Composition for preventing or treating cystic kidney disease, comprising USP1 inhibitor as active ingredient

Abstract

The present invention relates to: a composition for preventing or treating kidney disease, the composition comprising an inhibitor of ubiquitin specific protease 1 (USP1) as an active ingredient; and a composition for diagnosing kidney disease, the composition comprising, as an active ingredient, an agent for measuring the expression level of the USP1 protein or a gene encoding same. The present inventors confirmed that the expression level of the USP1 protein is significantly higher when suffering from a kidney disease, particularly cystic kidney disease, than in normal tissue, and found that abnormal cell proliferation and cyst formation in kidney tissues are effectively inhibited when the activity of USP1 is inhibited. Accordingly, USP1 may not only be a novel molecular marker for the diagnosis of cystic kidney disease, but may also be a useful therapeutic target for preventing or treating the disease. Therefore, the present invention provides a diagnostic and therapeutic composition targeting USP1, and thus can be effectively used for the early diagnosis and effective treatment of cystic kidney disease.

Description

A composition for the prevention or treatment of cystic kidney disease comprising a USP1 inhibitor as an active ingredient

[0001] The present invention relates to a technology for diagnosing the occurrence of kidney disease with high reliability or effectively inhibiting the progression of kidney disease by measuring the expression level of USP1 (Ubiquitin specific protease 1) or inhibiting its function.

[0002]

[0003] The kidneys are vital organs that play a crucial role in the human body. They filter waste products to produce urine, regulate electrolyte metabolism and body fluids, maintain pH balance, and secrete hormones that control blood pressure. Through these functions, the kidneys play a key role in maintaining homeostasis. If kidney function declines or is lost, the balance of the body's metabolism is disrupted, which can lead to serious health problems. In particular, since the kidneys are organs that do not heal naturally, the prevention and management of kidney diseases are extremely important.

[0004] Polycystic Kidney Disease (PKD) is a genetic disorder in which multiple cysts grow within the kidneys, causing them to enlarge abnormally and eventually replace normal kidney tissue. The cysts are filled with fluid and gradually grow larger over time. As the cysts grow, the kidneys progressively lose function and can eventually develop into end-stage renal failure. Furthermore, excessive secretion of the hormone vasopressin within the kidneys increases fluid accumulation within the cysts, causing them to enlarge further and exacerbating kidney function.

[0005] Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common form of Polycystic Kidney Disease (PKD) and is caused by defects in the PKD1 and PKD2 genes. ADPKD is inherited from parents to children and is one of the leading causes of renal failure, accounting for approximately 8–10% of patients with end-stage renal disease. In the early stages, symptoms are often minimal or mild; however, as time passes and cysts enlarge and kidney function gradually declines, various symptoms appear. The most common symptoms include hypertension, urinary tract infections, abdominal pain, and urination problems; in severe cases, a kidney transplant or dialysis may be necessary.

[0006] To date, no fundamental cure for cystic kidney disease has been developed. Conventional treatments for cystic kidney disease focus primarily on symptom relief and the management of complications, concentrating on reducing pain caused by cyst expansion or controlling secondary symptoms such as hypertension. Accordingly, the inventors have completed the invention of a composition for the prevention and treatment of kidney disease using a USP1 (Ubiquitin specific protease 1) inhibitor as a fundamental solution for cystic kidney disease.

[0007] Inhibiting USP1 inhibits the DNA repair ability of damaged cells, and certain cells can no longer repair damage, leading to cell death. For this reason, USP1 inhibitors are emerging as important research targets in strategies to block DNA repair pathways. Accordingly, the inventors have completed the invention of a composition for the prevention and treatment of cystic kidney disease containing a USP1 inhibitor as an active ingredient.

[0008]

[0009] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0010]

[0011] Cystic kidney disease presents a critical problem in that early diagnosis is delayed because there are few or no symptoms in the early stages. Although several drugs, including tolvaptan, are used to treat kidney disease, their effectiveness is limited and they may cause side effects such as electrolyte imbalance and hyponatremia, so new diagnostic and treatment methods are required.

[0012] Accordingly, the inventors made diligent research efforts to discover a novel molecular marker capable of diagnosing cystic kidney disease with high reliability, which is a gene whose expression level specifically changes in patients with cystic kidney disease. As a result, the inventors were the first to discover that USP1 is specifically overexpressed in cystic kidney disease, and experimentally demonstrated that the therapeutic effect of cystic kidney disease can be maximized by inhibiting USP1 expression.

[0013]

[0014] Accordingly, the objective of the present invention is to provide a composition for the prevention or treatment of kidney disease comprising an inhibitor of USP1 (Ubiquitin specific protease 1) as an active ingredient.

[0015] Another objective of the present invention is to provide a composition for diagnosing kidney disease comprising, as an active ingredient, a preparation for measuring the expression level of the USP1 protein or the gene encoding it.

[0016] Another objective of the present invention is to provide a screening method for a composition for the prevention or treatment of kidney disease.

[0017]

[0018] According to one aspect of the present invention, the present invention provides a composition for the prevention or treatment of kidney disease comprising an inhibitor of USP1 (Ubiquitin specific protease 1) as an active ingredient.

[0019] According to another aspect of the present invention, the present invention provides a method for preventing or treating kidney disease comprising the step of administering a USP1 inhibitor to a subject.

[0020] Conventional treatment methods for kidney diseases, particularly cystic kidney disease, have relied mainly on conservative treatment aimed at alleviating symptoms or delaying disease progression, and treatment methods capable of effectively inhibiting progression by targeting the underlying cause of the disease have been very limited.

[0021] Accordingly, the inventors have made diligent research efforts to discover a new therapeutic method capable of targeting the underlying cause of kidney disease by effectively inhibiting the progression of pathological changes and cyst formation in kidney tissue. As a result, they were the first to identify that inhibiting the activity of USP1 significantly inhibits abnormal cell proliferation and cyst formation in kidney tissue, and experimentally confirmed that USP1 inhibitors can be usefully utilized for the prevention or treatment of kidney disease.

[0022]

[0023] As used herein, the term “USP1 (ubiquitin specific protease 1)” refers to a deubiquitination enzyme belonging to the ubiquitin-specific protease family. USP1 is an enzyme that regulates the stability, degradation, intracellular localization, and function of proteins by cleaving and removing ubiquitin covalently bound to substrate proteins. USP1 is a type of cysteine ​​protease belonging to the ubiquitin-specific protease (USP) family and is known to be involved in the regulation of the ubiquitin signaling pathway. The USP1 protein comprises a catalytic domain having deubiquitination activity, and the catalytic domain includes an active site centered on a cysteine ​​residue to mediate a reaction that cleaves ubiquitin from a substrate protein to which ubiquitin is bound.

[0024] USP1 is involved in various cellular biological processes and plays a particularly important role in DNA damage response, cell cycle regulation, protein stability regulation, and cell proliferation regulation. Specifically, USP1 is involved in regulating the Fanconi anemia pathway and the DNA damage response pathway through the deubiquitination of FANCD2 and PCNA proteins.

[0025] In this specification, USP1 includes the USP1 protein, a gene or nucleic acid encoding the USP1 protein, the deubiquitase activity of USP1, and all homologs, variants, analogs, functional fragments, and derivatives that perform substantially the same biological function as USP1.

[0026]

[0027] As used herein, the term “kidney disease” refers to any disease caused by structural or functional abnormalities of the kidney. Specifically, said kidney disease includes various diseases involving damage to kidney tissue, inflammation, fibrosis, cyst formation, or a decline in kidney function. said kidney disease may appear in an acute or chronic form and may include diseases associated with abnormalities in the kidney’s filtration, reabsorption, or excretory functions. In this specification, kidney disease includes, but is not limited to, acute kidney injury, chronic kidney disease, glomerular disease, tubular disease, interstitial nephritis, renal fibrosis, diabetic nephropathy, and cystic kidney disease.

[0028]

[0029] In this specification, the term “prevention” means suppressing the occurrence of a disease or illness in subjects who have not been diagnosed with having such a disease or illness but are at risk of developing such a disease or illness.

[0030] In this specification, the term “treatment” means (a) inhibition of the progression of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. By effectively inhibiting the progression of abnormal cell proliferation and cyst formation appearing in the kidney tissue of patients with kidney disease, the composition of the present invention can alleviate, eliminate, or inhibit the progression of symptoms of kidney disease, thereby ultimately improving the disease state. Accordingly, the composition of the present invention may serve as a therapeutic composition for kidney disease in itself, or it may be applied as a therapeutic adjuvant when administered together with other pharmacological components. Accordingly, in this specification, the terms “treatment” or “therapeutic agent” include the meaning of “therapeutic aid” or “therapeutic adjuvant.”

[0031] In the present invention, the term “therapeutic effective amount” refers to the content of a composition in which the pharmacological component within the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and includes the meaning of “preventive effective amount.”

[0032]

[0033] According to a specific embodiment of the present invention, the aforementioned kidney disease is a cystic kidney disease.

[0034] As used in this specification, the term “cystic kidney disease” refers to a kidney disease characterized by the formation or proliferation of fluid-filled cysts within kidney tissue. The cysts may originate in the tubules or collecting ducts of the kidney, and as the cysts form and grow, the normal structure of the kidney may be deformed and kidney function may gradually decline.

[0035] The above-mentioned cystic kidney disease may involve the formation of cysts, abnormal proliferation of cyst-lining epithelial cells, structural deformation of kidney tissue, renal hypertrophy, and a decline in renal function. Specifically, in this specification, cystic kidney disease includes, but is not limited to, Autosomal Dominant Polycystic Kidney Disease, Autosomal Recessive Polycystic Kidney Disease (ARPKD), medullary cystic kidney disease, nephronophthisis, and other diseases associated with the formation of kidney cysts. More specifically, the above-mentioned cystic kidney disease is Autosomal Dominant Polycystic Kidney Disease.

[0036] The term “autosomal dominant polycystic kidney disease” as used in this specification refers to a type of polycystic kidney disease inherited in an autosomal dominant manner, which causes structural deformation and functional decline of the kidney as multiple cysts form and gradually grow within the kidney. The autosomal dominant polycystic kidney disease is mainly caused by mutations in the PKD1 or PKD2 genes, and said gene mutations induce abnormal proliferation of renal tubular epithelial cells and cyst formation, which can lead to the formation and expansion of cysts within the kidney.

[0037] In patients with autosomal dominant polycystic kidney disease, the continued growth of renal cysts can lead to kidney enlargement, decreased renal function, hypertension, and ultimately end-stage renal disease. The term autosomal dominant polycystic kidney disease in this specification is used interchangeably with autosomal dominant cystic kidney disease or autosomal dominant polycystic kidney disease.

[0038]

[0039] According to a specific embodiment of the present invention, the aforementioned inhibitor of USP1 is selected from the group consisting of an antibody or an antigen-binding fragment thereof that specifically binds to the USP1 protein, ML323, KSQ4279, IN-2, a compound of Formula 1 below, and pharmaceutically acceptable salts thereof:

[0040] [Chemical Formula 1]

[0041]

[0042] In the above chemical formula, R1 to R3 are each independently hydrogen or a halogen, and R4 and R5 are each independently C1-C5 alkyl.

[0043] As used herein, the term “inhibitor” refers to a substance that inhibits the expression or activity of USP1 at the protein or gene level, and refers to a substance that not only renders the activity or expression of USP1 undetectable or insignificant, but also significantly reduces the intrinsic biological function of USP1 to a measurable level.

[0044] In this specification, the term “decrease in expression” may mean a state in which the expression level of the USP1 protein is reduced by, for example, 20% or more compared to the control group, more specifically a state in which it is reduced by 30% or more, and more specifically a state in which it is reduced by 40% or more.

[0045] As used herein, the term “decrease in activity” means a significant decrease in the intrinsic in vivo function of the USP1 protein compared to a control group. Specifically, it includes cases where the physiological processes involving the USP1 protein are significantly inhibited or altered as a result of the decrease in the activity of the USP1 protein. Here, “decrease in activity” is a concept that encompasses not only a simple decrease in function but also the ultimate inhibition of activity resulting from a decrease in protein stability.

[0046]

[0047] In this specification, the term "antibody" refers to an immunoglobulin protein that specifically recognizes an antigen, comprising one or more variable domains that bind to an epitope of the antigen, and is produced by the immune system of a mammal. Both polyclonal and monoclonal antibodies may be used as antibodies that specifically recognize the USP1 protein used in the present invention, and specifically, polyclonal antibodies having cross-reactivity may be used.

[0048] The antibodies of the present invention may be prepared by methods commonly practiced in the art, for example, by a fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), a recombinant DNA method (U.S. Patent No. 4,816,567), or a phage antibody library method (Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:58, 1-597 (1991)). The process for preparing antibodies is described in detail in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984.

[0049] In this specification, the term “antigen binding fragment” refers to a portion of the entire immunoglobulin structure to which an antigen can bind, including, but not limited to, F(ab')2, Fab', Fab, Fv, and scFv.

[0050] In this specification, the term “specifically binding” has the same meaning as “specifically recognizing,” and means that an antigen and an antibody (or a fragment thereof) interact specifically through an immunological reaction.

[0051]

[0052] Specifically, in the aforementioned Chemical Formula 1, R1 to R3 are fluorine. More specifically, in the above Chemical Formula 1, R1 to R3 are fluorine and R4 is alkyl. Most specifically, in the above Chemical Formula 1, R1 to R3 are fluorine, R4 is alkyl, and R5 is isopropyl. The compound of Chemical Formula 1, in which R1 to R3 are fluorine, R4 is alkyl, and R5 is isopropyl, is I-138 (C 26 I-138 is an effective and reversible USP1 inhibitor that can be administered orally. It is being actively studied as an anticancer agent by inducing apoptosis in BRCA1-deficient cancer cells through the monoubiquitination of FANCD2 and PCNA, but no association with kidney disease has been reported.

[0053]

[0054] As shown in the examples described below, the inventors experimentally confirmed that USP1 inhibitors exhibit a significant apoptotic effect on cystic cell lines derived from patients with autosomal dominant cystic kidney disease. In particular, they were the first to identify that a compound represented by Formula 1, specifically I-138, effectively inhibits the activity of USP1, thereby significantly inhibiting the proliferation of said cystic cell lines and reducing cell viability.

[0055]

[0056] According to a specific embodiment of the present invention, the inhibitor of the aforementioned USP1 (Ubiquitin specific protease 1) is a nucleic acid molecule that inhibits the expression of a gene encoding the USP1 protein.

[0057] In this specification, the term “nucleic acid molecule that inhibits expression” means a nucleic acid molecule that specifically recognizes a target gene by including a complementary nucleic acid sequence that can hybridize with the target gene and can cause a modification in the nucleotide structure that causes a decrease in its function. More specifically, the nucleic acid molecule that inhibits the expression of the gene encoding the USP1 protein is an shRNA, siRNA, miRNA, ribozyme, PNA, antisense oligonucleotide, or gRNA (guideRNA) that specifically recognizes the nucleic acid sequence encoding the protein.

[0058] In this specification, the term “shRNA (small hairpin RNA)” refers to a single strand of 50 to 70 nucleotides forming a stem-loop structure in a non-compensated state, and means an RNA sequence that creates a tight hairpin structure to suppress the expression of a target gene through RNA interference. Typically, a long RNA of 19 to 29 nucleotides forms a double-stranded stem by base pairing complementarily on both sides of a loop region of 5 to 10 nucleotides, and is transduced into a cell via a vector containing a U6 promoter to ensure constant expression, and is usually transferred to a daughter cell to inherit the suppression of the target gene expression.

[0059] In this specification, the term “siRNA” refers to a short double-stranded RNA capable of inducing RNAi (RNA interference) phenomena through the cleavage of specific mRNA. It consists of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, and most preferably 20 to 70 bases, and both blunt and cohesive ends are possible as long as the expression of the target gene can be suppressed by the RNAi effect. The cohesive end structure can be either a structure with a protruding 3-terminal end or a structure with a protruding 5-terminal end.

[0060] In this specification, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is not expressed within a cell, has a short stem-loop structure, and inhibits target gene expression through complementary binding to the mRNA of the target gene.

[0061] In this specification, the term “ribozyme” refers to a type of RNA molecule that functions like an enzyme by recognizing a specific RNA base sequence and cleaving it. A ribozyme consists of a region that specifically binds to the base sequence complementary to the target mRNA strand and a region that cleaves the target RNA.

[0062] In this specification, the term “PNA (Peptide nucleic acid)” refers to a molecule that possesses the properties of both nucleic acids and proteins and is capable of binding complementarily to DNA or RNA. PNA is not found in nature but is artificially synthesized through chemical methods; it regulates the expression of target genes by forming a double strand through hybridization with natural nucleic acids of complementary base sequences.

[0063] In this specification, the term “antisense oligonucleotide” refers to a nucleic acid molecule that is a nucleotide sequence complementary to the sequence of a specific mRNA, which binds to the complementary sequence within the target mRNA and inhibits essential activities for its translation into protein, translocation into the cytoplasm, maturation, or any other overall biological function. Antisense oligonucleotides may be modified at one or more bases, sugars, or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). The oligonucleotide backbone may be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyls, cycloalkyls, short-chain heteroatomics, heterocyclic sugar sholphonates, etc.

[0064] In this specification, the term “gRNA (guideRNA)” refers to an RNA molecule used in a gene editing system that recognizes a target gene and specifically cleaves the recognized site by inducing a nuclease. A representative example of such a gene editing system is the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system.

[0065] According to another aspect of the present invention, the present invention provides a composition for diagnosing kidney disease comprising, as an active ingredient, a preparation for measuring the expression level of the USP1 protein or the gene encoding it.

[0066] As previously explained, the meaning of kidney disease that can be diagnosed by measuring the expression level of USP1 used in the present invention is omitted to avoid excessive duplication.

[0067] As seen in the examples described below, the inventors have identified that the expression level of the USP1 protein or the gene encoding it is significantly increased in kidney disease, particularly cystic kidney disease, compared to normal tissue. Accordingly, it has been identified that the presence or state of kidney disease, particularly cystic kidney disease, can be effectively diagnosed by measuring the expression level of the USP1 protein or the gene encoding it.

[0068]

[0069] As used in this specification, the term “diagnosis” means determining whether an individual currently has a specific disease.

[0070] As used in this specification, the term “diagnostic composition” refers to an integrated mixture or device comprising a means for measuring the expression level of the USP1 protein or the USP1 gene to determine whether an individual has developed kidney disease or to predict the likelihood of its development, and may also be expressed as a “diagnostic kit.”

[0071]

[0072] According to a specific embodiment of the present invention, a preparation for measuring the expression amount of the aforementioned protein is an antibody or an antigen-binding fragment thereof that specifically binds to the protein.

[0073] The present invention can diagnose kidney disease by detecting the USP1 protein according to an immunoassay method using an antigen-antibody reaction. Such immunoassay can be performed according to various immunoassay or immunostaining protocols developed in the past.

[0074] For example, when the method of the present invention is carried out according to a radioimmunoassay method, a radioactive isotope (e.g., C 14 , I 125 , P 32 and S 35An antibody labeled with ) may be used. In the present invention, the antibody that specifically recognizes the USP1 protein is a polyclonal or monoclonal antibody, specifically a monoclonal antibody.

[0075] The present invention may use aptamers that specifically bind to the USP1 protein instead of antibodies. As specified herein, the term “aptamer” means a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target substance with high affinity and specificity. General information on aptamers is disclosed in detail in Hoppe-Seyler F, Butz K “Peptide aptamers: powerful new tools for molecular medicine”. J Mol Med. 78(8):426-30(2000); and Cohen BA, Colas P, Brent R. “An artificial cell-cycle inhibitor isolated from a combinatorial library”. Proc Natl Acad Sci USA. 95(24):14272-7(1998).

[0076]

[0077] According to a specific embodiment of the present invention, a preparation for measuring the expression level of a gene encoding the aforementioned protein is a primer or probe that specifically binds to a nucleic acid molecule of the gene.

[0078] As used herein, the term “primer” refers to an oligonucleotide that acts as an initiator for synthesis under conditions in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, namely, in the presence of a polymerizing agent such as a nucleotide and DNA polymerase, and under suitable temperature and pH conditions. Specifically, the primer is a single strand of deoxyribonucleotide. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP, and dTMP), modified nucleotides, or non-natural nucleotides, and may also include ribonucleotides.

[0079] The primer of the present invention may be an extension primer that is annealed to a target nucleic acid and forms a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, and extends to the location where the immobilized probe is annealed and occupies the site where the probe is annealed.

[0080] The extension primer used in the present invention comprises a hybridization nucleotide sequence that is complementary to a specific base sequence of a target nucleic acid, e.g., the USP1 gene. The term “complementary” means that the primer or probe is sufficiently complementary to selectively hybridize to the target nucleic acid sequence under specific annealing or hybridization conditions, and encompasses both cases of being substantially complementary and perfectly complementary, specifically meaning the case of being perfectly complementary. In this specification, the term “substantially complementary sequence” includes not only perfectly matching sequences but also sequences that are partially mismatched with the sequence to the extent that they can serve as a primer by annealing to a specific sequence.

[0081] In this specification, the term “probe” means a natural or modified monomer or a linear oligomer having a bond comprising a deoxyribonucleotide and a ribonucleotide that can hybridize to a specific nucleotide sequence. Specifically, the probe is single-stranded for maximum efficiency in hybridization, and more specifically, is a deoxyribonucleotide. As a probe used in the present invention, a sequence perfectly complementary to a specific nucleotide sequence of the USP1 gene may be used, but a substantially complementary sequence may also be used to the extent that it does not interfere with specific hybridization.

[0082] Suitable conditions for hybridization can be determined by referring to the information disclosed in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

[0083] In this specification, the term “complementary” means that the nucleic acid molecule for expression inhibition is sufficiently complementary to selectively hybridize to the target nucleic acid sequence under specific annealing or hybridization conditions, and encompasses both substantially complementary and perfectly complementary; specifically, it means perfectly complementary. In this specification, the term “substantially complementary sequence” includes not only perfectly matching sequences but also sequences that are partially mismatched with the sequence to the extent that sequence-specific hybridization can occur upon annealing to a specific sequence.

[0084]

[0085] According to another aspect of the present invention, the present invention provides a screening method for a composition for the prevention or treatment of kidney disease comprising the following steps:

[0086] (a) a step of contacting a candidate substance with a biological sample comprising a cell expressing the USP1 (Ubiquitin specific protease 1) protein or a gene encoding it; and

[0087] (b) a step of measuring the activity or expression level of the USP1 protein or the gene encoding it in the sample,

[0088] If the activity or expression level of the above-mentioned USP1 protein or the gene encoding it is reduced, the above-mentioned candidate substance is determined to be a composition for the prevention or treatment of kidney disease.

[0089] The meaning of the USP1 protein or the gene encoding it, the reduction in activity, and the reduction in expression amount used in the present invention has already been described above, so to avoid excessive duplication, this is omitted.

[0090] In the present invention, the term “biological sample” refers to any sample obtained from mammals, including humans, containing cells expressing the USP1 protein or a gene encoding it, and includes, but is not limited to, tissues, organs, cells, or cell culture media. More specifically, the aforementioned biological sample is kidney tissue, kidney cells, or culture media thereof.

[0091]

[0092] The features and advantages of the present invention are summarized as follows:

[0093] (a) The present invention provides a novel molecular marker that can be used for the early diagnosis of cystic kidney disease by experimentally determining that the expression level of the USP1 (Ubiquitin specific protease 1) protein or the gene encoding it is significantly increased in kidney disease, particularly cystic kidney disease.

[0094] (b) The present invention provides a novel therapeutic target that can be usefully utilized for the prevention or treatment of cystic kidney disease by experimentally demonstrating that inhibiting the expression or activity of USP1 effectively inhibits abnormal cell proliferation and cyst formation in kidney tissue.

[0095]

[0096] Figure 1a is a graph showing the results of comparing the USP1 mRNA expression levels in normal kidney tissue and kidney tissue of ADPKD patients by analyzing the publicly available gene expression dataset (GSE7869). Figure 1b is a graph showing the results of comparing the USP1 mRNA expression levels in normal kidney tissue and kidney tissue of ADPKD patients by analyzing the publicly available gene expression dataset (GSE35831).

[0097] Figure 2 is a graph showing the results of comparing USP1 mRNA expression levels in normal kidney cells (RPTEC) and cystic cell lines derived from ADPKD patients (ADPKD#2 and ADPKD#4) through qPCR analysis.

[0098] Figure 3 is a graph showing the results of comparing the USP1 mRNA expression levels in kidney tissues of ADPKD mouse models (Pkd1 cKO and Cilk1 cKO) and normal control groups (Pkd1 cWT and Cilk1 cWT) through qPCR analysis.

[0099] Figure 4 is a graph showing the results of analyzing changes in cell viability in cystic cells derived from ADPKD patients treated with USP1 siRNA or control siRNA using a CCK-8 assay.

[0100] Figure 5a is a graph showing the change in cell viability in cystic cells derived from ADPKD patients treated with USP1 inhibitors (ML323, KSQ4279, I-138, and USP1-IN-2). Figure 5b is a graph showing the change in cell viability according to the concentration of I-138.

[0101]

[0102] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0103]

[0104] Examples

[0105] Example 1: USP1 expression in ADPKD patients

[0106] Experimental method

[0107] Gene expression analysis was performed using the gene expression dataset (GSE7869) derived from ADPKD patients, which is available in the Gene Expression Omnibus (GEO) database provided by the National Library of Medicine (NCBI), and the expression level of the deubiquitination enzyme USP1 was confirmed. The normal group in Figure 1a is a control group derived from normal tissues of three kidney cancer patients, and the cyst group is an experimental group collected from tissues of five ADPKD patients with PKD1 mutations, classified according to cyst size. Accordingly, the kidney tissue samples used for the control and experimental groups are as shown in Tables 1 to 5 below.

[0108] In addition, AW499935 and NM_003368 probes were used for USP1 expression analysis in the GEO dataset, and said probes provide expression signals based on different sequence detections of USP1 in the same sample.

[0109] In addition, USP1 expression in ADPKD patients was confirmed using the gene expression dataset (GSE35831) derived from ADPKD patients, which is available in the Gene Expression Omnibus (GEO) database provided by the National Library of Medicine (NCBI).

[0110]

[0111] GroupSample IDDescriptionNormal (normal tissue in kidney cancer patients, n=3)normal renal cortical tissue_1Normal renal cortical tissuenormal renal cortical tissue_2Normal renal cortical tissuenormal renal cortical tissue_3Normal renal cortical tissue

[0112]

[0113] GroupSample IDDescriptionMinimally cyst (PKD1 mutant ADPKD patients, n=5)minimally cystic tissue_1Minimally cystic renal tissueminimally cystic tissue_2Minimally cystic renal tissueminimally cystic tissue_3Minimally cystic renal tissueminimally cystic tissue_4Minimally cystic renal tissueminimally cystic tissue_5Minimally cystic renal tissue

[0114]

[0115] GroupSample IDDescriptionSmall cyst (PKD1 돌연변이 ADPKD 환자, n=5)PKD1 renal cyst_small cyst_1cyst < 1 mlPKD1 renal cyst_small cyst_2cyst < 1 mlPKD1 renal cyst_small cyst_3cyst < 1 mlPKD1 renal cyst_small cyst_4cyst < 1 mlPKD1 renal cyst_small cyst_5cyst < 1 ml

[0116]

[0117] GroupSample IDDescriptionMedium cyst (PKD1 돌연변이 ADPKD 환자, n=5)PKD1 renal cyst_medium cyst_1cyst 23 mlPKD1 renal cyst_medium cyst_2cyst 20 mlPKD1 renal cyst_medium cyst_3cyst 11 mlPKD1 renal cyst_medium cyst_4cyst 15 mlPKD1 renal cyst_medium cyst_5cyst 19 ml

[0118]

[0119] GroupSample IDDescriptionLarge cyst(PKD1 돌연변이 ADPKD 환자, n=3)PKD1 renal cyst_large cyst_1cyst 60 mlPKD1 renal cyst_large cyst_2cyst 50 mlPKD1 renal cyst_large cyst_3cyst 100 ml

[0120]

[0121] 실험 결과

[0122] Analysis of the publicly available gene expression dataset GSE7869 revealed that the expression level of USP1 mRNA was significantly increased in the kidney tissue of ADPKD patients compared to normal kidney tissue (Fig. 1a).

[0123] In addition, analysis of a separate gene expression dataset derived from ADPKD patients (GSE35831) also confirmed that the expression of USP1 was increased in the kidney tissue of ADPKD patients compared to normal kidney tissue (Fig. 1b).

[0124] These results suggest that USP1 expression is specifically increased in ADPKD and that USP1 is closely associated with the pathological progression of cystic kidney disease. Therefore, it was confirmed that USP1 is not only a useful molecular marker for the diagnosis of cystic kidney disease but can also be utilized as a therapeutic target.

[0125]

[0126] Example 2: USP1 expression in cystic cells derived from ADPKD patients

[0127] Experimental method

[0128] RPTEC (renal proximal tubule epithelial cells) were used as normal kidney cells, and ADPKD cell lines (ADPKD#2, ADPKD#4) derived from cysts of ADPKD patients and immortalized using hTERT were used.

[0129] RPTEC cells were cultured in DMEM:F12 medium supplemented with the hTERT Immortalized RPTEC growth kit (ATCC), and cystic cells derived from ADPKD patients were cultured in DMEM medium. All cells were cultured in a medium supplemented with 10% FBS and G418 in an incubator at 37 °C and 5% CO₂.

[0130] RNA was extracted from each cell using the NucleoSpin RNA / Protein kit (MACHEREY-NAGEL), and cDNA was synthesized by performing a reverse transcription reaction using M-MLV reverse transcriptase (Promega). Subsequently, quantitative PCR (qPCR) was performed using SYBR Green qPCR Master Mix (PCR Biosystems), and the results were analyzed using the LightCycler 96 System (Roche).

[0131]

[0132] Experimental results

[0133] Analysis results confirmed that the expression level of USP1 mRNA in ADPKD#2 and ADPKD#4, cystic cell lines derived from ADPKD patients, was significantly increased compared to RPTEC, a normal kidney cell (Fig. 2).

[0134] These results indicate that USP1 is specifically overexpressed in cystic cells derived from ADPKD patients, suggesting that USP1 may be involved in the pathological process of cystic kidney disease.

[0135]

[0136] Example 3: USP1 expression in an ADPKD mouse model

[0137] Experimental method

[0138] Conditional knockout mice were produced by crossing Pkd1 flox mice and Cilk1 flox mice with HoxB7-Cre mice that express Cre recombinase specifically to the kidney collecting duct.

[0139] RNA was extracted from the kidney tissue of the mouse, cDNA was synthesized, and qPCR analysis was performed to analyze the expression level of USP1.

[0140]

[0141] Experimental results

[0142] Analysis results confirmed that the expression level of USP1 mRNA in the kidney tissues of ADPKD model mice (Pkd1 cKO and Cilk1 cKO) was increased compared to normal controls (Pkd1 cWT and Cilk1 cWT) (Fig. 3).

[0143] These results suggest that increased expression of USP1 is associated with the pathological progression of ADPKD.

[0144]

[0145] Example 4: Changes in ADPKD cell viability following USP1 knockdown

[0146] Experimental method

[0147] ADPKD patient-derived cystic cell lines (ADPKD#2, ADPKD#4) were transfected with USP1 siRNA or control siRNA using Lipofectamine RNAiMAX (Invitrogen) to a final concentration of 40 nM, respectively. After culturing the cells for 48 and 72 hours, cell viability was evaluated using the CCK-8 assay (Dojindo).

[0148]

[0149] Experimental results

[0150] It was confirmed that cell viability was significantly reduced in ADPKD cells treated with USP1 siRNA compared to those treated with control siRNA (Fig. 4). This suggests that the expression of USP1 plays an important role in the survival and proliferation of ADPKD cells.

[0151]

[0152] Example 5: Inhibition of ADPKD cell viability by a USP1 inhibitor

[0153] Experimental method

[0154] USP1 inhibitors ML323, KSQ4279, I-138, and USP1-IN-2(C 26 H 22 Each cell was treated with F4N6O at a concentration of 50 μM and cultured for 72 hours. Subsequently, cell viability was evaluated using the CCK-8 assay. In addition, I-138, which showed the best effect, was treated at concentrations of 20, 30, 40, and 50 μM, cultured for 48 and 72 hours, and cell viability was measured.

[0155]

[0156] Experimental results

[0157] It was confirmed that the survival rate of ADPKD cells decreased when treated with USP1 inhibitors (Fig. 5a). In particular, I-138 was shown to reduce the survival rate of ADPKD cells in a concentration-dependent manner (Fig. 5b). These results suggest that USP1 inhibitors can effectively inhibit the proliferation of ADPKD cells and can be utilized as a treatment for cystic kidney disease.

[0158]

[0159] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composition for the prevention or treatment of kidney disease comprising an inhibitor of USP1 (Ubiquitin specific protease 1) as an active ingredient.

2. A composition according to claim 1, characterized in that the kidney disease is cystic kidney disease.

3. A composition according to claim 2, characterized in that the cystic kidney disease is autosomal dominant polycystic kidney disease.

4. A composition according to claim 1, wherein the inhibitor of USP1 (Ubiquitin specific protease 1) is selected from the group consisting of an antibody or an antigen-binding fragment thereof that specifically binds to the USP1 protein, ML323, KSQ4279, IN-2, a compound of Formula 1 below, and pharmaceutically acceptable salts thereof: [Chemical Formula 1] In the above chemical formula, R1 to R3 are each independently hydrogen or a halogen, and R4 and R5 are each independently C1-C5 alkyl.

5. A composition according to claim 4, characterized in that R1 to R3 in Chemical Formula 1 are fluorine.

6. A composition according to claim 5, characterized in that R4 in Chemical Formula 1 is an alkyl.

7. A composition according to claim 6, characterized in that R5 in Chemical Formula 1 is isopropyl.

8. A composition according to claim 1, characterized in that the inhibitor of USP1 (Ubiquitin specific protease 1) is a nucleic acid molecule that inhibits the expression of a gene encoding the USP1 protein.

9. A composition according to claim 8, wherein the nucleic acid molecule that inhibits the expression of the gene encoding the USP1 (Ubiquitin specific protease 1) protein is selected from the group consisting of shRNA, siRNA, miRNA, ribozyme, PNA, antisense oligonucleotide, and gRNA (guideRNA) that specifically recognizes the nucleic acid sequence encoding the protein.

10. A composition for diagnosing kidney disease comprising, as an active ingredient, a preparation for measuring the expression level of USP1 (Ubiquitin specific protease 1) protein or a gene encoding it.

11. A composition according to claim 10, characterized in that the kidney disease is cystic kidney disease.

12. A composition according to claim 11, characterized in that the cystic kidney disease is autosomal dominant polycystic kidney disease.

13. A composition according to claim 10, wherein the preparation for measuring the expression amount of the protein is an antibody that specifically binds to the protein or an antigen-binding fragment thereof.

14. A composition according to claim 10, wherein the agent for measuring the expression level of the gene encoding the protein is a primer or probe that specifically binds to the nucleic acid molecule of the gene.

15. A screening method for a composition for the prevention or treatment of kidney disease comprising the following steps: (a) a step of contacting a candidate substance with a biological sample comprising a cell expressing the USP1 (Ubiquitin specific protease 1) protein or a gene encoding it; and (b) a step of measuring the activity or expression level of the USP1 protein or the gene encoding it in the sample, If the activity or expression level of the above-mentioned USP1 protein or the gene encoding it is reduced, the above-mentioned candidate substance is determined to be a composition for the prevention or treatment of kidney disease.

16. A method according to claim 15, characterized in that the kidney disease is cystic kidney disease.

17. A method according to claim 16, characterized in that the cystic kidney disease is autosomal dominant polycystic kidney disease.

18. A method according to claim 15, wherein the biological sample is kidney tissue, kidney cells, or a culture medium thereof.

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