Novel ADP-ribosylcyclase and its inhibitors

Abstract

To provide a pharmaceutical composition containing an inhibitor against the expression or activation of a novel ADP-ribosyl cyclase or a naturally occurring variant thereof as an active ingredient for preventing or treating an ADP-ribosyl cyclase-mediated disease.SOLUTION: Prided is a pharmaceutical composition for preventing or treating an ADP-ribosyl cyclase-mediated disease that contains as an active ingredient an inhibitor of expression or activity of ADP-ribosyl cyclase having 90% or more homology with ADP-ribosyl cyclase containing a specific amino acid sequence. the ADP-ribosyl cyclase converting NAD+ to cyclic ADP-ribose (cADPR). In addition, the present disclosure relates to a composition for diagnosis of an ADP-ribosyl cyclase-mediated disease, the composition containing an agent for measuring a gene expression level or protein level of the ADP-ribosyl cyclase or a naturally occurring variant thereof.SELECTED DRAWING: Figure 7

Description

Detailed description of the invention

[0001] [Technical Field] The present invention is NAD + This invention relates to a novel ADP-ribosylcyclase that converts cyclic ADP-ribose and its inhibitor.

[0002] [Background technology] ADPRC is NAD + Cyclic ADP-ribose (cADPR) is synthesized from NADP + This enzyme synthesizes nicotinic acid adenine dinucleotide phosphate (NAADP) and is widely present in organisms ranging from plants to mammals. ADPRC is known to regulate various cellular functions by releasing calcium from intracellular calcium stores into the cytoplasm [Berridge MJ, Bootman MD, Roderick HL. Nat Rev Mol Cell Biol. 4, 517-529, 2003; Lee HC, Mol. Med. 12 317-323, 2006].

[0003] In a stable state, the intracellular calcium concentration is 10 -7 The calcium level remains below M and is maintained at the baseline. However, when cells are activated, intracellular calcium concentration increases to about 10 times the baseline level, and if the calcium concentration remains high in a pathological state, many cellular functions are affected, ultimately leading to the loss of normal cellular function.

[0004] In hypertension or diabetes mellitus accompanied by hypertension, obesity, dementia, hypothermia, and diseases accompanied by cell proliferation, abnormalities occur in the regulation of intracellular calcium metabolism, and intracellular calcium concentrations are maintained at levels higher than normal [Resnick LM.Am.J.Hypertens.6:123S-134S,1993].

[0005] As an example, in patients with chronic diseases such as hypertension, various genetic, environmental, or secondary causes from certain diseases cause the calcium concentration in vascular smooth muscle cells to remain consistently higher than normal. This, in turn, induces contraction of the vascular smooth muscle, increasing peripheral vascular resistance, and consequently, blood pressure remains elevated above normal levels, leading to hypertension.

[0006] This elevated blood pressure is known to secondarily lead to the proliferation and hypertrophy of vascular smooth muscle cells and fibrous tissue, further increasing peripheral vascular resistance and blood pressure, and impairing the function of organs such as the cardiovascular system, brain, and kidneys, ultimately leading to death from myocardial infarction, angina pectoris, stroke, and chronic heart failure [Cowley AW Jr. Physiol Rev. 72:231-300, 1992].

[0007] It is known that increased calcium levels, mediated by CD38 activity (a type of ADPRC), specifically cADPR, play a crucial role in insulin secretion from the pancreas. Furthermore, diabetes induction increases angiotensin production, and this peptide hormone is also well known to induce kidney disease, hypertension, and heart disease, as well as activate ADPRCs.

[0008] Therefore, ADPRCs, while present in immune cells, cardiac muscle cells, pancreatic beta cells, renal muscle cells, and nerve cells, regulate intracellular calcium concentration and various physiological activities in association with various hormone receptors. If there is an abnormality in the expression or regulation of ADPRC activity, abnormalities in the regulation of physiological phenomena (immunity, renal function, insulin secretion, cardiovascular function) may occur.

[0009] These ADPRCs are activated by various hormones, and this enzyme uses the substrate NAD +It produces cADPR as a product, which increases intracellular calcium in most organs. Furthermore, abnormal increases in calcium due to ADPRC activity have been known to be a contributing factor to various biological pathologies, including insulin secretion [Kim BJ, Park KH, Yim CY, Takasawa S, Okamoto H, Im MJ, Kim UH. Diabetes. 57:868-878, 2008], cell hypertrophy [Gul R, Park JH, Kim SY, Jang KY, Chea JK, Ko JK, Kim UH. Cardiovasc Res. 81:582-591, 2009], and cell proliferation [Kim SY, Gul R, Rah SY, Kim SH, Park SK, Im MJ, Kwon HJ, Kim UH. Am J Physiol Renal Physiol. 294:F989-F989, 2008].

[0010] To date, the most well-studied ADPRC is CD38, a T-cell surface antigen, which is widely expressed in various organs in addition to immune cells.

[0011] However, it has been revealed that ADPRCs different from CD38 are expressed in the heart, kidneys, and brain [Partida-Sanchez S, Cockayne DA, Monard S, Jacobson EL, Oppenheimer N, Garvy B, Kusser K, Goodrich S, Howard M, Harmsen A, Randall TD, Lund FE. Nat Med 7:1209-16, 2001].

[0012] Therefore, regulating tissue-specific ADPRC expression may be useful in treating diseases induced by increased intracellular calcium. However, the mechanism of ADPRC activity is still unknown.

[0013] The matters described above as background technology are merely for the purpose of enhancing understanding of the background of the present invention and should not be understood as constituting prior art already known to those with ordinary skill in this art.

[0014] [Summary of the Invention] [Problems the invention aims to solve] The inventors of this invention discovered a new type of ADPRC enzyme, distinct from existing ADPRCs (CD38, CD157) known from mammals, and confirmed its activity, thereby completing the present invention.

[0015] Therefore, an object of the present invention is to provide a novel ADP-ribosyl cyclase (ADPRC) or a naturally occurring variant thereof.

[0016] Another object of the present invention is to provide nucleic acid molecules encoding the ADP-ribosylcyclase or naturally occurring variants thereof.

[0017] Another object of the present invention is to provide a vector containing the nucleic acid molecule.

[0018] Another object of the present invention is to provide a host cell containing the vector.

[0019] A further object of the present invention is to provide a vector comprising the ADP-ribosylcyclase or a naturally occurring variant thereof, a nucleic acid molecule encoding it, or the nucleic acid molecule. + The objective is to provide a catalyst composition that converts to cyclic ADP-ribose (cADPR).

[0020] Still another object of the present invention is to provide a pharmaceutical composition for preventing or treating an ADP-ribosyl cyclase-mediated disease, comprising an expression inhibitor or an activity inhibitor of the ADP-ribosyl cyclase or a naturally-occurring mutant thereof as an active ingredient.

[0021] Still another object of the present invention is to provide a food composition for preventing or ameliorating an ADP-ribosyl cyclase-mediated disease, comprising an expression inhibitor or an activity inhibitor of the ADP-ribosyl cyclase or a naturally-occurring mutant thereof as an active ingredient.

[0022] Still another object of the present invention is to provide an animal model in which the hetero type gene of the ADP-ribosyl cyclase or a naturally-occurring mutant thereof is deficient.

[0023] Still another object of the present invention is to provide a method for providing information regarding the diagnosis of an ADP-ribosyl cyclase-mediated disease.

[0024] Still another object of the present invention is to provide a diagnostic composition for an ADP-ribosyl cyclase-mediated disease, comprising a preparation for measuring the gene expression level of the ADP-ribosyl cyclase or a naturally-occurring mutant thereof or a preparation for measuring the protein level.

[0025] Still another object of the present invention is to provide a diagnostic kit for an ADP-ribosyl cyclase-mediated disease, comprising a preparation for measuring the gene expression level of the ADP-ribosyl cyclase or a naturally-occurring mutant thereof or a preparation for measuring the protein level.

[0026] Still another object of the present invention is to provide a method for screening a preventive or therapeutic substance for the ADP-ribosyl cyclase-mediated disease. 并

[0027] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

[0028] [Means for Solving the Problem] According to one aspect of the present invention, the present invention provides an ADP-ribosyl cyclase (ADPRC) containing the amino acid sequence of the Array Catalog First Array or a naturally occurred variant thereof.

[0029] The inventor has + been earnestly endeavoring to discover a novel ADP-ribosyl cyclase in addition to the conventionally known ADP-ribosyl cyclase, which is an enzyme that converts NAD

[0030] In the present specification, the term "ADP-ribosyl cyclase" or "ADPRC (ADP-ribosyl cyclase)" means an enzyme that converts NAD + into cyclic ADP-ribose (cADPR).

[0031] In the present specification, the term "variant of ADP-ribosyl cyclase" means a variant in which a part of the amino acid sequence of the ADP-ribosyl cyclase is substituted, deleted, or added naturally or artificially, and means a variant of ADP-ribosyl cyclase having the activity of a catalyst that converts NAD + [[ID=​​​​​​​​ According to a preferred embodiment of the present invention, naturally occurring mutants of the ADP-ribosylcyclase of the present invention are naturally occurring mutants selected from the group consisting of species variants, species homologs, isoforms, allelic variants, conformation variants, splice variants, and point mutant variants.

[0034] The naturally occurring mutants of the ADP-ribosylcyclase of the present invention preferably have 50% or more homology, more preferably 60% or more homology, even more preferably 70% or more homology, particularly preferably 80% or more homology, and most preferably 90% or more homology to the ADP-ribosylcyclase containing the amino acid sequence of sequence catalog 1.

[0035] According to a preferred embodiment of the present invention, naturally occurring variants of the ADP-ribosylcyclase of the present invention originate from organisms selected from the group consisting of mammals, birds, reptiles, amphibians, and fish.

[0036] According to one embodiment of the present invention, naturally occurring mutants of the ADP-ribosylcyclase of the present invention were found to have more than 70% homology in mammals, more than 60% in birds, reptiles and amphibians, and more than 50% in fish compared to ADP-ribosylcyclase containing the amino acid sequence of sequence catalog 1.

[0037] According to one embodiment of the present invention, naturally occurring mutants of the ADP-ribosylcyclase of the present invention showed high homology to ADP-ribosylcyclase containing the amino acid sequence of sequence catalog 1 in humans and rats (96%), chimpanzees (93%), guinea pigs and horses (91%), dogs, goats and sheep (90%), rabbits (89%), pigs (88%), cattle (78%), chickens (70%), frogs (63%), and turkeys (62%), confirming that it is an enzyme with a very high specific gravity for interspecies-conserved sequences (Figure 4).

[0038] According to a preferred embodiment of the present invention, a naturally occurring mutant of the ADP-ribosylcyclase of the present invention comprises an amino acid sequence selected from the group consisting of sequences 2 to 21 of the sequence catalog.

[0039] According to yet another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the ADP-ribosylcyclase or a naturally occurring variant thereof, a vector containing the nucleic acid molecule, or a host cell containing the vector.

[0040] The nucleic acid molecules of the present invention may be isolated or recombinant, and may include single-stranded and double-stranded DNA and RNA, as well as corresponding complementary sequences. “Isolated nucleic acid” in the case of nucleic acid isolated from a naturally occurring source is the nucleic acid isolated from peripheral genetic sequences present in the genome of the individual from which the nucleic acid was isolated. Nucleic acids synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, may be understood as isolated nucleic acid molecules of nucleic acids produced by such procedures. An isolated nucleic acid molecule may appear as a distinct fragment or as a component of a larger nucleic acid construct. Nucleic acids are “operably ligated” when they are positioned in a functional relationship with other nucleic acid sequences. For example, the DNA of a pre-sequence or secretory leader is operably ligated to the DNA of a polypeptide when expressed as a preprotein, which is the pre-secretion form of the polypeptide; a promoter or enhancer is operably ligated to a coding sequence when it affects the transcription of a polypeptide sequence; or a ribosome binding site is operably ligated to a coding sequence when it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences to be linked are located adjacent to each other, and in the case of secretory readers, this means they are adjacent and located within the same reading frame. However, enhancers do not need to be located adjacent to each other. Linking is achieved by ligation at convenient restriction enzyme sites. If such sites are not present, synthetic oligonucleotide adapters or linkers are used by conventional methods.

[0041] In a preferred embodiment of the present invention, the nucleic acid molecule is cDNA.

[0042] In this specification, the term “vector” means a carrier into which a nucleic acid sequence can be inserted for introduction into a replicable cell. The nucleic acid sequence may be exogenous or heterologous. Examples of vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art can construct vectors using standard recombination techniques (e.g., Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994).

[0043] In this specification, the term “expression vector” means a vector containing a nucleic acid sequence that encodes at least a portion of the gene product to be transcribed. In some cases, the RNA molecule is subsequently translated as a protein, polypeptide, or peptide. Expression vectors may contain various regulatory sequences. Along with regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide other functions.

[0044] In this specification, the term “host cell” means any transformable organism, including eukaryotes and prokaryotes, that is capable of replicating the vector or expressing the genes encoded by the vector. The host cell may be transfected or transformed by the vector, which means the process by which an exogenous nucleic acid molecule is transmitted or introduced into the host cell.

[0045] The host cells of the present invention may preferably be bacterial cells, yeast, animal, or human cells (such as CHO cells, HeLa cells, HEK293 cells, MES13 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, NIH3T3 cells, etc.), but are not limited thereto.

[0046] According to another aspect of the present invention, the present invention comprises the ADP-ribosylcyclase or a naturally occurring variant thereof, a nucleic acid molecule encoding it, or a vector containing the nucleic acid molecule, NAD + The present invention provides a catalyst composition that converts to cyclic ADP-ribose (cADPR).

[0047] The catalyst composition of the present invention is NAD + It efficiently catalyzes the process by which it is converted to cADPR.

[0048] According to yet another aspect of the present invention, the present invention provides a composition comprising an inhibitor of expression or activity of ADP-ribosylcyclase or a naturally occurring variant thereof.

[0049] According to a preferred embodiment of the present invention, the composition of the present invention is a pharmaceutical composition for the prevention or treatment of ADP-ribosylcyclase-mediated diseases.

[0050] The pharmaceutical composition of the present invention may include (a) the expression inhibitor or activity inhibitor; and (b) a pharmaceutically acceptable carrier.

[0051] According to a preferred embodiment of the present invention, the composition of the present invention is a food composition for the prevention or improvement of ADP-ribosylcyclase-mediated diseases.

[0052] According to yet another aspect of the present invention, the present invention provides a method for preventing or treating an ADP-ribosylcyclase-mediated disease, comprising the step of administering a pharmaceutically effective amount of the pharmaceutical composition to a subject.

[0053] According to yet another aspect of the present invention, the present invention provides an inhibitor of expression or activity of ADP-ribosylcyclase or a naturally occurring variant thereof, for use in therapy.

[0054] The types of ADP-ribosylcyclase-mediated diseases that the present invention aims to prevent or treat are not limited, but are preferably diabetes mellitus or kidney disease, and more preferably renal failure, nephropathy, nephritis, renal fibrosis, or nephrosclerosis.

[0055] According to a preferred embodiment of the present invention, the renal failure is chronic renal failure, acute renal failure, or mild renal failure before dialysis.

[0056] According to a preferred embodiment of the present invention, the nephropathy is nephropathy syndrome, lipoid nephropathy, diabetic nephropathy, immunoglobin A nephropathy (IgA nephropathy), analgesic nephropathy, or hypertensive nephropathy.

[0057] According to one embodiment of the present invention, the composition of the present invention showed a significant decrease in kidney weight relative to body weight, a significant increase in creatine scavenging rate, and a significant decrease in urinary albumin levels, confirming its potential as a candidate for the treatment of kidney diseases, including chronic renal failure and diabetic nephropathy (Figure 6); further, it lowered ADP-ribosylcyclase activity and cADPR concentration to normal levels (Figure 7); lowered the expression levels of TGF-β1, fibronectin, and collagen IV to normal levels (Figure 8); and also possessed the ability to restore glomerular thickening, inflammatory cell infiltration, and atypical epithelial cell formation to normal levels, thereby restoring histopathological changes in the kidney (Figure 9); and in a hypertension model, the creatine scavenging rate was significantly increased, confirming its applicability to hypertensive nephropathy (Figure 11B), thus demonstrating its potential as a treatment for various kidney diseases caused by kidney dysfunction or damage.

[0058] In this specification, the term "expression inhibitor" means a substance that inhibits the expression of ADP-ribosylcyclase, and such an inhibitor can be easily prepared by a person skilled in the art based on the structure and function of ADP-ribosylcyclase.

[0059] The expression inhibitor of the present invention is not limited, but is preferably one selected from the group consisting of antisense oligonucleotides, siRNAs, shRNAs, miRNAs, ribozymes, DNAzymes, and PNAs (protein nucleic acids) that bind complementarily to the mRNA of the ADP-ribosylcyclase or its naturally occurring mutant gene.

[0060] According to a preferred embodiment of the present invention, the siRNA comprises the nucleotide sequence of sequence catalog sequence 22.

[0061] In this specification, the term "activity inhibitor" means a substance that inhibits the activity of the expressed ADP-ribosylcyclase protein, and is preferably one selected from the group consisting of compounds, peptides, peptide analogs (mimetics), aptamers, and antibodies that specifically bind to the ADP-ribosylcyclase or its naturally occurring mutant protein.

[0062] According to a preferred embodiment of the present invention, the compound is selected from the group consisting of 4,4'-dihydroxyazobenzene, 2-(1,3-benzoxazol-2-ylamino)-1-methylquinazoline-4(1H)-one, and dicaffeoylquinic acid (DCQA).

[0063] The 4,4'-dihydroxyazobenzene of the present invention may preferably be represented by the following chemical formula 1:

[0064] [ka]

[0065] The 2-(1,3-benzoxazole-2-ylamino)-1-methylquinazoline-4(1H)-one of the present invention may preferably be represented by the following chemical formula 2:

[0066] [ka]

[0067] The dicaffeoylquinic acid (DCQA) of the present invention may be represented by any one compound selected from the group consisting of the following chemical formulas 3 to 8 (1,4-DCQA, 3,4-DCQA, 3,5-DCQA, 4,5-DCQA, 1,3-DCQA, and 1,5-DCQA):

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] [ka]

[0072] [ka]

[0073] [ka]

[0074] According to one embodiment of the present invention, it was confirmed that the novel ADP-ribosylcyclase expression inhibitor or activity inhibitor according to the present invention contributes to suppressing kidney disease through a mechanism that modulates the expression or activity of ADPRC.

[0075] Therefore, the novel ADP-ribosylcyclase expression inhibitor or activity inhibitor of the present invention can be used as a clinically useful cell-specific selective inhibitor, and further as an agent for the prevention, improvement, and / or treatment of renal diseases.

[0076] The composition of the present invention can be used not only as a novel ADPRC expression inhibitor or activity inhibitor, which is the active ingredient, but also with existing known therapeutic agents for ADPRC-related diseases.

[0077] The pharmaceutical composition of the present invention may be formulated in appropriate dosage forms with a pharmaceutically acceptable carrier. 'Pharmaceutically acceptable' means a carrier that is physiologically acceptable and does not typically cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness when administered to humans.

[0078] The pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical compositions of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0079] The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally, for example by intravenous infusion, local infusion, or intraperitoneal infusion.

[0080] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity, and a skilled, ordinary physician can easily determine and prescribe a dosage that is effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg.

[0081] The pharmaceutical compositions of the present invention can be manufactured in unit volume form or by being contained in multi-volume containers by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains. The dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may further contain a dispersant or stabilizer.

[0082] When the composition of the present invention is manufactured as a food composition, the active ingredients include, in addition to the ADP-ribosylcyclase expression inhibitor or activity inhibitor, ingredients that are normally added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of carbohydrates mentioned above include monosaccharides, such as glucose and fructose; disaccharides, such as maltose, sucrose, and oligosaccharides; and polysaccharides, such as dextrin and cyclodextrin, which are common sugars, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavorings (thaumatin, stevia extract [e.g., rebaudioside A, glycyrrhizin, etc.]) and synthetic flavorings (saccharin, aspartame, etc.) can be used as flavorings.

[0083] For example, when the food composition of the present invention is manufactured as a beverage, in addition to the ADP-ribosylcyclase expression inhibitor or activity inhibitor of the present invention, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Du Chong extract, jujube extract, licorice extract, and the like.

[0084] The food composition of the present invention has an excellent effect in improving kidney disease by regulating various pathological morphology-related factors of the kidney (meaningful reduction in kidney weight relative to body weight, meaningful increase in creatine scavenging rate, meaningful decrease in urinary albumin levels; reduction of TGF-β1, fibronectin, and collagen IV expression levels to normal levels; and restoration of glomerular hypertrophy, inflammatory cell infiltration, and atypical epithelial cell formation to normal levels).

[0085] According to yet another aspect of the present invention, the present invention provides an animal model lacking a heterotype gene of ADP-ribosyl cyclase (ADPRC) or a naturally occurring variant thereof.

[0086] According to a preferred embodiment of the present invention, the animal is a mammal other than a human, a bird, a reptile, an amphibian, or a fish.

[0087] In yet another aspect of the present invention, the present invention provides a method for determining the presence or absence of ADP-ribosylcyclase-mediated diseases, comprising the steps of: (a) inducing a specific disease in an animal model lacking a heterotype gene of ADP-ribosylcyclase or a naturally occurring variant thereof, and in a wild-type animal model; and (b) confirming the differences between the animal models.

[0088] Using the aforementioned animal models, it is possible to determine whether specific diseases (e.g., diabetes, kidney disease, hypertension, obesity, etc.) develop through the mediation of ADP-ribosylcyclase or independently of ADP-ribosylcyclase, by comparing the differences between the animal models (e.g., differences in body weight, kidney weight, blood pressure, creatine scavenging rate, blood glucose, inflammation, and glomerular thickening).

[0089] According to yet another aspect of the present invention, the present invention provides a method for providing information regarding the diagnosis of ADP-ribosylcyclase-mediated diseases, comprising the following steps: 1) A step of measuring the expression or activity level of ADP-ribosylcyclase or its naturally occurring mutant containing the amino acid sequence of sequence 1 of the sample sequence catalog isolated from the subject; and 2) A step in which the expression or activity level of ADP-ribosylcyclase or its naturally occurring mutants from step 1) is compared with a normal control group to determine whether an individual is at risk of developing a cyclase-mediated disease.

[0090] In this specification, the term “diagnosis” means confirmation of the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is confirmation of whether an ADP-ribosylcyclase-related or mediated disease is currently present or likely to develop.

[0091] ADP-ribosylcyclase is activated by various hormones, and this enzyme is activated by the substrate NAD + ADP-ribosylcyclase produces cADPR, a product that increases intracellular calcium in most organs. Furthermore, the abnormal increase in calcium due to ADP-ribosylcyclase activity is known to be a factor in various biological pathologies, including insulin secretion, hypertrophy, and cell proliferation. Therefore, measuring the expression or activity level of ADP-ribosylcyclase can provide useful information for diagnosing ADP-ribosylcyclase-mediated diseases induced by increased intracellular calcium.

[0092] According to yet another aspect of the present invention, the present invention provides a diagnostic composition for ADP-ribosylcyclase-mediated diseases, comprising a formulation for measuring the gene expression level or protein level of ADP-ribosylcyclase or a naturally occurring variant thereof, comprising the amino acid sequence of sequence catalog sequence 1.

[0093] According to a preferred embodiment of the present invention, the formulation for measuring gene expression levels of the present invention is a primer or probe that specifically binds to a gene encoding ADP-ribosylcyclase or a naturally occurring variant thereof.

[0094] According to a preferred embodiment of the present invention, the formulation for measuring protein levels of the present invention is an antibody or antigen-binding fragment thereof that specifically binds to ADP-ribosylcyclase or a naturally occurring variant thereof.

[0095] Since the aforementioned antibody or its antigen-binding fragment specifically binds to ADP-ribosylcyclase or its naturally occurring variants, it is possible to accurately measure the amount of ADP-ribosylcyclase or its naturally occurring variants contained in a sample.

[0096] The diagnostic composition of the present invention allows for the quantification of ADP-ribosylcyclase or its naturally occurring variants by analyzing the antigen against the antibody using an antigen-antibody binding reaction. The antigen-antibody binding reaction is preferably selected from the group consisting of conventional ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), sandwich assay, Western blot on polyacrylamide gel, immunoblot assay, and immunohistochemical staining, but is not limited thereto.

[0097] For the fixation body for the antigen-antibody binding reaction, a well plate made of nitrocellulose membrane, PVDF membrane, polyvinyl resin, or polystyrene resin, and a slide glass made of glass may be used, but is not limited to these.

[0098] The secondary antibody is preferably labeled with a conventional chromogen that causes a color reaction. Any label selected from the group consisting of fluorescent substances (fluoresceins) and dyes (dyes) such as HRP (horseradish peroxidase), alkaline phosphate degrading enzyme (Alkaline phosphatase), colloidal gold, FITC (Poly L-lysine-fluorescein isothiocyanate), and RITC (Rhodamine-B-isothiocyanate) may be used. The substrate that induces color development is preferably used in accordance with the label that causes the color reaction. It is preferable, but not limited to, any one selected from the group consisting of TMB (3,3',5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)], and OPD (ophenylenediamine).

[0099] According to yet another aspect of the present invention, the present invention provides a diagnostic kit for ADP-ribosylcyclase-mediated diseases comprising a formulation for measuring the gene expression level or protein level of ADP-ribosylcyclase or a naturally occurring variant thereof, comprising the amino acid sequence of sequence catalog sequence 1.

[0100] The diagnostic kit of the present invention may comprise one or more other component compositions, solutions, or apparatus suitable for the analytical method, as well as instructions for use thereof.

[0101] According to yet another aspect of the present invention, the present invention provides a method for screening substances for the prevention or treatment of ADP-ribosylcyclase-mediated diseases, comprising the following steps: 1) A step of treating cells expressing ADP-ribosylcyclase or a naturally occurring variant thereof containing the amino acid sequence of sequence number 1 of the sequence catalog with a candidate substance; and 2) A step of measuring the gene expression level or protein level of the ADP-ribosylcyclase or its naturally occurring variant by processing the candidate substance; and 3) A step in which the candidate substance is selected as a preventive or therapeutic substance for ADP-ribosylcyclase-mediated diseases if the gene expression level or protein level decreases compared to a control group that is not treated with the candidate substance.

[0102] The gene expression levels of ADP-ribosylcyclase or its naturally occurring variants are measured, but are not limited to, one or more methods selected from the group consisting of immunoprecipitation, radioimmunoassay (RIA), enzyme immunosorbent assay (ELISA), immunohistochemistry, RT-PCR, Western blotting, and fluid cell analysis (FACS).

[0103] The protein levels of ADP-ribosylcyclase or its naturally occurring variants are measured, but are not limited to, one or more methods selected from the group consisting of SDS-PAGE, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and protein chips.

[0104] [Effects of the invention] The features and advantages of this invention can be summarized as follows: (i) The present invention provides a pharmaceutical composition for the prevention or treatment of ADP-ribosylcyclase-mediated diseases, comprising as an active ingredient an inhibitor of the expression or activity of a novel ADP-ribosylcyclase or a naturally occurring variant thereof.

[0105] (ii) The present invention also provides a diagnostic composition for ADP-ribosylcyclase-mediated diseases, comprising a preparation for measuring the gene expression level or protein level of ADP-ribosylcyclase or its naturally occurring variant.

[0106] (iii) The compositions of the present invention have the effect of suppressing the increase in intracellular calcium in kidney cells due to the expression or increased activity of a new ADP-ribosylcyclase mediated by angiotensin II, and can be usefully used as therapeutic agents for ADP-ribosylcyclase-mediated diseases, particularly kidney diseases.

[0107] [Brief explanation of the drawing] [Figure 1] This figure shows the measurement of the NAD-glycohydrolase (NADase) activity of the novel ADPRC in HEK293 cells and MES13 cells.

[0108] [Figure 2] This figure shows the evaluation of the cADPR synthesis ability of purified FLAG-ADPRC.

[0109] [Figure 3] This figure shows the measurement of intracellular cADPR production by a novel ADPRC when MES13 cells were stimulated with angiotensin II.

[0110] [Figure 4] Results of comparing the interspecies sequence homology of the ADP-ribosylcyclase of the present invention using alignment (mouse as reference, human (96%), rat (96%), dog (90%), pig (88%), rabbit (89%), sheep (90%), chicken (70%), cattle (78%), chimpanzee (93%), horse (91%), frog (63%), goat (90%), turkey (62%), guinea pig (91%), Echinococcus granulosus (27%), Schistosoma haematobium (22%), Tricinella spiralis (22%), Drosophila melanogaster (19%), zebrafish (56%)).

[0111] [Figure 5] This figure shows the effect of a new inhibitor on suppressing the NAD glycohydrolase (NADase) activity of a new ADPRC.

[0112] [Figure 6] This figure shows the effects of dicaffeolylquinic acid (DCQA) measured in blood glucose (Figure 6A), the ratio of kidney weight to body weight (Figure 6B), creatinine scavenging rate (Figure 6C), and urinary albumin level (Figure 6D) in a mouse model of diabetic kidney disease.

[0113] [Figure 7] This figure shows the effects of DCQA on ADPRC activity (Figure 7A) and cADPR concentration (Figure 7B) in kidney tissue of a mouse model of diabetic kidney disease.

[0114] [Figure 8] This figure shows the effects of DCQA on changes in the expression of TGF-β1, fibronectin, and collagen IV in kidney tissue of a mouse model of diabetic kidney disease.

[0115] [Figure 9] This figure shows the histopathological changes observed in kidney tissue of a mouse model of diabetic kidney disease using H&E (Hematoxylin and Eosin) staining.

[0116] [Figure 10] This figure shows the blood glucose (Figure 10A), kidney weight-to-body weight ratio (Figure 10B), and creatinine cleanup rate (Figure 10C) measured in normal mice and ADPRC heterozygous (ADPRC(+ / -)) mice in diabetic kidney disease models.

[0117] [Figure 11] This figure shows the effects of DCQA on blood pressure (Figure 11A) and creatinine cleanup rate (Figure 11B) in normal mice and hypertensive mouse models.

[0118] [Modes for carrying out the invention] The present invention will be described in more detail below with reference to examples. These examples are merely for the purpose of illustrating the present invention in more detail, and it will be obvious to those with ordinary skill in the art that the scope of the present invention is not limited by these examples.

[0119] The present invention can also be configured as follows. [1] ADP-ribosyl cyclase (ADPRC) or a naturally occurring variant thereof containing the amino acid sequence of sequence 1 in the sequence catalog. [2] The naturally occurring mutant of ADP-ribosylcyclase described in [1] is characterized in that the naturally occurring mutant is selected from the group consisting of species variants, species homologs, isoforms, allelic variants, conformation variants, splice variants, and point mutant variants. [3] The ADP-ribosylcyclase or its naturally occurring variant according to [1], characterized in that the naturally occurring variant of ADP-ribosylcyclase originates from organisms selected from the group consisting of mammals, birds, reptiles, amphibians and fish. [4] The naturally occurring mutant of ADP-ribosylcyclase described in [1], characterized in that the naturally occurring mutant of ADP-ribosylcyclase contains an amino acid sequence selected from the group consisting of sequence 2 to sequence 21 of the sequence catalog. [5] The ADP-ribosylcyclase or its variant is NAD + ADP-ribosylcyclase or a naturally occurring variant thereof, characterized by converting to cyclic ADP-ribose (cADPR), as described in [1]. [6] A nucleic acid molecule encoding ADP-ribosylcyclase of [1] or a naturally occurring variant thereof. A vector containing nucleic acid molecules [7] and [6]. Host cells containing the vectors [8] and [7]. [9] [1] ADP-ribosylcyclase or its naturally occurring variant, the nucleic acid molecule of [6] or the vector of [7], NAD+ A catalyst composition that converts to cyclic ADP-ribose (cADPR).

[10] A pharmaceutical composition for the prevention or treatment of ADP-ribosylcyclase-mediated diseases, comprising as an active ingredient an inhibitor of expression or activity of ADP-ribosylcyclase or a naturally occurring mutant thereof containing the amino acid sequence of sequence number 1 of the sequence catalog.

[11] The pharmaceutical composition according to

[10] , characterized in that the inhibitor of ADP-ribosylcyclase or its naturally occurring mutant is selected from the group consisting of antisense oligonucleotide, siRNA, shRNA, miRNA, ribozyme, DNAzyme, and PNA (protein nucleic acid).

[12] The pharmaceutical composition according to

[11] , characterized in that the siRNA comprises the nucleotide sequence of sequence catalog sequence 22.

[13] The pharmaceutical composition according to

[10] , characterized in that the inhibitor of the activity of ADP-ribosylcyclase or a naturally occurring mutant thereof is selected from the group consisting of compounds, peptides, peptide analogs (mimetics), aptamers, and antibodies.

[14] The pharmaceutical composition according to

[13] , characterized in that the compound is selected from the group consisting of 4,4'-dihydroxyazobenzene, 2-(1,3-benzoxazol-2-ylamino)-1-methylquinazoline-4(1H)-one, and dicaffeoylquinic acid.

[15] The pharmaceutical composition according to

[10] , characterized in that the ADP-ribosylcyclase-mediated disease is a kidney disease.

[16] The pharmaceutical composition according to

[15] , characterized in that the kidney disease is renal failure, nephropathy, nephritis, renal fibrosis, or nephrosclerosis.

[17] The pharmaceutical composition according to

[16] , characterized in that the renal failure is chronic renal failure, acute renal failure, or mild renal failure before dialysis.

[18] The pharmaceutical composition according to

[16] , characterized in that the nephropathy is Nephropathy Syndrome, Lipoid Nephropathy, Diabetic Nephropathy, IgA Nephropathy, Analgesic Nephropathy, or Hypertensive Nephropathy.

[19] A food composition for the prevention or improvement of ADP-ribosylcyclase-mediated diseases, comprising as an active ingredient an inhibitor of expression or activity of ADP-ribosylcyclase or a naturally occurring mutant thereof containing the amino acid sequence of sequence number 1 of the sequence catalog.

[20] A non-human animal model lacking the heterotype gene of ADP-ribosyl cyclase (ADPRC) or a naturally occurring variant thereof.

[21] Method for confirming the presence or absence of ADP-ribosylcyclase-mediated diseases, including the following stages: (a) the stage of inducing a specific disease in the animal model of

[20] and the wild-type animal model; and (b) A step to confirm the differences between the animal models.

[22] Methods for providing information on the diagnosis of ADP-ribosylcyclase-mediated diseases, including the following stages: 1) A step of measuring the expression or activity level of ADP-ribosylcyclase or its naturally occurring variant containing the amino acid sequence of sequence 1 of the sample sequence catalog isolated from the subject; and, 2) A step in which the expression or activity level of ADP-ribosylcyclase or its naturally occurring mutants from step 1) is compared with that of a normal control group to determine whether an individual is at risk of developing a cyclase-mediated disease.

[23] A diagnostic composition for ADP-ribosylcyclase-mediated diseases, comprising a preparation for measuring the gene expression level or protein level of ADP-ribosylcyclase or a naturally occurring variant thereof containing the amino acid sequence of sequence catalog 1.

[24] A diagnostic kit for ADP-ribosylcyclase-mediated diseases comprising a preparation for measuring the gene expression level or protein level of ADP-ribosylcyclase or its naturally occurring variants, which contain the amino acid sequence of sequence catalog 1.

[25] A screening method for substances that can prevent or treat ADP-ribosylcyclase-mediated diseases, including the following steps: 1) A step of treating cells expressing ADP-ribosylcyclase or a naturally occurring variant thereof containing the amino acid sequence of sequence number 1 of the sequence catalog with the candidate substance; 2) A step of measuring the gene expression level or protein level of the ADP-ribosylcyclase or its naturally occurring variant by processing the candidate substance; and 3) A step in which the candidate substance is selected as a preventive or therapeutic substance for ADP-ribosylcyclase-mediated diseases if the gene expression level or protein level decreases compared to a control group that is not treated with the candidate substance.

[0120] [Examples] Example 1: NAD-glycohydrolase (NADase) enzyme activity of a novel ADPRC To confirm the activity of ADP-ribosylcyclase (ADPRC) of sequence 1 in the sequence catalog, the cDNA sequence encoding ADPRC was ligated into the FLAG-CMV-2 vector to create a FLAG-ADPRC plasmid. Overexpression of the new ADPRC was induced in HEK293 cells or MES13 cells using a transfection reagent. The overexpressed new ADPRC was lysed in lysis buffer. 45 μl of the lysed sample was treated with 5 μl of 2 mM ε-NAD (Nicotinamide 1,N6-ethenoadenine dinucleotide) and reacted at 37°C for 1 hour. After the reaction, 50 μl of 10% trichloroacetic acid was added to stop the enzyme-substrate reaction. After centrifuging for 10 minutes, 80 μl of the supernatant was added to 720 μl of 0.1 M sodium phosphate buffer, and the absorbance was measured using a fluorescence analyzer at excitation 297 nm and emission 410 nm. The results are shown in Figure 1.

[0121] Example 2: Evaluation of the cADPR synthesis ability of a new ADPRC The cADPR synthesis ability of the aforementioned ADPRC was evaluated by the method reported by Graeff R et al. [Graeff R, Lee HC. Biochem. J. 361:379-384, 2002].

[0122] Specifically, HEK293 cells were overexpressed with the FLAG-ADPRC plasmid using a phenotypic infection reagent, and then lysed with lysis buffer. The lysed sample was purified to remove the overexpressed FLAG-ADPRC using a FLAG-agarose column. The purified sample was treated with 100 μM β-NAD and reacted at 37°C for 1 hour. After the reaction, cADPR was extracted by treating with trichloroacetic acid to a final concentration of 0.6 M. Then, 50 μl of a mixed solution of ADPR cyclase (0.3 μg / ml), nicotinamide (30 mM), and sodium phosphate (100 mM) was added to 0.1 ml of the extract and 0.1 ml of cADPR standard solution, and the mixture was reacted at room temperature for 30 minutes.

[0123] To this mixture, ethanol (2%), alcohol dehydrogenase (100 μg / ml), resazurin (20 μM), diaphorase (10 μg / ml), FMN (10 μM), nicotinamide (10 mM), bovine serum albumin (BSA, 0.1 mg / ml), and sodium phosphate (100 mM) were added, and the mixture was allowed to react for 2 to 4 hours. The absorbance of the reaction product was measured using a fluorescence spectrophotometer in the range of 544 nm to 590 nm. The results are shown in Figure 2.

[0124] Example 3: Changes in intracellular cADPR concentration in MES13 cells induced by ADPRC The intracellular cADPR concentration changes induced by the aforementioned ADPRC were prepared by the method reported by Graeff R et al. [Graeff R, Lee HC. Biochem. J. 361:379-384, 2002].

[0125] Specifically, MES13 cells were treated with a novel small interfering RNA (sequence catalog sequence 22) of ADPRC as a phenotypic infection reagent to suppress ADPRC expression. After treating the cells with suppressed ADPRC expression with 150 nM angiotensin II for 60 seconds, cADPR was extracted using 0.6 M trichloroacetic acid. Then, 50 μl of a mixed solution of ADPR cyclase (0.3 μg / ml), nicotinamide (30 mM), and sodium phosphate (100 mM) was added to 0.1 ml of the extract and 0.1 ml of cADPR standard solution, and the mixture was allowed to react at room temperature for 30 minutes.

[0126] To this mixture, ethanol (2%), alcohol dehydrogenase (100 μg / ml), resazurin (20 μM), diaphorase (10 μg / ml), FMN (10 μM), nicotinamide (10 mM), bovine serum albumin (BSA, 0.1 mg / ml), and sodium phosphate (100 mM) were added, and the mixture was allowed to react for 2 to 4 hours. The absorbance of the reaction product was measured using a fluorescence spectrophotometer in the range of 544 nm to 590 nm. The results are shown in Figure 3.

[0127] Example 4: Effect of a novel inhibitor on suppressing the NAD glycohydrolase (NADase) activity of a new ADPRC. To find a novel inhibitor of ADP-ribosylcyclase (ADPRC) activity in sequence 1 of the sequence catalog, we are investigating 4,4'-dihydroxyazobenzene (4-DHAB, TCI (Japan)), 2,2'-dihydroxyazobenzene (2-DAB, Sigma-Aldrich (USA)), 2-(3,4-dihydroxyphenyl)-3,5,7-dihydroxychromen-4-one (Quercetin, Sigma-Aldrich (USA)), San4825 (kannt A, Sicka K, Kroll K, Kadereit D, Gogelein [H. Naunyn. Schmiedebergs. Arch. Pharmacol. 385:717-727, 2012] (synthesized in China) and newly discovered 2-(1,3-benzoxazole-2-ylamino)-1-mitylquinazoline-4(1H)-one (2-BMQ, synthesized in China) and 1,4-dicaffeoylquinic acid (1,4-DCQA, Biopurify in China, hereafter DCQA) were reacted in 40 μl of new ADPRC with 5 μl of inhibitor in chilled water for 15 minutes. After the reaction, 5 μl of 2 mM ε-NAD (Nicotinamide 1,N6-ethenoadenine dinucleotide) was added and the mixture was reacted at 37°C for 1 hour. After the reaction, 50 μl of 10% trichloroacetic acid was added to stop the enzyme-substrate reaction. After centrifuging for 10 minutes, 80 μl of the supernatant was added to 720 μl of 0.1 M sodium phosphate buffer, and the absorbance was measured using a fluorescence analyzer at excitation 297 nm and emission 410 nm. The results are shown in Figure 5. As shown in Figure 5, compared to the control group, 4-DHAB inhibited NAD glycohydrolase (NADase) activity by 42.40%, 2-BMQ by 36.49%, and DCQA by a remarkable 79.64%.

[0128] Example 5: Effects of DCQA on blood glucose, kidney weight-to-body weight ratio, creatine cleanup rate, and urinary albumin levels in a mouse model of kidney disease. Blood glucose, kidney-to-body weight ratio, creatinine scavenging rate, and urinary albumin levels in a mouse model of renal disease were measured using the method reported by Kim SY et al. [Kim SY, Park KH, Gul R, Jang KY, Kim UH. Am.J. Physiol. Renal Physiol. 296:F291-F297, 2009].

[0129] Specifically, C57BL / 6J mice were divided into two groups: one group received 0.2 ml of 50 mM citrate buffer (pH 4.8) intraperitoneally (control group and DCQA group), and the other group received 200 μl of streptozotocin (STZ, Sigma-Aldrich (USA)) dissolved at 5 mg / ml in 50 mM citrate buffer intraperitoneally (STZ group and STZ+DCQA group). Blood glucose was measured from the second day after STZ injection, and mice with a blood glucose level of 300 mg / dL were classified into the STZ group and the STZ+DCQA group. For mice with elevated blood glucose, DCQA, which showed the best inhibitory effect on ADPRC as newly described in Example 5, was dissolved at 9 mg / ml in dimethyl sulfoxide, diluted to 9 μg / ml in saline, and then administered by intraperitoneal injection of 100 μl daily for 6 weeks. On the last day of week 6, the mice were placed in a metabolic cage, and urine was collected over a 24-hour period.

[0130] After obtaining urine over 24 hours, the body weight and blood glucose levels of the mice were measured (Figure 6A). Furthermore, to confirm its potential as a treatment for kidney disease, serum was obtained and the kidneys were removed after sacrificing the mice with an anesthesia. After measuring the ratio of kidney weight to body weight using the removed kidneys (Figure 6B), some kidneys were fixed in 10% formalin for fluorescent and H&E staining, while the remaining kidneys were subjected to multiple section ADPR cyclase activity and cADPR concentration measurements. Creatinine levels in urine and serum were measured using a creatinine measurement kit (BioAssay System, USA) to calculate the creatinine cleanup rate (Figure 6C), and urine albumin levels were measured using an albumin measurement kit (BioAssay System, USA) (Figure 6D). The results are shown in Figure 6.

[0131] As shown in Figure 6, in a mouse model of diabetes induced by STZ, the STZ+DCQA group showed a significant decrease in kidney weight relative to body weight (Figure 6B), a significant increase in creatine cleanup rate (Figure 6C), and a significant decrease in urinary albumin levels (Figure 6D) compared to the control group (STZ), even though blood glucose levels did not decrease (Figure 6A). This confirmed its potential as a candidate for the treatment of kidney diseases such as chronic renal failure and diabetic nephropathy.

[0132] Example 6: Effects of DCQA on ADPRC activity and cADPR concentration in kidney tissue of a mouse model of kidney disease. ADPRC activity and cADPR concentration in kidney tissue of a mouse model of renal disease were measured using the method reported by Kim SY et al. [Kim SY, Park KH, Gul R, Jang KY, Kim UH. Am.J. Physiol. Renal Physiol. 296:F291-F297, 2009].

[0133] Specifically, C57BL / 6J mice were divided into two groups: one receiving 0.2 ml of 50 mM citrate buffer (pH 4.8) intraperitoneally (control group and DCQA group), and the other receiving 200 μl of streptozotocin (STZ, Sigma-Aldrich (USA)) dissolved at 5 mg / ml in 50 mM citrate buffer intraperitoneally (STZ group and STZ+DCQA group). Blood glucose was measured from the second day after STZ injection, and mice with a blood glucose level of 300 mg / dL were classified into the STZ group and the STZ+DCQA group. For mice with elevated blood glucose, DCQA, which showed the best inhibitory effect on the novel ADPRC in Example 5, was dissolved at 9 mg / ml in dimethyl sulfoxide, diluted to 9 μg / ml in saline, and then administered 100 μl daily via intraperitoneal injection for 6 weeks. To confirm its potential as a treatment for kidney disease, serum was obtained and kidneys were removed after sacrificing animals using anesthesia.

[0134] After dissolving a portion of the kidney tissue excised from each group in lysis buffer, 45 μl of the lysis sample was treated with 5 μl of 2 mM NGD (Nicotinamide guanine dinucleotide), and the reaction was carried out at 37°C for 1 hour. After the reaction, 50 μl of 10% trichloroacetic acid was added to stop the enzyme-substrate reaction. The supernatant was centrifuged for 10 minutes, and 80 μl was added to 720 μl of 0.1 M sodium phosphate buffer. The absorbance was then measured using a fluorescence analyzer (Hitachi, Japan) at excitation 297 nm and emission 410 nm.

[0135] Furthermore, cADPR was extracted by treating a portion of the kidney tissue excised from each group with 0.2 ml of 0.6 M trichloroacetic acid. Then, 50 μl of a mixed solution of ADPR cyclase (0.3 μg / ml), nicotinamide (30 mM), and sodium phosphate (100 mM) was added to 0.1 ml of the extract and 0.1 ml of cADPR standard solution, and the mixture was reacted at room temperature for 30 minutes. To this mixture, ethanol (2%), alcohol dehydrogenase (100 μg / ml), resazurin (20 μM), diaphorase (10 μg / ml), FMN (10 μM), nicotinamide (10 mM), bovine serum albumin (BSA, 0.1 mg / ml), and sodium phosphate (100 mM) were added, and the mixture was reacted for 2 to 4 hours. The absorbance of the reactants was measured using a fluorescence spectrophotometer in the range of 544 nm to 590 nm. The results are shown in Figure 7.

[0136] As shown in Figure 7A, the DCQA of the present invention reduced the ADP-ribosylcyclase activity increased by STZ to below the control level, and as shown in Figure 7B, the DCQA of the present invention reduced the cADPR concentration increased by STZ to the control level. This confirms that the DCQA has the ability to reduce the activity of ADPRC and the concentration of cADPR, which are generated by renal dysfunction or damage associated with kidney disease.

[0137] Example 7: Effects of 1,4-DCQA on changes in TGF-β1, fibronectin, and collagen IV expression in kidney tissue of a mouse model of kidney disease. In a mouse model of renal disease, changes in the expression of TGF-β1, fibronectin, and collagen IV in renal tissue were investigated using the method reported by Kim SY et al. [Kim SY, Park KH, Gul R, Jang KY, Kim UH. Am.J. Physiol. Renal Physiol. 296:F291-F297, 2009].

[0138] Specifically, C57BL / 6J mice were divided into two groups: one group received 0.2 ml of 50 mM citrate buffer (pH 4.8) intraperitoneally (control group and DCQA group), and the other group received 200 μl of streptozotocin (STZ, Sigma-Aldrich (USA)) dissolved at 5 mg / ml in 50 mM citrate buffer intraperitoneally (STZ group and STZ+DCQA group). Blood glucose was measured from the second day after STZ injection, and mice with a blood glucose level of 300 mg / dL were used as the STZ group and STZ+DCQA group. For mice with elevated blood glucose, DCQA, which showed the best inhibitory effect on the novel ADPRC in Example 5, was dissolved at 9 mg / ml in dimethyl sulfoxide, diluted to 9 μg / ml in saline, and then 100 μl was injected intraperitoneally daily for 6 weeks. To confirm its potential as a treatment for kidney disease, serum was obtained and kidneys were removed after sacrificing animals using anesthesia. A portion of the removed kidney was fixed in 10% formalin. The kidney tissue fixed in 10% formalin was placed on a slide using a tissue sectioning device, washed with TTBS (Tris-buffered saline (TBS) with 0.1% Tween 20) buffer, and then reacted with TTBS buffer containing 1% bovine serum albumin (BSA) for 1 hour. Primary antibodies (TGF-β1 (Santa Cruz, USA), fibronectin (Santa Cruz, USA), collagen IV (Abcam, UK)) were diluted 1:200 in TTBS buffer containing 1% bovine serum albumin (BSA) and reacted at 4°C for more than 12 hours. After washing the tissue treated with the primary antibody in TTBS buffer three times, the FITC-labeled secondary antibody was diluted 1:200 in TTBS buffer and reacted at room temperature in a dark place for 1 hour. After the secondary antibody reaction, the tissue was washed three times with TTBS buffer, and then a coverslip was attached using mounting solution. The stained kidney tissue was observed using a fluorescence microscope (Carl Zeiss, Germany) that observes green fluorescence. The results are shown in Figure 8.

[0139] As shown in Figure 8, DCQA of the present invention reduced the expression levels of TGF-β1, fibronectin, and collagen IV, which were increased by STZ, to control levels, confirming that DCQA has the ability to reduce the expression levels of TGF-β1, fibronectin, and collagen IV, which are generated by kidney dysfunction or damage associated with kidney disease.

[0140] Example 8: Histopathological changes in kidney tissue of a mouse model of kidney disease using H&E (Hematoxylin and Eosin) staining. In a mouse model of kidney disease, histopathological changes in kidney tissue were assessed using the method reported by Shu B et al. [Shu B, Feng Y, Gui Y, Lu Q, Wei W, Xue X, Sun X, He W, Yang J, Dai C. Cell. Signal. 42:249-258, 2018].

[0141] Specifically, C57BL / 6J mice were divided into two groups: one group received 0.2 ml of 50 mM citrate buffer (pH 4.8) intraperitoneally (control group and DCQA group), and the other group received 200 μl of streptozotocin (STZ, Sigma-Aldrich (USA)) dissolved at 5 mg / ml in 50 mM citrate buffer intraperitoneally (STZ group and STZ+DCQA group). Blood glucose was measured from the second day after STZ injection, and mice with a blood glucose level of 300 mg / dL were used as the STZ group and STZ+DCQA group. For mice with elevated blood glucose, DCQA, which showed the best inhibitory effect on the novel ADPRC in Example 5, was dissolved at 9 mg / ml in dimethyl sulfoxide, diluted to 9 μg / ml in saline, and 100 μl was administered intraperitoneally daily for 6 weeks. To confirm its potential as a treatment for kidney disease, serum was obtained after sacrificing animals using anesthesia, and the kidneys were removed. A portion of the removed kidney was fixed in 10% formalin. The kidney tissue fixed in 10% formalin was placed on a slide using a freezing tissue sectioning device, washed with running water for 5 minutes, and stained with hematoxylin for 5 minutes. After staining, it was washed with running water for 5 minutes, quickly dipped twice in 157 mM hydrochloric acid, removed, and washed with running water for 5 minutes. Quickly dipped once in 0.25% ammonia water, removed, washed again with running water for 5 minutes, stained with eosin for approximately 30 seconds, and then reacted with 70% ethanol for 30 seconds, 80% ethanol for 30 seconds, 90% ethanol for 30 seconds, primary 100% ethanol for 30 seconds, secondary 100% ethanol for 30 seconds, and tertiary 100% ethanol for 30 seconds. Finally, the tissue was reacted with primary xylene for 5 minutes, then with secondary xylene for more than 5 minutes, and then a coverslip was attached using mounting solution. The stained kidney tissue was observed under a light microscope (Lieca, Germany). The results are shown in Figure 9.

[0142] As shown in Figure 9, DCQA of the present invention restored glomerular thickening, inflammatory cell infiltration, and atypical epithelial cell formation, which were increased by STZ, to levels similar to those of the control group, confirming that DCQA has the ability to restore histopathological changes in the kidney that occur due to kidney dysfunction or damage associated with kidney disease.

[0143] Example 9: Comparison of blood glucose, kidney-to-body weight ratio, and creatinine cleanup rate in normal mice and ADPRC heterozygous (ADPRC(+ / -)) mice in renal disease models. Blood glucose, kidney-to-body weight ratio, and creatine cleanup rate in a mouse model of renal disease were measured using the method reported by Kim SY et al. [Kim SY, Park KH, Gul R, Jang KY, Kim UH. Am.J. Physiol. Renal Physiol. 296:F291-F297, 2009].

[0144] Specifically, 129S1 / SvImJ mice (wild type, WT) and ADPRC hetero-knockout (ADPRC(+ / -)) mice generated by The Jackson Laboratory (USA) were intraperitoneally injected with 200 μl of streptozotocin (STZ) dissolved at 5 mg / ml in 50 mM citrate buffer (pH 4.8). Blood glucose levels were measured from the second day after injection, and mice with a blood glucose level of 300 mg / dL were used. Six weeks after the rise in blood glucose, the mice were placed in metabolic cages, and urine samples were collected over 24 hours.

[0145] After collecting urine over 24 hours, the body weight and blood glucose levels of the mice were measured (Figure 10A). After sacrificing the mice using an anesthesia, serum was obtained and the kidneys were removed. The ratio of kidney weight to body weight was measured using the removed kidneys (Figure 10B). Creatinine levels in urine and serum were measured using a creatinine measurement kit (BioAssay System, USA), and the creatinine cleanup rate was calculated (Figure 10C). The results are shown in Figure 10.

[0146] As shown in Figure 10, in a diabetic mouse model induced by STZ, while (ADPRC(+ / -)) blood glucose levels were not reduced compared to the control group (Figure 10A), the kidney weight relative to body weight in the ADPRC(+ / -)+STZ group of the present invention showed a significant reduction compared to the WT+STZ group (Figure 10B). Furthermore, the reduction in creatinine scavenging rate by STZ was not reduced in the ADPRC(+ / -)+STZ group (Figure 10C), confirming the importance of a new ADPRC for kidney diseases such as chronic renal failure and diabetic nephropathy.

[0147] Example 10: Effects of DCQA on blood pressure and creatinine cleanup rate in normal mice and hypertensive mouse models Blood pressure and creatinine cleanup rate in hypertensive mouse models were measured using methods reported by Allagnat et al. [Allagnat F, Haefliger JA, Lambelet M, Longchamp A, Berard X, Mazzolai L, Corpataux JM, Deglise S.Eur.J.Vasc.Endovasc.Surg.51:733-742,2016] and Kim SY et al. [Kim SY, Park KH, Gul R, Jang KY, Kim UH.Am.J.Physiol.Renal Physiol.296:F291-F297,2009].

[0148] Specifically, C57BL / 6J mice were orally administered 0.2 ml daily after dissolving 8 mg of L-NAME (Nω-nitro-l-arginine-methyl-ester, Sigma-Aldrich, USA) in 1 ml of saline. On days 7 and 14 of oral administration, the mice's blood pressure was measured from the tail to confirm an increase in blood pressure. Then, DCQA, which showed the best inhibitory effect on the novel ADPRC in Example 5, was dissolved at 9 mg / ml in dimethyl sulfoxide, diluted to 9 μg / ml in saline, and 100 μl of this DCQA was injected intraperitoneally daily for 7 days, and L-NAME was administered orally. Six days after DCQA treatment, the mice were placed in metabolic cages, and urine was collected over 24 hours.

[0149] After collecting urine over 24 hours, the blood pressure of the mice was measured (Figure 11A). Serum was obtained by sacrificing the mice using an anesthesia.

[0150] Creatinine levels in urine and serum were measured using a creatinine measurement kit (BioAssay System, USA), and the creatinine cleanup rate was calculated (Figure 11B). The results are shown in Figure 11.

[0151] As shown in Figure 11A, DCQA was found to have no blood pressure-lowering effect in the hypertension model. However, the increase in creatinine cleansing rate observed with DCQA suggests its potential as a candidate treatment for kidney diseases such as hypertensive nephropathy (Figure 11B).

[0152] <References> 1. Berridge MJ, Bootman MD, Roderick HL. Calcium signaling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 4:517-529, 2003. 2. Lee HC. Structure and enzymatic functions of human CD38. Mol Med. 12:317-323, 2006. 3. Resnick LM. Ionic basis of hypertension, insulin resistance, vascular disease, and related disorders. The mechanism of “Syndrome X”. Am. J. Hypertens. 6:123S-134S, 1993. 4. Cowley AW Jr. Long-term control of arterial blood pressure. Physiol. Rev. 72:231-300, 1992. 5. Kim BJ, Park KH, Yim CY, Takasawa S, Okamoto H, Im MJ, Kim UH. Generation of nicotininc acid adenine dinucleotide phosphate and cyclic ADP-ribose by glucagon-like peptide-1 evokes Ca 2+ signal that is essential for insuline secreation in mouse pancreatic islets. Diabetes. 57: 868-878, 2008. 6. Gul R, Park JH, Kim SY, Jang KY, Chea JK, Ko JK, Kim UH. Inhibition of ADP-ribosyl cyclase attenuates antiotensin II-induced cardiac hypertrophy. Cardiovasc. Res. 81: 582-591, 2009. 7. Kim SY, Gul R, Rah SY, Kim SH, Park SK, Im MJ, Kwon HJ, Kim UH. Molecular mechanism of ADP-ribosyl cyclase activation in angiotensin II signaling in murine mesangial cells. Am. J. Physiol. Renal. Physiol. 294: F989-F989, 2008. 8. Partida-Sanchez S, Cockayne DA, Monard S, Jacobson EL, Oppenheimer N, Garvy B, Kusser K, Goodrich S, Howard M, Harmsen A, Randall TD, Lund FE. Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo. Nat Med 7:1209-1216, 2001. 9. Graeff R, Lee HC. A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity. Biochem. J. 361:379-384, 2002. 10. Kim SY, Park KH, Gul R, Jang KY, Kim UH. Role of kidney ADP-ribosyl cyclase in diabetic nephropathy. Am. J. Physiol. Renal Physiol. 296:F291-F297, 2009. 11. Shu B, Feng Y, Gui Y, Lu Q, Wei W, Xue X, Sun X, He W, Yang J, Dai C. Blockade of CD38 diminishes lipopolysaccharide-induced macrophage classical activation and acute kidney injury involving NF-κB signaling suppression. Cell. Signal. 42:249-258, 2018. 12. Allagnat F, Haefliger JA, Lambelet M, Longchamp A, Berard X, Mazzolai L, Corpataux JM, Deglise S. Nitric oxide deficit drives intimal hyperplasia in mouse models of hypertension. Eur. J. Vasc. Endovasc. Surg. 51:733-742, 2016. 13. Kannt A, Sicka K, Kroll K, Kadereit D, Gogelein H. Naunyn. Schmiedebergs. Arch. Pharmacol. 385:717-727, 2012. Having described in detail certain aspects of the present invention, it will be clear to those with ordinary skill in the art that such specific technologies are merely preferred examples and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents. [Brief explanation of the drawing]

[0153] [Figure 1] This figure shows the measurement of the NAD-glycohydrolase (NADase) activity of the novel ADPRC in HEK293 cells and MES13 cells. [Figure 2] This figure shows the evaluation of the cADPR synthesis ability of purified FLAG-ADPRC. [Figure 3] This figure shows the measurement of intracellular cADPR production by a novel ADPRC when MES13 cells were stimulated with angiotensin II. [Figure 4]This is the result of comparing the interspecies sequence homology of the ADP-ribosylcyclase of the present invention using alignment (with mouse as the reference, human (96%), rat (96%), dog (90%), pig (88%), rabbit (89%), sheep (90%), chicken (70%), cattle (78%), chimpanzee (93%), horse (91%), frog (63%), goat (90%), turkey (62%), guinea pig (91%), Echinococcus granulosus (27%), Schistosoma haematobium (22%), Tricinella spiralis (22%), Drosophila melanogaster (19%), zebrafish (56%)). [Figure 5] This figure shows the effect of a new inhibitor on suppressing the NAD glycohydrolase (NADase) activity of a new ADPRC. [Figure 6] This figure shows the effects of dicaffeolylquinic acid (DCQA) on blood glucose (Figure 6A), the ratio of kidney weight to body weight (Figure 6B), creatinine scavenging rate (Figure 6C), and urinary albumin level (Figure 6D) in a mouse model of diabetic kidney disease. [Figure 7] This figure shows the effects of DCQA on ADPRC activity (Figure 7A) and cADPR concentration (Figure 7B) in kidney tissue of a mouse model of diabetic kidney disease. [Figure 8] This figure shows the effects of DCQA on changes in the expression of TGF-β1, fibronectin, and collagen IV in kidney tissue of a mouse model of diabetic kidney disease. [Figure 9] This figure shows the histopathological changes observed in kidney tissue from a mouse model of diabetic kidney disease using H&E (Hematoxylin and Eosin) staining. [Figure 10] This figure shows the blood glucose levels (Figure 10A), kidney weight-to-body weight ratio (Figure 10B), and creatinine cleanup rate (Figure 10C) measured in normal mice and ADPRC heterozygous (ADPRC(+ / -)) mice in diabetic kidney disease models. [Figure 11]This figure shows the effects of DCQA on blood pressure (Figure 11A) and creatinine cleanup rate (Figure 11B) in normal mice and hypertensive mouse models.

Claims

1. It contains an ADP-ribosylcyclase activity inhibitor as an active ingredient, The aforementioned ADP-ribosylcyclase is NAD ＋ A pharmaceutical composition for the prevention or treatment of kidney disease, which converts to cyclic ADP-ribose (cADPR), The aforementioned kidney disease is renal failure or nephropathy. The ADP-ribosylcyclase comprises an amino acid sequence selected from the group consisting of sequence 1 to 3 and sequence 10 of the sequence catalog. The pharmaceutical composition wherein the ADP-ribosylcyclase activity inhibitor is dicaffeoylquinic acid.

2. The pharmaceutical composition according to claim 1, characterized in that the renal failure is chronic renal failure, acute renal failure, or mild renal failure before dialysis.

3. The pharmaceutical composition according to claim 1, characterized in that the nephropathy is Nephropathy Syndrome, Lipoid Nephropathy, Diabetic Nephropathy, IgA Nephropathy, Analgesic Nephropathy, or Hypertensive Nephropathy.

4. It contains an ADP-ribosylcyclase activity inhibitor as an active ingredient, The aforementioned ADP-ribosylcyclase is NAD ＋ Convert to cyclic ADP-ribose (cADPR), The ADP-ribosylcyclase comprises an amino acid sequence selected from the group consisting of sequence 1 to 3 and sequence 10 of the sequence catalog. The ADP-ribosylcyclase activity inhibitor is dicaffeoylquinic acid, in a food composition for the prevention or improvement of kidney disease, such as renal failure or nephropathy.

5. Methods for determining whether kidney disease, including the stages described below, is mediated by ADP-ribosylcyclase or is independently of ADP-ribosylcyclase: (a) A step of inducing kidney disease in an animal model lacking a heterotype gene for ADP-ribosylcyclase and a wild-type animal model, wherein the ADP-ribosylcyclase comprises an amino acid sequence selected from the group consisting of sequence 1 to 3 and sequence 10 of the sequence catalog; and (b) A step to confirm the differences between the animal models, Here, The aforementioned differences include differences in body weight, kidney weight, creatine cleanup rate, or blood glucose levels. The aforementioned kidney disease is renal failure or nephropathy. The animals mentioned above are mammals other than humans, birds, reptiles, amphibians, or fish.

6. Methods for providing information on the diagnosis of kidney disease, including the following stages: 1) A step of measuring the expression or activity level of ADP-ribosylcyclase in a sample isolated from a subject, wherein the ADP-ribosylcyclase is NAD ＋ A step of converting to cyclic ADP-ribose (cADPR), where the ADP-ribosylcyclase comprises an amino acid sequence selected from the group consisting of sequence 1 to 3 and sequence 10 of the sequence catalog; and, 2) A step in which the expression or activity level of ADP-ribosylcyclase in step 1) is compared with that of a normal control group to determine that the individual is at risk of developing kidney disease. Here, the aforementioned kidney disease is renal failure or nephropathy.

7. The preparation includes a preparation for measuring the gene expression level of ADP-ribosylcyclase or a preparation for measuring the protein level, The aforementioned ADP-ribosylcyclase is NAD ＋ Convert to cyclic ADP-ribose (cADPR), The ADP-ribosylcyclase comprises an amino acid sequence selected from the group consisting of sequences 1 to 3 and sequence 10 of the sequence catalog, in a diagnostic composition for kidney disease. Here, the aforementioned kidney disease is renal failure or nephropathy.

8. The preparation includes a preparation for measuring the gene expression level of ADP-ribosylcyclase or a preparation for measuring the protein level, The aforementioned ADP-ribosylcyclase is NAD ＋ Convert to cyclic ADP-ribose (cADPR), The aforementioned ADP-ribosylcyclase contains an amino acid sequence selected from the group consisting of sequences 1 to 3 and sequence 10 of the sequence catalog, in a diagnostic kit for kidney disease. Here, the aforementioned kidney disease is renal failure or nephropathy.

9. A screening method for substances that prevent or treat kidney disease, including the following stages: 1) A step of treating isolated cells expressing ADP-ribosylcyclase with a candidate substance, wherein the ADP-ribosylcyclase is NAD ＋ The process involves converting to cyclic ADP-ribose (cADPR), wherein the ADP-ribosylcyclase comprises an amino acid sequence selected from the group consisting of sequence 1 to 3 and sequence 10 of the sequence catalog; 2) A step of measuring the gene expression level or protein level of the ADP-ribosylcyclase by processing the candidate substance; and 3) When the gene expression level or protein level decreases compared to a control group that is not treated with the candidate substance, the candidate substance is selected as a substance for the prevention or treatment of kidney disease. Here, the aforementioned kidney disease is renal failure or nephropathy.

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