinatorial libraries are well known in the art. For example, ER Fel der, Chimia 1994, 48, 512-541; Gallop et al., Med. Chem. 1994, 37, 123. 3-1251; RA Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al., Nature 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84; Carell et al., Chem. Biol.
  • the present inventors prepared human LOX-1 (LOX-1) complexed with acetylated LDL.
  • the three-dimensional (3-D) crystal structure of the ligand-binding fragment was determined.
  • Disclosed herein is the crystal structure of this LOX-1 ligand binding fragment-acetylated LDL binary complex.
  • the present specification also discloses other structural data that can be inferred from this structural data. Such information on the crystal structure is at a remodeling level that has not been attainable in the past, the force is at the native level, and further, at a level that has not been attained at the past, If something is made possible, an excellent effect will be achieved.
  • the present invention further elucidated the crystal and structure of the ligand-binding domain of LOX-1 that retains a dimeric structure due to a disulfide bond in the same form as that present on the cell surface.
  • the LOX-1 crystal structure which retains the natural dimer structure, has a cavitation structure existing at the dimer interface and a LOX-1 crystal having a dramatic amino acid substitution near the cavities.
  • a cavitation structure existing at the dimer interface and a LOX-1 crystal having a dramatic amino acid substitution near the cavities.
  • the cavities present at the dimer interface become target sites for LOX-1 specific inhibitors, and this can be used to screen for modulators such as inhibitors.
  • the present invention provides a method for producing a crystal of a LOX-1 ligand binding fragment or a homolog or variant thereof, comprising the steps of: a) expressing a LOX-1 ligand binding fragment; Unwinding the obtained protein to provide a solution containing the obtained protein; b) adding a buffer having a buffer region to the solution at an acidic pH; c) adding calcium or zinc to the solution. And d) obtaining crystals by a vapor diffusion method.
  • the present invention also provides, in another aspect, a method for producing crystals of a dimer of a LOX-1 ligand-binding fragment or a homolog or a variant thereof, comprising the steps of: a) LOX-1 ligand A step of providing a solution containing the protein obtained by unwinding the protein obtained by expressing the binding fragment or a homologue or variant thereof, wherein the LOX-1 is a cysteine residue required for disulfide bonding. And b) obtaining crystals by a vapor diffusion method.
  • Buffers having an acidic pH as the buffer region are known in the art and include, but are not limited to, citric acid, acetic acid, and the like. Preferably, citric acid is used.
  • Sources of calcium or zinc ions are also known in the art, such as, but not limited to, calcium chloride or zinc chloride.
  • Steam diffusion methods are also known in the art. In the vapor diffusion method, droplets of a solution containing 1101 proteins are equilibrated against an external solution of about 100 times the volume of the precipitant, and the droplets of the protein solution are suspended on a cover glass.
  • the non-dropping method and the sitting drop method are commonly used for the method of leaving the sample in a recessed place.
  • the droplets in which the protein solution and the external solution are mixed at a certain ratio are vapor-balanced with respect to the external solution in a closed system, and thus are diffused. This causes crystallization.
  • the LOX-1 ligand-binding fragment preferably contains an NECK region, more preferably CTLD, and NECK.
  • the region includes 14 residues (positions 129 to 273 of SEQ ID NO: 4).
  • the present invention provides methods for determining the atomic coordinates of a LOX-1 ligand binding fragment.
  • a three-dimensional computer model generated with the data is provided.
  • the atomic coordinates may be the atomic coordinates of the LOX-1 ligand binding fragment described in FIG. 7, or a variant thereof. More preferably, the atomic coordinates include the atomic coordinates of acetylated LDL, and may be the atomic coordinates of acetylated LDL-conjugated LOX-1 ligand binding fragment shown in FIG. 7 or a modified version thereof.
  • Such a three-dimensional structure computer model can be generated by a technique well-known in the art as described above in this specification. Therefore, for example, those skilled in the art can easily provide the model of the present invention by inputting the data described in FIG.
  • the present invention provides a computer model of a three-dimensional structure generated from data of atomic coordinates of a LOX-1 ligand binding fragment that forms a dimer.
  • the atomic coordinates may be the atomic coordinates of the LOX-1 ligand binding fragment described in FIG. 17 or a variant thereof. More preferably, the atomic coordinates include the atomic coordinates of the part that undergoes dimerization, and the atomic coordinates of the dimerized LOX-1 ligand binding fragment atomic coordinates or a modified form thereof shown in FIG. There may be.
  • Such a three-dimensional structure computer model generation can be generated by a technique well-known in the art as described above in this specification. Therefore, for example, those skilled in the art can easily provide the model of the present invention by inputting the data described in FIG.
  • the invention provides a data array comprising the atomic coordinates of a LOX-1 ligand binding fragment, wherein the data array comprises a three-dimensional structure by using a three-dimensional molecular modeling algorithm.
  • the atomic coordinates are a force that is the atomic coordinates described in FIG. 7 or a modified version thereof.
  • the atomic coordinates include the atomic coordinates of the acetylated LDL and are the acetylated LDL-conjugated LOX-1 ligand binding fragment atomic coordinates or a variant thereof as described in FIG.
  • the present invention also provides a data array comprising the atomic coordinates of a LOX-1 ligand binding fragment or a homolog or variant thereof in dimeric form, wherein
  • the data array provides a data array that can present a three-dimensional structure by using a three-dimensional molecular modeling algorithm.
  • the atomic coordinates are a force that is the atomic coordinates described in FIG. 17, or a homolog or a variant thereof.
  • the present invention provides a computer-readable medium coded with the atomic coordinates of a LOX-1 ligand binding fragment.
  • this atomic coordinate is a force or a variant thereof that is the atomic coordinates described in FIG.
  • the atomic coordinates include the atomic coordinates of the acetylated LDL and are the acetylated LDL-complexed LOX-1 ligand binding fragment atomic coordinates or a variant thereof shown in FIG. .
  • the present invention also provides a computer-readable recording medium that encodes the atomic coordinates of a LOX-1 ligand binding fragment or a homolog or variant thereof in a dimeric form. .
  • the present invention provides a program for providing a method for analyzing the three-dimensional structure of a LOX-1 ligand-binding fragment, comprising: A) data encoding atomic coordinates of a LOX-1 ligand-binding fragment; And B) provide a program including an application code for causing a computer to execute a three-dimensional structure analysis.
  • Such a program may execute a technique such as three-dimensional structure refinement or modeling, as described above and herein.
  • the atomic coordinates are the atomic coordinates described in FIG. 7 or a variant thereof.
  • the atomic coordinates include the atomic coordinates of acetylated LDL, and are the acetylated LDL complexed LOX-1 atomic coordinates shown in FIG. 7 or a modified version thereof.
  • the present invention relates to a program for providing a method for analyzing a three-dimensional structure of a LOX-1 ligand-binding fragment or a homologue or a variant thereof in a dimeric form,
  • the atomic coordinates are the forces that are the atomic coordinates described in FIG. 17 or a modified version thereof.
  • the application includes: A) a step of applying a three-dimensional molecular modeling algorithm to the atomic coordinates to obtain a three-dimensional molecular model; and B) a three-dimensional molecular model of the candidate compound.
  • the step of comparing a molecular model with the three-dimensional molecular model may be performed by a computer. Applications for comparing such models are well known in the art and can be readily implemented by one of ordinary skill in the art with the description provided herein above.
  • the present invention provides an isolated and purified protein having a structure defined by the atomic coordinates described in FIG.
  • the present inventors are the first to elucidate the three-dimensional structure of acetylated LDL-conjugated LOX-1 ligand binding fragment using high-resolution X-ray crystallography.
  • the LOX-1 ligand binding fragment has not been crystallized and its structural analysis has not been performed. Therefore, it can be said that a protein having a crystal structure as described in FIG. 7 has a conventional powerful structure.
  • a protein having a crystal structure as described in FIG. 7 has a conventional powerful structure.
  • a protein comprises the activity of LOX-1.
  • Such activity can be measured using techniques well known in the art.
  • Such an activity can be, for example, an LDL binding activity.
  • a crystal of a LOX-1 ligand binding fragment is provided.
  • the crystals may preferably be complexed with acetylated LDL.
  • the crystals have a P2 22 orthorhombic form.
  • the crystal of the present invention has an amino acid sequence represented by SEQ ID NO: 2, a sequence containing one or more substitutions, additions or deletions in the amino acid sequence, or at least about 30% or more of the amino acid sequence. Has homology.
  • the crystals are complexed with acetylated LDL.
  • the crystal of the present invention has a calcium ion or a zinc ion in the asymmetric unit.
  • the crystal of the present invention contains one molecule in the asymmetric unit.
  • the crystals of the present invention are in the P4 22 tetragonal form.
  • This P4 2 2 Preferably, in the tetragonal form, in embodiments, the crystals are complexed with acetylated LDL.
  • the crystalline system of the present invention may be in the P22 22 orthorhombic form.
  • This crystal is also, in a preferred embodiment, complexed with the acetylated LDL. More preferred ⁇
  • the crystal has two molecules in the asymmetric unit.
  • the present invention provides a molecule or a molecular complex comprising a binding pocket of a LOX-1 ligand binding fragment or a part thereof, or a homologue or variant of these binding pockets having a similar three-dimensional shape. I do.
  • the present invention also provides a computer readable format comprising the structural coordinates of a LOX-1 (eg, human LOX-1) ligand binding fragment or the LOX-1 ligand conjugate fragment that forms a dimer.
  • a recording medium is provided, which contains all or part of the binding pocket of the LOX-1 ligand binding fragment.
  • Such recording media encoded with these data, can be used to display molecules or molecular complexes (such as homologous homologues in such binding pockets or similar shapes) on a computer screen or similar visual device. (Including the binding pocket) can be displayed.
  • the present invention also provides a method for designing, evaluating, and identifying a compound that binds to all or a part of the binding pocket or the dimer-forming structure. Such a compound may be a candidate inhibitor of LOX-1 or a homolog thereof.
  • a compound may be a candidate inhibitor of LOX-1 or a homolog thereof.
  • the present invention also provides novel classes of compounds useful as inhibitors of LOX-1 or a homolog thereof or a dimer thereof, and pharmaceutical compositions thereof.
  • the present invention also provides a method for determining at least a portion of a three-dimensional structure of a molecule or molecular complex comprising features that are at least somewhat structurally similar to LOX-1 or a LOX-1 ligand binding fragment.
  • the present invention also provides methods for crystallizing LOX-1 or LOX-1 ligand binding fragments and related complexes.
  • Such a crystal has never been able to be provided by conventional techniques.
  • the crystallization of the LOX-1 ligand-binding fragment was successful and the structural analysis was successfully performed only by using the conditions provided in the Examples. Therefore, such a crystal of the present invention is a force that cannot be obtained by the conventional technology, and it can be said that the present invention has a remarkable effect.
  • the present invention has succeeded in crystallization by intensive study using such conditions by appropriately modifying such conditions.
  • the present inventors have provided a crystal suitable for X-ray crystallography, comprising acetylated LDL and a complexed LOX-1 ligand-binding fragment.
  • the present inventors have provided, for the first time, information on the shape and structure of the active site binding pocket of an acetylated LDL-conjugated LOX-1 ligand binding fragment.
  • Binding pockets have important applications in areas such as drug discovery.
  • the association of natural ligands or substrates with their corresponding receptor or enzyme binding pockets is the basis of many biological mechanisms of action.
  • many drugs exert their biological effects through association with a receptor or enzyme binding pocket. Such an association can occur at all or any part of the binding pocket. Understanding such associations will help guide the design of drugs that better associate with the target receptor or enzyme, thereby improving the biological effect. Therefore, this information can be obtained from the acetylated LD L-complexed LOX-1 is useful for designing candidate inhibitors of the binding pocket of a ligand-binding fragment or a homolog thereof.
  • the present invention provides a protein comprising an active pocket of a LOX-1 ligand binding fragment or a homolog or variant thereof.
  • this active pocket may be a complex formed with acetylated LDL.
  • the active pocket is an amino acid residue described in FIG.
  • the active pocket is an amino acid residue or SEQ ID NO:
  • the active pocket is defined by the atomic coordinates of the amino acid residue shown in FIG. 7 or the amino acid residue corresponding to positions 208 to 231 of SEQ ID NO: 4.
  • the active site pocket contains a binding ligand (eg, LDL or a variant thereof (eg, acetyl LDL).
  • a binding ligand eg, LDL or a variant thereof (eg, acetyl LDL).
  • the protein is for at least one or two or more molecules selected from the group consisting of oxidized LDL, acetylated LDL, polyinosinic acid, phosphatidylserine, phosphatidylinositol, apoptotic cells, bacteria and platelet force. Has binding activity.
  • the present invention relates to an isolated and purified protein or homologue or modification thereof in dimeric form, having a structure defined by the atomic coordinates shown in FIG. Provide body.
  • the present invention relates to an amino acid sequence represented by SEQ ID NO: 36 (amino acid numbers 129 to 273 of SEQ ID NO: 4), or one or more Provided is a crystal of a LOX-1 ligand-binding fragment or a homolog or variant thereof in a dimeric form having a sequence containing substitution, addition or deletion, or at least about 30% homology with the amino acid sequence.
  • the present invention also provides a protein comprising a cavity structure present at the dimer interface of a LOX-1 ligand-binding fragment or a homolog or variant thereof.
  • This protein includes the sequences shown in SEQ ID NOs: 35 and 36.
  • the present invention provides a LOX-1 ligand-binding fragment or a homolog or a variant thereof, which forms an active pocket or a dimer of an LOX-1 ligand-binding fragment or a homolog or a variant thereof.
  • a nucleic acid molecule encoding an amino acid sequence of a protein, and a recording medium on which the information is recorded.
  • One of skill in the art once knowing the amino acid sequence of a protein, can easily obtain a nucleic acid molecule having a nucleic acid sequence encoding such a sequence.
  • the present invention provides a method for obtaining a homolog of a LOX-1 ligand binding fragment, comprising the steps of: A method comprising comparing
  • the LOX-1 may be complexed with acetylated LDL.
  • the atomic coordinates include the atomic coordinates described in FIG. 7, but any atomic coordinates that can be reasonably modeled from the atomic coordinates described in FIG. 7 may be used. It is possible.
  • the present invention provides a method for obtaining a dimeric homolog of a LOX-1 ligand-binding fragment, comprising the steps of: Comparing with the atomic coordinates of the dimer interface of Preferably, the dimer has a structure defined by the atomic coordinates described in FIG.
  • the present invention provides a method for obtaining a homolog of LOX-1 or a LOX-1 ligand binding fragment.
  • the method comprises the following steps: A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of LOX-1 or LOX-1 ligand binding fragment to obtain a three-dimensional molecular model; and B) three-dimensional molecular model of the candidate compound. Comparing the molecular model to a three-dimensional molecular model of the LOX-1 or LOX-1 ligand binding fragment.
  • a technique as described above in this specification can be used.
  • the comparison step is also well known in the art, Can also use techniques such as those described herein above.
  • the LOX-1 ligand binding fragment is complexed with acetylated LDL.
  • the atomic coordinates include the atomic coordinates described in FIG.
  • the present invention provides a method for obtaining a dimeric homologue of a LOX-1 ligand binding fragment, comprising the following steps: A) LOX-1 ligand binding fragment Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the dimer to obtain a three-dimensional molecular model; and B) converting the three-dimensional molecular model of the candidate conjugate into a dimer of the LOX-1 ligand binding fragment.
  • a method comprising comparing with a three-dimensional molecular model of the body.
  • the dimer has a structure defined by the atomic coordinates described in FIG.
  • the present invention does not form or form a LOX-1 or LOX-1 ligand binding fragment homolog, or dimer, identified by the method of the invention above. Provides LOX-1 ligand binding fragment homologs.
  • the present invention provides a method for producing a LOX-1 or LOX-1 ligand-binding fragment homolog, or LOX, which does not form or form a dimer, as identified by the methods of the invention above.
  • the present invention provides a method for obtaining a modified form of LOX-1 or a LOX-1 ligand-binding fragment.
  • the method comprises the following steps: A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of LOX-1 or LOX-1 ligand binding fragment to obtain a three-dimensional molecular model; and B) three-dimensional molecular model of the candidate compound. Comparing the molecular model with the three-dimensional molecular model of the LOX-1 or LOX-1 ligand-binding fragment, and introducing a mutation based on a predetermined value.
  • a technique as described above in this specification can be used as described above in this specification.
  • the predetermined parameter may be a parameter relating to the physical properties of the protein, for example, hydrogen bonding, van der ⁇ Interactions include, but are not limited to, Ruska, ionic, non-ionic, and electrostatic interactions.
  • the LOX-1 or LOX-1 ligand binding fragment is complexed with acetylated LDL.
  • the atomic coordinates include the atomic coordinates described in FIG.
  • the present invention provides a method for obtaining a dimeric variant of a LOX-1 ligand binding fragment, comprising the following steps: A) LOX-1 ligand binding fragment Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the dimer to obtain a three-dimensional molecular model; and B) converting the three-dimensional molecular model of the candidate compound to the third order of the dimer of the LOX-1 ligand-binding fragment. Comparing with the original molecular model to introduce a mutation based on a predetermined parameter.
  • the dimer has a structure defined by the atomic coordinates set forth in FIG.
  • the variant has enhanced LOX-1 activity.
  • whether or not LOX-1 activity is enhanced can be determined, for example, by checking whether such a variant using LDL as a ligand significantly increases the binding activity as compared with wild-type LOX-1. Can be determined. In the present specification, in such a determination, LOX-1 activity can be determined under the same conditions as those of wild-type LOX-1.
  • the variant has a reduced LOX-1 activity.
  • whether or not LOX-1 activity is reduced can be determined, for example, by examining whether or not acetyl LDL is a ligand and the ability to significantly reduce the binding of such modified LDL to wild-type LOX-1. Can be determined.
  • the present invention relates to LOX-1 ligand binding that forms a variant or dimer of LOX-1 or a LOX-1 ligand binding fragment identified by a method of the invention. Fragment variants are provided.
  • the present invention relates to a modified LOX-1 or LOX-1 ligand-binding fragment or a modified LOX-1 ligand-binding fragment that forms a dimer, which is identified by the method of the present invention.
  • the Molecular Similarity application allows comparison between different structures, different conformations of the same structure, and different parts of the same structure.
  • the procedure used in Molecular Similarity to compare structures is divided into four steps: 1) loading the structures to be compared; 2) defining the atomic equivalents in these structures; 3) fitting operations And 4) analyze the results.
  • Each structure is specified by a name.
  • One structure is identified as a target (ie, a fixed structure); all remaining structures are working structures (ie, variable structures).
  • Atomic equivalents are defined by user input in QUANTA, and for the purposes of the present invention, equivalent atoms for all conserved residues between the two structures being compared are replaced with protein skeleton atoms (N, C H, C, and O) are defined by the present inventors. We also consider only robust adaptation operations.
  • the working structure is translated and rotated to obtain the best fit with the target structure.
  • the fitting operation uses an algorithm that calculates the optimal translation and rotation applied to the variable structure, so that the root-mean-square difference in fitting for a particular pair of equivalent atoms is absolutely lowest. This number is given in Angstroms, Reported by QUANTA.
  • OA when superimposed on the relevant skeleton atom described by the structural coordinates listed in FIG.
  • root mean square deviation refers to the square root of the arithmetic mean of the squares of the mean force deviations. It is a way of expressing deviation or variation from a trend or purpose.
  • root mean square deviation is defined as the deviation of LOX-1 or its binding pocket moiety in the backbone of the protein, even in the backbone of the protein, by the structural coordinates of LOX-1 as described herein. To be defined.
  • the present invention relates to a molecule or molecular complex comprising all or part of the amino acid residue set forth in Figure 7 or the binding pocket at positions 191-240 of SEQ ID NO: 4, Or 3.
  • a homologue of the molecule or molecular complex comprising a binding pocket having a root mean square deviation from the backbone atom of the amino acid of 3.OA or less, preferably 2.5A or less.
  • An alternative more preferred embodiment of the invention is an acetylated LDL-conjugated LOX-1 molecule.
  • a computer readable medium includes a data storage material encoded with computer readable data that can represent a graphic three dimensional representation.
  • the present invention relates to computer-readable data.
  • a computer readable data storage medium including a loaded data storage material is provided and a machine programmed with instructions for using the data is used
  • the computer readable data storage medium may be a molecule or a data storage medium.
  • a three-dimensional representation of the molecule complex can be displayed, wherein the molecule or molecule complex is the amino acid residue described in FIG. 7 or the position 191-240 of SEQ ID NO: 4, or the molecule or molecule.
  • the binding pocket defined by the structural coordinates of the homologue of the complex, wherein the homologue has a root mean square deviation from the skeletal atom of the amino acid of 2.5 A or less. Includes binding pockets with differences.
  • the computer-readable data storage medium stores data encoded using a first set of computer-readable data including a Fourier transform of the structural coordinates described in FIG.
  • the first set may include at least one of the computer-readable data and a corresponding set of structural coordinates. It can be combined with a second set of computer readable data, including the X-ray diffraction pattern of the molecule or molecular complex, to determine a portion.
  • the system 10 includes a computer 11, including a central processing unit (“CPU”) 20, a working memory 22 (which may be, for example, RAM (random access memory) or “core” memory), a mass storage memory 24 (eg, , One or more disk drives or CD-ROM drives), one or more cathode ray tube (“CRT") display terminals 26, one or more keyboards 28, one or more input lines 30, one or more output lines 40, all of which are interconnected by a conventional bidirectional system bus 50.
  • CPU central processing unit
  • working memory 22 which may be, for example, RAM (random access memory) or “core” memory
  • mass storage memory 24 eg, One or more disk drives or CD-ROM drives
  • CRT cathode ray tube
  • the input hardware 36 connected to the computer 11 by the input line 30 can be implemented in various ways.
  • the computer readable data of the present invention may be entered through the use of a modem 32 connected by a telephone line or a dedicated data line 34.
  • input hardware 36 may include a CD-ROM drive or disk drive 24.
  • a keyboard 28 can also be used as an input device.
  • the output hardware 46 connected to the computer 11 by the output line 40 can be similarly implemented by ordinary equipment.
  • output hardware 46 may include a CRT display terminal 26 for displaying a graphical representation of the binding pocket of the present invention using a program such as QUANTA described herein.
  • Output hardware may also include a printer 42, or a disk drive 24, capable of producing hardcopy output to save system output for later use.
  • the CPU 20 interfaces with various input and output devices 36, 46, associates mass storage 24 with data access and access to and from the working memory 22, and arranges them. Is determined. Numerous program powers can be used to process the computer readable data of the present invention. Such programs are designed with reference to the computer-based method of drug discovery as described herein. Components of the hardware system 10 as appropriate throughout the following description of the data storage medium are also within the scope of the present invention.
  • FIG. 13 illustrates a cross-section of a magnetic data storage medium 100 that may be coded with computer readable data that may be executed by a system such as system 10 of FIG.
  • the medium 100 may be a conventional floppy diskette or hard disk having a suitable substrate 101, which may be conventional, and a suitable coating 102, which may be conventional, on one or both sides. This includes magnetic domains (invisible! / ⁇ ) whose polarity or orientation can be changed magnetically.
  • Media 100 may also have an opening (not shown) for receiving the axis of a disk drive or other data storage device 24.
  • the magnetic domains of the coating 102 of the medium 100 encode computer readable data as described herein in a conventional manner for implementation by a system such as the system 10 of FIG. As such, they are polarized or oriented.
  • FIG. 14 illustrates the traversal of an optically readable data storage medium 110 that may also be coded using such a computer readable data or set of instructions, which may be performed by a system such as system 11 of FIG. Surface.
  • Medium 110 is a conventional compact disk read-only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk that is optically readable and magneto-optically writable. obtain.
  • the medium 100 preferably has a suitable substrate 111, which may be conventional, and usually a suitable coating 112, which may be conventional, on one side of the substrate 111.
  • the coating 112 is impressed with a plurality of pits 113 to be reflective and to encode computer readable data.
  • the array of pits is read by reflecting off the laser light to the surface of the coating 112.
  • a protective coating 114 preferably substantially transparent, is provided on the surface of the force coating 112.
  • the coating 112 has no pits 113, but its polarity or orientation is magnetic when heated by a laser (not shown) above a certain temperature. It has a plurality of magnetic domains, which can be changed in a dynamic way. The orientation of this section can be read by measuring the polarization of the laser light reflected from the coating 112. This array of sections codes the data as described above.
  • data capable of representing the three-dimensional structure of all or a portion of LOX-1 eg, a LOX-1 ligand binding fragment
  • the graphic is stored on a machine-readable recording medium capable of displaying a three-dimensional display.
  • Such data can be used for various purposes, such as drug discovery.
  • the structure encoded by the data can be evaluated computationally for its ability to associate with the chemical. Chemicals that associate with LOX-1 or LOX-1 ligand binding fragments can inhibit LOX-1. Such substances are candidate drugs.
  • the structure encoded by the data may be displayed on a computer screen in a graphical three-dimensional display. This allows for a visual inspection of the structure as well as a visual inspection of the association of the structure with the chemical.
  • the present invention relates to a method for assessing the ability of a chemical to associate with any of the molecules or molecular complexes described above.
  • the method includes the following steps: a) performing a fitting operation between the chemical substance and the binding pocket of the molecule or molecular complex using computational means; and b) determining the result of this fitting operation. Analyzing to quantify the association between the chemical and the binding pocket.
  • the term "chemical” refers to a compound, a complex of at least two compounds, and to such compounds. Or a complex fragment.
  • the present invention provides a medicament identified by the method of the present invention, and a method of treatment and prevention using the same.
  • prevention refers to the treatment of a disease or disorder before the occurrence of such condition so that the condition does not occur.
  • treatment refers to the prevention of a disease or disorder from occurring when such a disease or disorder is brought into such a state. More preferably, it means reducing, and more preferably reducing.
  • the pharmaceutical composition of the present invention comprises LOX-1 or LOX-1 ligand-binding fragment and Z or a homolog and Z or an inhibitor obtained by the screening method of the present invention; and, if necessary, any pharmaceutically It contains an acceptable carrier, adjuvant or vehicle. Such compositions may optionally contain other additional agents.
  • Lipinski factor is an index for identifying a compound having a good in vivo absorption profile.
  • Lipinski's five principles e.g., with a hydrogen donor number of 5 or less, a hydrogen bond allowable number of 10 or less, a molecular weight of 500 or less, and a logP of Compounds with a calculated value of 5 or less are selected (for details, see Lipinski, CA et al., Adv. Drug Deliv. Rev. 2001 Mar 1; 46 (1-3): 3-26).
  • the term "pharmaceutically acceptable carrier” refers to a substance that is used when producing a medicament or an animal drug, and does not adversely affect the active ingredient.
  • Such pharmaceutically acceptable carriers include, for example, antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, Delivery vehicles include, but are not limited to, diluents, excipients and Z or pharmaceutical adjuvants.
  • the pharmaceutical composition of the present invention may be in any form as long as it is suitable for transfer to living organisms. May be provided. Such preparation forms include, for example, liquid preparations, injections, and sustained release preparations.
  • the method of administration is oral, parenteral (e.g., intravenous, intramuscular, subcutaneous, intradermal, mucosal, rectal, vaginal, trunk local, dermal, intraarterial) Administration, intrasynovial administration, intrasternal administration, intrathecal administration, intralesional injection, and intracranial injection or infusion techniques), and direct administration to the affected area.
  • Formulations for such administration may be provided in any form.
  • Such preparation forms include, for example, solutions, injections and sustained-release preparations.
  • compositions of the present invention when administered systemically, may be in the form of a pyrogen-free, orally acceptable aqueous solution.
  • the preparation of such pharmaceutically acceptable protein solutions is within the skill of those in the art, as long as considerable attention is paid to pH, isotonicity, stability and the like.
  • the solvent used for the formulation of a medicament in the present invention may have either aqueous or non-aqueous properties.
  • the vehicle may be used to modify or maintain the formulation's pH, osmolality, viscosity, clarity, color, sterility, stability, isotonicity, disintegration rate, or odor.
  • the compositions of the present invention may include other formulation materials to modify or maintain the rate of release of the active ingredient, or to facilitate absorption or penetration of the active ingredient.
  • the present invention when formulated as a pharmaceutical composition, may optionally comprise a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed. , Mack Publishing Company, 1990) and a selected composition having the desired degree of purity, and can be prepared for storage in the form of a lyophilized cake or aqueous solution.
  • a physiologically acceptable carrier excipient or stabilizer
  • excipient or stabilizer Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed. , Mack Publishing Company, 1990
  • a selected composition having the desired degree of purity
  • Such suitable pharmaceutically acceptable agents include, but are not limited to, antioxidants, preservatives, coloring agents, flavorings, and diluents, emulsifiers, suspending agents. Agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and Z or agricultural or pharmaceutical adjuvants.
  • a medicament of the invention will be administered in the form of a composition of the active ingredient of the invention together with one or more physiologically acceptable carriers, excipients or diluents.
  • a suitable vehicle may be water for injection, physiological solution, or artificial cerebrospinal fluid, including those common in compositions for parenteral delivery. It is possible to supplement the quality.
  • Such acceptable carriers, excipients or stabilizers are non-toxic to recipients, and are preferably inert at the dosages and concentrations employed, and include the following: Include: phosphates, citrates, or other organic acids; antioxidants (eg, ascorbic acid); low molecular weight polypeptides; proteins (eg, serum albumin, gelatin or immunoglobulin); Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA) Sugar alcohols (eg, mannitol or sorbitol); (Eg, sodium); and Z or a non-ionic surfactant (eg, Tween, pluronic or polyethylene glycol (PEG)).
  • phosphates, citrates, or other organic acids eg, ascorbic acid
  • proteins eg, serum albumin
  • An injection can be prepared by a method well known in the art. For example, after dissolving in an appropriate solvent (saline, buffer such as PBS, sterile water, etc.), filter sterilize it with a filter, etc., and then fill an aseptic container (eg, ampoule, etc.) to give an injection.
  • an appropriate solvent saline, buffer such as PBS, sterile water, etc.
  • filter sterilize it with a filter, etc. and then fill an aseptic container (eg, ampoule, etc.) to give an injection.
  • the injection may contain a conventional pharmaceutical carrier, if necessary.
  • a method of administration using a non-invasive catheter may also be used.
  • Exemplary suitable carriers include neutral buffered saline, or saline mixed with serum albumin.
  • the medicament of the invention is formulated as a lyophilizate using suitable excipients (eg, sucrose).
  • compositions include a Tris buffer at pH 7.0-5.5 or an acetate buffer at pH 4.0-5.5, which may further comprise sorbitol or a suitable alternative thereof. May be included.
  • the pH of the solution should also be chosen based on the relative solubility of the active ingredients of the present invention at various pHs.
  • the compound identified in the present invention is provided in a sustained release form.
  • the sustained release dosage form can be any form known in the art as long as it can be used in the present invention.
  • Such a form may be, for example, a preparation in the form of a rod (pellet, cylinder, needle, etc.), tablet, disk, sphere, sheet.
  • Methods for preparing sustained release forms are known in the art, and are described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopeia, and in other countries.
  • Methods for producing a sustained-release preparation include, for example, a method using the dissociation of a drug from a complex, a method using an aqueous suspension injection, a method using an oily injection or an oily suspension. And a method for preparing an emulsion injection solution (oZw type, wZo type emulsion injection solution, etc.).
  • the active ingredient of such medicament may be, for example, between about OlmgZkg body weight and about 100 mgZkg body weight per day, preferably about 0.5 mgZkg body weight per day. It can be administered between about 75 mgZkg body weight.
  • Such dosages will vary depending on the disease or disorder associated with LOX-1 being treated, whether preventive or therapeutic, the age, size, sex, medical history, concomitant medication, etc. of the subject, but those of ordinary skill in the art will appreciate Appropriate dosages can be determined appropriately taking into account such variables.
  • the frequency of administration of the pharmaceutical composition of the present invention also takes into account the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, and course of treatment. Then, those skilled in the art can easily determine. For example, administration may be by force, administered about once to about 5 times per day, or by a continuous infusion method. Such administration can be used for chronic or acute treatment.
  • the amount of active ingredient that may be combined with the carrier materials to provide a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation contains about 5% to about 95% (wZw) of the active conjugate. Preferably, such preparations contain from about 20% to about 80% active compound.
  • composition of the present invention contains one or more additional pharmaceutical compositions or a combination of preventive agents as an active ingredient of a medicament
  • additional pharmaceutical compositions or a combination of preventive agents as an active ingredient of a medicament
  • both the compound species identified in the present invention and the additional medicinal agent are used. Should be given at a dosage level of between about 10-100% of the dosage normally administered in a monotherapy regimen, more preferably between about 10-80%.
  • Structural coordinates shown in Figure 7 or Figure 17 also In obtaining structural information about other crystallizing molecules that belong (e.g., NK cell receptor, lymphocyte IGE receptor, dendritic cell receptor CLEC-1 and CLEC-2) or molecular complexes, Can be used to assist. This can be achieved by well-known techniques, including any of a number of molecular replacements.
  • other crystallizing molecules e.g., NK cell receptor, lymphocyte IGE receptor, dendritic cell receptor CLEC-1 and CLEC-2
  • molecular complexes can be used to assist. This can be achieved by well-known techniques, including any of a number of molecular replacements.
  • the present invention provides methods that utilize molecular substitution to obtain structural information about a molecule or molecular complex of unknown structure.
  • the method includes the following steps: a) crystallizing the molecule or molecular complex having an unknown structure; b) obtaining an X-ray diffraction pattern from the crystallized molecule or molecular complex; c) a step of applying at least a part of the structural coordinates shown in FIG. 7 to the X-ray diffraction pattern to obtain a three-dimensional electron density map of the molecule or the molecular complex whose structure is unknown.
  • Molecular replacement provides an accurate estimate of the topology of an unknown structure. Phase is a factor in the equations used to solve crystal structures that cannot be determined directly. Obtaining accurate values of the phase by methods other than molecular replacement is a time consuming process involving repeated cycles of estimation and refinement, and greatly hinders the elucidation of the crystal structure. However, when the crystal structure of a protein containing at least the homologous portion has been solved, the topology of the known structure provides a basis for predicting the phase of the unknown structure.
  • this method uses the method shown in FIG. And preparing a preliminary model of a molecule or molecular complex whose structural coordinates are unknown by orienting and positioning the relevant portion of LOX-1 or LOX-1 ligand binding fragment described in US Pat. The phase can then be calculated from this model and combined with the observed X-ray diffraction pattern amplitude to create an electron density map of a structure with unknown coordinates. This, in turn, applies to any well-known model building and structural refinement techniques. Can provide a final and accurate structure of an unknown crystallized molecule or molecular complex
  • the molecular replacement method is used to obtain structural information about a molecule or molecular complex, wherein the complex comprises at least one LOX.
  • the complex comprises at least one LOX.
  • LOX Contains 1 or LOX-1 ligand binding fragment or homolog.
  • the structural coordinates of LOX-1 provided by the present invention are based on the structure of LOX-1 or LOX-1 ligand-binding fragment or other crystal forms of LOX-1 complex, and the mechanism of Z or dimer formation. It is particularly useful for elucidating
  • the structural coordinates of the LOX-1 or LOX-1 ligand binding fragment provided by the present invention are the same as those of the mutant LOX-1 or LOX-1 ligand binding fragment (this may be, if necessary, It may be crystallized as a complex with a chemical substance).
  • the crystal structure of a series of such complexes can then be solved by molecular replacement and compared to the crystal structure of wild-type LOX-1.
  • Candidate forces for modification sites within the various binding sites of the enzyme can thus be identified. This information provides an additional means to determine the most efficient binding interaction (eg, increased hydrophobic interaction) between LOX-1 or LOX-1 ligand binding fragment and a chemical or compound
  • the structural coordinates are also particularly useful for elucidating the crystal structures of LOX-1 or LOX-1 homologs co-complexed with various chemicals.
  • This approach allows the determination of the optimal site for the interaction between a chemical containing a candidate inhibitor of LOX-1 and LOX-1 or a LOX-1 ligand binding fragment. For example, high-resolution X-ray diffraction data collected from crystals exposed to different types of solvents show that each type of solvent molecule is present. Enables you to determine where you are. Small molecules that bind tightly to these sites can then be designed and synthesized and tested for their LOX-1 inhibitory activity.
  • the present invention provides a method for identifying a compound that can bind to LOX-1 or a LOX-1 ligand binding fragment or a homolog thereof.
  • a method for identifying a compound that can bind to LOX-1 or a LOX-1 ligand binding fragment or a homolog thereof involves the following steps: a) Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of LOX-1 or LOX-1 ligand binding fragment or the atomic coordinates of a homolog or variant thereof. Determining the spatial coordinates of the active site pocket of LOX-1 or LOX-1 ligand binding fragment; and b) the space of the active site pocket of the LOX-1 or LOX-1 ligand binding fragment or a homolog or variant thereof.
  • Active site pockets can be identified by the methods described in the present invention. Examples of such active pockets are as described herein above. Identification of the above-mentioned compounds capable of binding can also be performed using techniques known in the art. Such methods are described herein above.
  • the linkage can be anywhere on the protein, but preferably is an active site pocket. The ability to bind to a protein is determined by calculating interactions (eg, hydrogen bonding, van der Waals forces, ionic, non-ionic, electrostatic interactions, etc.). Can be.
  • the LOX-1 or LOX-1 is used.
  • the LOX-1 ligand binding fragment may be complexed with acetylated LDL. More preferably, the atomic coordinates include the atomic coordinates described in FIG.
  • the present invention relates to a method for identifying a compound capable of binding to a LOX-1 ligand binding fragment or a homolog or a variant thereof, comprising the following steps: A) LOX — Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the dimer of one ligand binding fragment or its homolog or variant to form a dimer of LOX 1 ligand binding fragment Determining the spatial coordinates of the dimer interface; and B) electronically determining the set of candidate conjugates relative to the spatial coordinates of the dimer interface forming the dimer of the LOX-1 ligand binding fragment.
  • the atomic coordinates include the atomic coordinates described in FIG.
  • the compounds presented herein may enhance or reduce LOX-1 activity.
  • the polypeptide that forms the dimer of the LOX-1 ligand binding fragment comprises the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 35 and the amino acid sequence represented by Z or SEQ ID NO: 36. More preferably, in the polypeptide forming the dimer of the LOX-1 ligand binding fragment, the tributophan (W) at position 150 in the sequence shown in SEQ ID NO: 4 is advantageously conserved.
  • the polypeptide forming the dimer of the LOX-1 ligand-binding fragment preferably has a conserved portion containing a tryptophan (W) at position 150 in the sequence shown in SEQ ID NO: 4. It is. Without wishing to be bound by theory, it is likely that such a cavity-forming moiety is required for dimer formation.
  • the homologue or variant may be one obtained by the method of the present invention.
  • the above-mentioned LOX-1 or LOX-1 ligand-binding fragment includes mammals such as human, staple, and hidge, or human LOX-1 or LOX-1 ligand-binding fragment.
  • the spatial coordinates determined in the above are defined by the atomic coordinates of the amino acid residues shown in FIG. 7 or the amino acid residues at positions 191 to 240 of SEQ ID NO: 4 or corresponding thereto.
  • the spatial coordinates include the atomic coordinates of the salt ion (C1—), the phosphate ion, and the binding ligand (such as LDL or a variant thereof) described in FIG.
  • the present invention provides LOX-1 by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of LOX-1 or LOX-1 ligand binding fragment or a homolog thereof.
  • the present invention provides a method for identifying a compound capable of binding to a LOX-1 ligand binding fragment or a homolog or a variant thereof.
  • the method consists of the following steps: A) Applying a 3D molecular modeling algorithm to the atomic coordinates of LOX-1 or LOX-1 ligand binding fragment or its homologs or variants to obtain a 3D molecular model B) a step of inputting the coordinate data of the three-dimensional molecular model into a data structure and searching for a distance between atoms of LOX-1 or LOX-1 ligand binding fragment; and C) hydrogen in the candidate compound.
  • Identifying candidate species that theoretically form a stable complex with the active site pocket of the three-dimensional molecular model of LOX-1 or LOX-1 ligand binding fragment By obtaining such a model, the step of searching for the distance between atoms, and the step of identifying the candidate compound species are well known in the art, and are performed by appropriately using the above-described computer analysis in the present specification. be able to.
  • the present invention provides a method for comparing LOX-1 by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of a LOX-1 ligand-binding fragment that forms a dimer or a homolog thereof.
  • the third order of the LOX-1 ligand binding fragment that forms a dimer based on the optimal hydrogen bond between the two structures, comparing the distance between the heteroatoms that form the site pocket A method comprising identifying a candidate compound species that theoretically forms a stable complex with a dimer interface that forms a dimer of the original molecular model.
  • the LOX-1 or LOX-1 ligand binding fragment may be complexed with acetylated LDL.
  • the atomic coordinates include the atomic coordinates described in FIG. 7 or FIG. 17 (dimer formation).
  • the homologue or variant is one obtained by the method of the present invention.
  • the LOX-1 or LOX-1 ligand binding fragment used is a mammalian or human LOX-1 or LOX-1 such as a human, a mouse, a hedge, a mouse, and the like. Includes ligand binding fragments.
  • the candidate compound has an activity of inhibiting the LDL-binding activity of LOX-1.
  • the candidate compound has an activity of activating the LDL-binding activity of LOX-1.
  • the present invention relates to a conjugate identified by the method of the present invention.
  • a conjugate identified by the method of the present invention may be an antagonist or antagonist of LOX-1, or an inhibitor.
  • LOX-1 an antagonist or antagonist of LOX-1
  • an inhibitor an inhibitor that inhibits the atomic coordinates of the atomic coordinates.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound identified by the method of the present invention as an active ingredient.
  • Methods for making pharmaceutical compositions are well-known in the art and are as described herein above.
  • the pharmaceutical composition of the present invention may be for treating or preventing a disease or disorder associated with LOX-1.
  • diseases or disorders are known in the art, and include, for example, diseases relating to cardiovascular diseases caused by arteriosclerosis such as arteriosclerosis, angina pectoris, myocardial infarction, and cerebral infarction. , But not limited to them.
  • the present invention provides a database encoding data including the name and structure of the compound identified by the method of the present invention.
  • Methods for generating a database that encodes such data are well known in the art and may be performed using methods as described herein above.
  • the present invention provides a recording medium including a database, which codes data including the name and structure of the compound identified by the method of the present invention.
  • a recording medium including a database, which codes data including the name and structure of the compound identified by the method of the present invention.
  • Such recording media can be in any form, for example, MO, CD-R, CD-RW, CD-ROM, DVD-RAM, DVD-R, DVD-RW ⁇ DVD + RW ⁇ DVD-ROM ⁇ It may be a memory card.
  • the present invention provides a transmission medium including a database, which codes data including the name and structure of the compound identified by the method of the present invention.
  • a transmission medium including a database, which codes data including the name and structure of the compound identified by the method of the present invention.
  • a transmission medium those well-known in the art can be used, and examples include, but are not limited to, the Internet, an intranet, a LAN, and a WAN.
  • the present invention provides a method for preparing a pharmacophore model of a ligand of a LOX-1 ligand binding fragment, comprising: a ) a LOX-1 ligand binding fragment and the LOX-1 ligand binding fragment and acetyl Providing a complex with the conjugated LDL; b) comparing the NMR of the LOX-1 ligand binding fragment with the NMR of the complex; c) assigning an atom (preferably an amino acid position) with a modification
  • Techniques for providing single NMR and complex NMR can utilize methods known in the art and can be performed in accordance with the disclosure herein. NMR atomic assignments are also well known in the art and the methods outlined herein can be used.
  • the present invention provides a method for preparing a pharmacophore model of a ligand of a LOX-1 ligand-binding fragment, comprising: a ) a LOX-1 ligand-binding fragment that forms a dimer or a modification thereof. Providing a modified form of the LOX-1 ligand binding fragment without forming the dimer and dimer; b) NMR of the LOX-1 ligand binding fragment or the modified form thereof, which forms the dimer, LOX-1 ligand binding fragment without dimer formation Comparing the NMR of the variant of the reagent with the NMR; c) assigning the atom with the change.
  • Techniques for providing single NMR and complex NMR can use known methods in the relevant field and can be performed according to the disclosure of the present specification. NMR atom assignment is also well known in the art, and the methods outlined herein can be used.
  • the present invention provides a pharmacophore model identified by the method of the present invention.
  • a pharmacophore model can be created using the atomic assignments obtained by comparison of the NMRs presented herein.
  • the present invention provides the use of the pharmacophore model of the present invention in screening of drug candidate molecules.
  • the present invention provides a compound or a salt thereof identified by screening using the pharmacophore model of the present invention. Once a pharmacophore model is determined for such a compound, those skilled in the art can select and synthesize a compound that can be synthesized as appropriate.
  • the present invention provides a compound defined by the pharmacophore identified in the present invention.
  • a compound can be identified by using the pharmacophore data identified in the present invention by applying techniques well known in the art.
  • the present invention provides use of the pharmacophore model of the present invention in screening for a drug candidate molecule.
  • a computer analysis technique as described in the present specification can be used.
  • the present invention provides a drug candidate molecule identified by screening using the pharmacophore model of the present invention.
  • the molecule has activity to inhibit LOX-1 activity of LOX-1.
  • the present invention provides a pharmaceutical composition comprising the drug candidate molecule.
  • the present invention provides a method for preparing a pharmaceutical composition for treating or preventing a disease or disorder associated with LOX-1.
  • This method is based on A) LOX-l A step of obtaining a three-dimensional molecular model by applying a three-dimensional molecular modeling algorithm to the atomic coordinates or the atomic coordinates of a homologue or a variant thereof; B) the three-dimensional molecular model and a candidate to be included in the pharmaceutical composition. Evaluating the interaction of the compound with the library; C) selecting a compound species having an activity of regulating the activity of LOX-1 among the candidate conjugates; andD) selecting the compound species. Mixing with a pharmaceutically acceptable carrier.
  • the above A) to C) can be performed by using a technique described in another place in this specification.
  • the mixing step can also be performed using techniques well known in the art, for example, such a method can be performed using techniques as described in the Japanese Pharmacopoeia.
  • the LOX-1 or LOX-1 ligand binding fragment may be complexed with acetylated LDL.
  • the atomic coordinates include the atomic coordinates described in FIG.
  • the homolog or the variant obtained by the method of the present invention can be used.
  • the present invention provides a method for preparing a pharmaceutical composition for treating or preventing a disease or disorder associated with LOX-1, comprising: A) a dimer forming LOX-1 ligand binding fragment Obtaining a three-dimensional molecular model by applying a three-dimensional molecular modeling algorithm to the atomic coordinates of or a homologue or variant thereof; B) the three-dimensional molecular model and a candidate to be included in the pharmaceutical composition. E) evaluating the interaction of the compound with the library; C) selecting from among the candidate compounds a compound having an effect of regulating the activity of the LOX-1; and D) selecting the compound. And a pharmaceutically acceptable carrier.
  • the mixing step can also be performed using techniques well known in the art, for example, such a method can be performed using techniques as described in the Japanese Pharmacopoeia.
  • the LOX-1 or LOX-1 ligand binding fragment may be dimeric or capable.
  • the atomic coordinates include the atomic coordinates described in FIG.
  • the homolog or the variant obtained by the method of the present invention can be used.
  • the LOX-1 or LOX-1 ligand-binding fragment used in the present invention is a mammal such as a human, a mouse, a mouse, a mouse, or a human LOX-1 or LOX-1. Includes ligand binding fragments.
  • the method further includes a step of synthesizing the compound species to produce the compound species in a large amount.
  • mass synthesis methods once a method for synthesizing the compound species, are known to those of skill in the art using methods well known in the art.
  • the method further includes a step of conducting a biological test for LOX-1 activity on the conjugated species contained in the pharmaceutical composition of the present invention.
  • a biological test may be an in vitro or in vivo or animal experiment. Methods for performing such biological tests are well-known in the art.
  • the present invention provides a method for treating or preventing a disease or disorder associated with LOX-1 or LOX-1 ligand binding fragment.
  • This method comprises the steps of: A) obtaining a three-dimensional molecular model by applying a three-dimensional molecular modeling algorithm to the atomic coordinates of LOX-1 or LOX-1 ligand-binding fragment or to the atomic coordinates of a homolog or variant thereof. B) a step of identifying a means for modulating the activity of LOX-1 or LOX-1 ligand binding fragment based on the three-dimensional molecular model; and C) the ability of the modulating means to affect the disease or disorder or its ability. Administering to a potential subject.
  • the LOX-1 or LOX-1 ligand binding fragment may be complexed with acetylated LDL.
  • the atomic coordinates include the atomic coordinates described in FIG.
  • the homologue or variant may be obtained by the method of the present invention.
  • the present invention provides a method for treating or preventing a disease or disorder related to LOX-1, comprising: A) an atom of a LOX-1 ligand binding fragment that forms a dimer Applying a three-dimensional molecular modeling algorithm to the coordinates or atomic coordinates of a homolog or variant thereof to obtain a three-dimensional molecular model; B) regulating the activity of LOX-1 based on the three-dimensional molecular model And C) administering said modulating means to a subject capable of or possibly having the disease or disorder.
  • the LOX-1 or LOX-1 ligand binding The fragment may be dimeric or capable of dimerization.
  • the atomic coordinates include the atomic coordinates described in FIG.
  • the homologue or variant may be obtained by the method of the present invention.
  • the LOX-1 or LOX-1 ligand-binding fragment is a mammal such as a human, a mouse, a mouse, a rat, or the like, or a human LOX-1 or LOX-1 ligand-binding fragment. including. In mammals such as humans, pests, sheep, mice, and rats, diseases caused by abnormal LOX-1 or LOX-1 ligand binding fragment by regulating the activity of LOX-1 derived from self Or the ability to treat disability.
  • the present invention provides a program for causing a computer to execute a method for obtaining a homolog of LOX-1 or a LOX-1 ligand-binding fragment.
  • the present invention provides a computer-readable recording medium recording a program for causing a computer to execute a method for obtaining a homologue of LOX-1 or a LOX-1 ligand binding fragment.
  • the present invention provides a computer practicing the method of obtaining a homologue of LOX-1 or LOX-1 ligand binding fragment.
  • the method executed by the program comprises A) comparing the atomic coordinates of the candidate compound with the atomic coordinates of the LOX-1 or LOX-1 ligand binding fragment.
  • the method of running the program comprises: A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of LOX-1 or LOX-1 ligand binding fragment to obtain a three-dimensional molecular model; B) comparing the three-dimensional molecular model of the candidate conjugate with the three-dimensional molecular model of the LOX-1 or LOX-1 ligand binding fragment.
  • the technique for obtaining such a three-dimensional molecular model and the technique for causing a computer to execute the comparison step are well known in the art, and a program cited elsewhere in this specification can also be used.
  • the present invention provides a program for causing a computer to execute a method for obtaining a variant of LOX-1 or a LOX-1 ligand-binding fragment.
  • the present invention provides a computer-readable recording medium recording a program for causing a computer to execute a method for obtaining a variant of LOX-1 or a LOX-1 ligand-binding fragment.
  • the present invention provides a computer for performing the method of obtaining a variant of LOX-1.
  • the method executed by the program includes: A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of LOX-1 or LOX-1 ligand binding fragment to obtain a three-dimensional molecular model; And B) comparing the three-dimensional molecular model of the candidate compound with the three-dimensional molecular model of the LOX-1 or LOX-1 ligand binding fragment, and introducing a mutation based on predetermined parameters.
  • the technique for obtaining such a three-dimensional molecular model and the technique for causing a computer to execute the mutation introduction step are well known in the art, and a program referred to elsewhere in this specification can also be used.
  • the predetermined parameters include, for example, polarity, hydrophobicity, hydrophilicity, chemical property, chemical structure, molecular weight, hydrogen bond, van der Waals force, ionic interaction, nonionic interaction, complex Examples include, but are not limited to, formability, receptor-ligand interaction, electrostatic interaction, and the like.
  • the present invention provides a program for causing a computer to execute a method for identifying a compound capable of binding to LOX-1 or a LOX-1 ligand-binding fragment or a homolog or a variant thereof. I do.
  • the present invention relates to a computer storing a program for causing a computer to execute a method for identifying a compound capable of binding to LOX-1 or LOX-1 ligand binding fragment or a homolog or variant thereof.
  • Provided is a readable recording medium.
  • the present invention provides a computer for performing a method of identifying a compound capable of binding LOX-1 or a homolog or variant thereof.
  • the method executed by the program comprises: A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of LOX-1 or LOX-1 ligand binding fragment, or to the atomic coordinates of a homolog or variant thereof.
  • the active site pocket of LOX-1 or LOX-1 ligand binding fragment Determining the spatial coordinates of the LOX-1 or LOX-1 ligand binding fragment or the spatial coordinates of the active site pocket of the homolog or variant thereof. Screening the spatial coordinates of the set to identify compounds capable of binding to the LOX-1 or LOX-1 ligand binding fragment or homolog or variant thereof. Techniques for determining such spatial coordinates and for causing a computer to execute an electronic screening process are well known in the art, and programs cited elsewhere in this specification can be used. .
  • the present invention provides a program for causing a computer to execute a method for identifying a compound capable of binding to LOX-1, comprising: A) a dimer-forming LOX-1 ligand Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the binding fragment or its homologues or variants, the spatial coordinates of the dimer interface that forms the dimer of the LOX-1 ligand-binding fragment are calculated. Determining; and B) a set of electronically conjugated candidates relative to the spatial coordinates of the dimer interface forming the dimer of the LOX-1 ligand binding fragment or a homolog or variant thereof.
  • the present invention provides a computer-readable recording medium on which a program for causing a computer to execute a method for identifying a compound capable of binding to LOX-1 is recorded, the method comprising: A) forming a dimer Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the LOX-1 ligand-binding fragment or of its homologue or variant to form a dimer of the LOX-1 ligand-binding fragment Determining the spatial coordinates of the interface; and B) electronically determining the spatial coordinates of the dimeric interface forming the dimer of the LOX-1 ligand binding fragment or a homolog or variant thereof.
  • the present invention provides a computer for performing a method for identifying a compound capable of binding to LOX-1, comprising: A) dimer forming LOX-1 Applying a three-dimensional molecular modeling algorithm to the atomic coordinates of the ligand-binding fragment or its homologs or variants, the spatial coordinates of the dimer interface that forms the dimer of the LOX-1 ligand-binding fragment B) a set of candidate conjugates electronically with respect to the spatial coordinates of the dimer interface forming the dimer of the LOX-1 ligand binding fragment or a homolog or variant thereof.
  • a computer which includes a step of screening the spatial coordinates of the compound to identify a compound capable of binding to the LOX-1.
  • the technique of obtaining such a three-dimensional molecular model and the technique of causing a computer to execute the mutation introduction step are well known in the art, and a program cited elsewhere in the present specification can also be used.
  • the predetermined parameter includes, for example, polarity, hydrophobicity, hydrophilicity, chemical property, chemical structure, molecular weight, hydrogen bond, van der Waalska, ionic interaction, nonionic interaction, complex forming ability. , Receptor-ligand interaction, electrostatic interaction, and the like, but are not limited thereto.
  • the present invention provides a method for comparing LOX-1 or LOX-1 by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of LOX-1 or LOX-1 ligand binding fragment or a homolog thereof.
  • the present invention provides a method for comparing LOX-1 or LOX-1 by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of LOX-1 or LOX-1 ligand binding fragment or a homolog thereof.
  • a computer-readable recording medium on which a program for causing a computer to execute a method for identifying a compound capable of binding to a ligand binding fragment or a homolog or a variant thereof is recorded.
  • the present invention provides a method for comparing LOX-1 or LOX-1 or LOX-1 by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of LOX-1 or LOX-1 ligand binding fragment or a homolog thereof.
  • the method executed by the program comprises: A) applying a three-dimensional molecular modeling algorithm to the atomic coordinates of LOX-1 or a LOX-1 ligand binding fragment or to the atomic coordinates of a homolog or variant thereof. , A step of obtaining a three-dimensional molecular model; B) a step of inputting coordinate data of the three-dimensional molecular model into a data structure to search for a distance between atoms of LOX-1 or LOX-1 ligand binding fragment; and C) a candidate. By comparing the distance between the heteroatom that forms a hydrogen bond in the compound and the heteroatom that forms the active site pocket in the three-dimensional molecular model, the optimal hydrogen between the two structures is determined.
  • Identifying a candidate compound species that theoretically forms a stable complex with the active site pocket of the three-dimensional molecular model of LOX-1 or LOX-1 ligand binding fragment based on the binding are performed by a computer. Programs well known in the art and cited elsewhere herein can be used for IJ.
  • the present invention provides a method for binding LOX-1 by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of a dimer-forming LOX-1 ligand binding fragment or a homolog thereof.
  • a program is provided that includes the step of identifying candidate compound species that theoretically bind to the interface.
  • the present invention may bind LOX-1 by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of a dimer forming LOX-1 ligand binding fragment or homolog thereof.
  • a computer-readable recording medium having recorded thereon a program for causing a computer to execute a method for identifying a compound, comprising: A) atomic coordinates of a LOX-1 ligand-binding fragment forming a dimer or a phase thereof.
  • the present invention provides a compound capable of binding to LOX-1 by comparing a three-dimensional molecular model of a candidate compound with a three-dimensional molecular model of a dimer-forming LOX-1 ligand-binding fragment or a homolog thereof.
  • a computer for performing a method for identifying a compound comprising: A) three-dimensional molecular modeling on the atomic coordinates of a LOX-1 ligand-binding fragment that forms a dimer, or the atomic coordinates of a homolog or variant thereof. Applying an algorithm to obtain a three-dimensional molecular model; B) inputting coordinate data of the three-dimensional molecular model into a data structure to search for a distance between atoms of a LOX-1 ligand binding fragment; and C) The ratio of the distance between the heteroatom forming a hydrogen bond in the candidate compound and the heteroatom forming the active site pocket in the three-dimensional molecular model.
  • a computer includes the steps of: Techniques for determining such spatial coordinates and causing a computer to perform an electronic screening process are well known in the art, and a program cited elsewhere in this specification can be used.
  • the above-mentioned recording medium may be any recording medium as long as the above-described program can be recorded.
  • a medium includes MO, CD-R, CD-RW, Examples include, but are not limited to, CD-ROM, DVD-RAM, DVD-R, DVD-RW, DVD + RW, DVD-ROM, and memory card.
  • the computer to be used may be any computer as long as the above program can be executed.
  • Windows registered trademark
  • UNIX registered trademark
  • Mac OS-based LINUX-based but not limited to.
  • a human LOX-1 CTLD (143-273) having the following sequence was incorporated into a multi-cloning site (Ndel-Xhol) of a histidine-tag fusion protein expression vector pET28a manufactured by Novagen and introduced into Escherichia coli BL21 (DE3). Mass expression was performed:
  • Escherichia coli transformed with the LOX-1 expression vector was cultured at 37 ° C in 8 L of M9 minimal medium containing 50 ⁇ g Zml of kanamycin, and had an OD value of 660 nm of SO.5. Then, IPTG was added to a final concentration of ImM, culture was continued at 37 ° C for 4 hours, and the cells were collected by centrifugation at 3,500 X g for 30 minutes.
  • the recovered cells were suspended in 5 ml of lysis buffer (50 mM Tris / HCl (pH 8.0), 400 mM KC1, 0.1% Triton X-100) per gram of cells, and the CompleteMini protease inhibitor (Roche, Add 0.5 tablets per lg of cells and disrupt the cells using an Astrasson sonicator at 4 ° C and centrifuge (10,000 ⁇ g, 30 minutes, 4 ° C). ) To collect the pellet. The obtained pellet is washed twice by repeating resuspension, sonication, and centrifugation twice with a lysis buffer, recovered as an insoluble inclusion body, and subjected to the next solubilization operation. Stored at 80 ° C until nausea.
  • lysis buffer 50 mM Tris / HCl (pH 8.0), 400 mM KC1, 0.1% Triton X-100
  • the insoluble inclusion body was suspended in 10 ml of a solubilization buffer (100 mM Tris / HCl (pH 8.0), 6 M guanidine hydrochloride, 5 mM DTT) at 30 mg (as the amount of LOX-1 CTLD protein) (final protein concentration). 3mgZml), remove the precipitate by centrifugation, and let stand at room temperature for 4 hours.
  • a solubilization buffer 100 mM Tris / HCl (pH 8.0), 6 M guanidine hydrochloride, 5 mM DTT
  • DTT was added to a final concentration of 50 mM, and added to 1 L of refolding buffer (1 OmM Tris / HCl (pH 8.5), 400 mM arginine, 5 mM reduced glutathione, 0.5 mM oxidized daltathione). While stirring, the solution was slowly dropped at 4 ° C. at a speed of 20-30 lZmin using an FPLC solution pump. After completion of the dropping, PMSF was refined to a final concentration of 0. ImM, and the mixture was further stirred at 4 ° C for 12 hours to perform protein refolding.
  • refolding buffer 1 OmM Tris / HCl (pH 8.5), 400 mM arginine, 5 mM reduced glutathione, 0.5 mM oxidized daltathione.
  • the column was washed with a column equilibration buffer (50 mM Tris / HCl (pH 7.5), 100 mM NaCl, 100 mM imidazole), and the concentration of imidazole was increased with a linear gradient to 500 mM to elute proteins.
  • the eluted protein solution was concentrated at about 2 m with Centricon-10 (manufactured by Millipore), ligated with 10 cut units of bovine thrombin protease per mg protein, and reacted at 4 ° C for 5 hours for histidine protein.
  • the thrombin was cleaved, and then thrombin was removed by passing through a benzamidine sepharose column (Pharmacia), and PMSF was added to a final concentration of 0.1 ImM to stop the protease reaction.
  • the protein solution was then applied to a Superdex75 gel filtration column (Pharmacia) equilibrated with gel filtration column equilibration buffer (10 mM Tris / HC1 ( ⁇ 7.5), NaCl 50 mM), and fractions with the correct molecular weight were collected. I took it out.
  • Example 2 Example 2
  • Solubilization and refolding procedures were performed in the same manner as for LOX-1 CTLD.
  • the composition of the dialysis buffer was 25 mM Tris / HCl (pH 7.5) and 100 mM NaCl, and the composition of the gel filtration column equilibration buffer was 10 mM. Except that Tris / HCl (pH 7.5), NaCl 400 mM, and the concentration of the Ni-chelating Sepharose column, when concentrating the eluted protein solution, adding NaCl to the final concentration of 400 mM, also perform the power. And LOX 1 CTLD.
  • the extracellular region of hLOX-1 or the DNA fragment encoding CTLD was prepared by a conventional PCR method. At both ends, Nrul was added on the 5 'side and an EcoRV restriction enzyme site was added on the 3' side. After extracting the PCR product, treating it with both restriction enzymes, insert it into pBS, a cloning vector, and confirm that the gene sequence is correct. Confirmed.
  • the nucleotide sequence and amino acid sequence of the extracellular region of hLOX-1 are shown in SEQ ID NOS: 5 and 6, respectively, and the nucleotide sequence and amino acid sequence of hLOX-1 CTLD are shown in SEQ ID NOs: 1 and 2, respectively.
  • a gene encoding the protein whose sequence has been confirmed is excised with a restriction enzyme, and is known to undergo biotinylation in Escherichia coli, and a plasmid vector encoding a polypeptide, PinPoint Xa (manufactured by Promega). At the above restriction enzyme site. Next, after transformation into Escherichia coli JM109, which is an expression host, a transformant that correctly incorporated the target gene was selected.
  • a colony of Escherichia coli JM109 transformed with the target plasmid was inoculated at a final concentration of 5 ml of LB medium containing 100 / zg / ml of ampicillin and 2 M of biotin, and cultured at 37 ° C with shaking. . Subsequently, this culture solution was inoculated in a ratio of 1: 100 (volume ratio) to 50 ml of LB medium containing 100 gZml of ampicillin and 2 ⁇ of biotin at a final concentration, and cultured for 1 hour. IPTG was added to 100 M to induce the expression of the target fusion protein, and the cells were further cultured with stirring for 4 hours.
  • the culture solution 1001 after the induction treatment was placed in a 1.5 ml centrifuge tube, and centrifuged at 15,000 rpm for several minutes to collect the cells.
  • the collected cells were disrupted by sonication, and the supernatant (soluble surface) and the precipitate (insoluble surface) obtained by centrifugation at 20,000 g for 30 minutes were suspended in SDS sample buffer. Treated at ° C for 4 minutes. Next, the proteins were separated by 12% SDS PAGE and then electrically transferred to a nitrocellulose membrane.
  • the trocellulose membrane was stained with Ponceso-S to confirm the position of the protein band, and then placed in TBS-Tween (20 mM Tris, 150 mM NaCl, pH 7.6, 0.1% Tween 20). And gently stirred at room temperature for 60 minutes. Next, the reaction was carried out at room temperature for 30 minutes in streptavidin-labeled alkaline phosphatase. Subsequently, the nitrocellular membrane after the reaction was washed with TBS-Tweeen, and an NB TZBCIP solution, which is a substrate for alkaline phosphatase, was added thereto. The solution was incubated at room temperature until the band of the biotinylated protein was detected. I responded.
  • TBS-Tween 20 mM Tris, 150 mM NaCl, pH 7.6, 0.1% Tween 20. And gently stirred at room temperature for 60 minutes. Next, the reaction was carried out at room temperature for 30 minutes in streptavidin-labeled alkaline phosphata
  • DTT was added to a final concentration of 50 mM, and added to 1 L of refolding buffer (1 OmM Tris / HCl (pH 8.5), 400 mM arginine, 5 mM reduced glutathione, 0.5 mM oxidized daltathione). While stirring, the solution was slowly dropped at 4 ° C. at a speed of 20-30 lZmin using an FPLC solution pump. After completion of the dropping, PMSF was refined to a final concentration of 0. ImM, and the mixture was further stirred at 4 ° C for 12 hours to perform protein refolding.
  • refolding buffer 1 OmM Tris / HCl (pH 8.5), 400 mM arginine, 5 mM reduced glutathione, 0.5 mM oxidized daltathione.
  • the protein solution is then applied to a Superdex 75 gel filtration column (Pharmacia) equilibrated with a gel filtration column equilibration buffer (10 mM Tris / HCl (pH 7.5), NaCl 50 mM) to fractionate the correct molecular weight fraction. did.
  • a protein refolded by a conventional refolding method using cyclic carbohydrate cycloamylose and a surfactant was prepared as follows.
  • biotinylated CTLD The refolded biotinylated extracellular domain, biotinylated CTLD, was immobilized on streptavidin beads, and the binding of fluorescently labeled DilAcL DL to acetylated LDL, one of the ligands, was confirmed. Fluorescence was observed on the beads in which the biotinylated extracellular region or the biotinylated CTLD region had been immobilized, indicating that both recovered the ligand binding ability.
  • the histidine-tagged LOX-1 CTLD protein is also dissolved in a 0.1% TFA (trifluoroacetic acid) solution and the protein with the tag removed is dissolved in a 30% acetonitrile solution containing 0.1 TFA.
  • TFA trifluoroacetic acid
  • the dissolved sinapinic acid solution was added and mixed.
  • the dissolved sample was analyzed by a MALDI-TOF mass spectrometer (Voyager Elite, manufactured by Perspective Biosystems).
  • results of protein purity assay by mass spectrometry of refolded LOX-1 CTLD The histidine-tagged LOX-1 CTLD protein with the tag removed, obtained by the refolding method of the present invention, gave a mass spectroscopy spectrum with a mZz ratio (mass Z charge) t of about 50, a narrow width and a narrow width. The result of this narrow width indicates that the product is purified to a higher degree of purity with less contaminants than the conventional refolding method using cyclic carbohydrate cycloamylose (Fig. 2).
  • Example 1 The protein power obtained in Example 1 was also removed from the tag, and dissolved in 20 mM Tris HCl buffer (pH 7.0) and 50 mM NaCl to a concentration of 0. ImM. The measurement of the two-dimensional correlation spectrum was performed at 25 ° C. by using ⁇ —N HSQC (Heteronuclear Single Quantum Coherence correlation spectroscopy).
  • ⁇ —N HSQC Heteronuclear Single Quantum Coherence correlation spectroscopy
  • the 1 H signal derived from the main chain of the protein was distributed over a wide range from 7 ppm to 1 lppm on a 15 N HSQC two-dimensional correlation spectrum!
  • the signals overlap each other within a narrow range of 7 ppm to 8.5 ppm, and the line width of each signal is It was wider than the properly refolded protein.

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Abstract

Pour définir en détail le mode de liaison d'un récepteur de LDL oxydée LOX-1 à la LDL oxydée, l'invention a pour objet de définir la stéréostructure du domaine de liaison à la LDL oxydée de LOX-1. Plus particulièrement, elle a pour objet de définir la stéréostructure d'un domaine de liaison au CTLD utilisé comme structure dimère conservant la même configuration que sur la couche de surface cellulaire. Le problème pour définir le domaine de liaison à la LDL oxydée (CTLD: domaine de type lectine de type C) de LOX-1 utilisé dans la cristallisation ainsi qu'une protéine contenant le domaine de liaison à la LDL oxydée de LOX-1 et un domaine NECK (région reliant une partie transmembranaire au CTLD) se présentant sous la forme d'un dimère peut être résolu par obtention de divers cristaux au moyen d'échantillons obtenus par déroulement de protéines exprimées à large échelle dans Escherichia coli selon la méthode présentée dans la description (ce procédé consiste de préférence à ajouter une solution tampon comprenant une zone tampon dans la plage de pH acide dans une solution, puis à ajouter un ion calcium ou zinc à cette solution, ou à ajouter une solution tampon comprenant une zone tampon dans la plage de pH neutre dans la solution dans le cas du dimère LOX-1), et par analyse des cristaux ainsi obtenus.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007254397A (ja) * 2006-03-23 2007-10-04 National Agriculture & Food Research Organization Ａｇｅを認識する分子

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