Pharmaceutical composition for reducing blood cholesterol, for preventing or treating cardiovascular and metabolic diseases, and for anti-inflammatory use

Abstract

The present invention relates to pharmaceutical compositions for lowering blood cholesterol, preventing or treating cardiometabolic diseases, and anti-inflammatory purposes, which contain as an active ingredient an inhibitor of the binding between CAP1 and PCSK9, an inhibitor of the binding between CAP1 and resistin, or an inhibitor of CAP1 gene expression. The present invention has the effect of reducing blood LDL cholesterol levels by inhibiting the binding between CAP1 and PCSK9, the binding between CAP1 and resistin, or the expression of the CAP1 gene. Therefore, the present invention can be useful as a pharmaceutical composition for treating abnormal blood cholesterol levels and various cardiometabolic diseases caused by such levels, such as dyslipidemia, stroke, and arteriosclerosis, and for suppressing inflammation.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for reducing blood cholesterol, preventing or treating cardiometabolic diseases, or anti-inflammatory, containing as an active ingredient a binding inhibitor between CAP1 and PCSK9, a binding inhibitor between CAP1 and resistin, or an expression inhibitor of the CAP1 gene, and a diagnostic composition for hypercholesterolemia or cardiometabolic diseases containing a preparation for measuring the binding level between CAP1 and PCSK9 or resistin, etc.

[0002] This application claims priority based on Korean Patent Application No. 10-2020-0026073 filed on March 2, 2020, and all contents disclosed in the specification and drawings of the application are incorporated herein by reference.

Background Art

[0003] PCSK9 (Proprotein convertase subtilisin / kexin type-9) determines plasma LDL cholesterol levels by regulating the internalization and lysosomal degradation of LDL (low-density lipoprotein) receptors, thereby emerging as a promising therapeutic target. PCSK9 inhibitors have shown a reduction in plasma LDL cholesterol levels and improved cardiovascular outcomes. However, the exact mechanism by which PCSK9 determines the fate of LDL receptors remains unclear. The LDL receptor enters cells while bound to LDL cholesterol, separates from LDL cholesterol in the endosome, and recycles to the cell surface, while LDL cholesterol is sent to the lysosome for degradation. In contrast, when bound to PCSK9, the LDL receptor is internalized and guided to the lysosome for degradation through an unknown mechanism. PCSK9 is a proteinase K-like serine protease, but after autocatalytic cleavage, the terminal portion of the pro-domain (amino acids 32-152) covers the catalytic triad, preventing further proteolytic activity. The catalytic domain of PCSK9 binds to the LDL receptor, and another part of PCSK9, namely the C-terminal cysteine rich domain (CRD), has been suspected to interact with a putative membrane-bound protein that escorts the protein complex to the lysosomal degradation side.

[0004] On the one hand, CAP1 is known to regulate the dynamics of actin filaments, which are important for cell morphology, movement, and endocytosis. Previous research results have reported that CAP1 is a receptor for human resistin. However, it is completely unknown whether CAP1 interacts with PCSK9 or resistin and is involved in the regulation of LDL cholesterol concentration in various cardiometabolic diseases including dyslipidemia, stroke, and atherosclerosis.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The inventors of the present invention have found that CAP1 (Adenylyl cyclase-Associated Protein 1) consisting of the amino acid sequence of SEQ ID NO: 1 directly binds to PCSK9 (proprotein convertase subtilisin / kexin type-9) that determines the fate of the LDL receptor and induces the degradation of the receptor. When the binding between CAP1 and PCSK9 or the binding between CAP1 and resistin is suppressed, or the expression of CAP1 is suppressed, the degradation of the LDL receptor is inhibited, thereby reducing the level of LDL cholesterol. At the same time, it has been found that inflammation is suppressed, and thus the present invention has been completed.

[0006] Therefore, an object of the present invention is to provide a composition for reducing blood cholesterol, comprising, as an active ingredient, a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 (proprotein convertase subtilisin / kexin type-9) consisting of the amino acid sequence of SEQ ID NO: 2.

[0007] Another object of the present invention is to provide a composition for reducing blood cholesterol, comprising, as an active ingredient, an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0008] Still another object of the present invention is to provide a composition for preventing, ameliorating or treating cardiovascular and metabolic diseases, comprising (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; or (iii) a mixture of (i) and (ii).

[0009] Still another object of the present invention is to provide a pharmaceutical composition for anti-inflammation or a health functional food composition, comprising as an active ingredient at least one selected from the group consisting of (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and resistin consisting of the amino acid sequence of SEQ ID NO: 3; and (iii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0010] Still another object of the present invention is to provide a composition for diagnosing hypercholesterolemia or cardiovascular and metabolic diseases, comprising a preparation for measuring the binding level between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2 or resistin consisting of the amino acid sequence of SEQ ID NO: 3.

[0011] Still another object of the present invention is to provide a method for diagnosing hypercholesterolemia or cardiovascular and metabolic diseases or a method for providing information for diagnosis, comprising the step of measuring the binding level between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2 or resistin consisting of the amino acid sequence of SEQ ID NO: 3 in a sample of a patient.

[0012] Also, an object of the present invention is to provide a method for screening a therapeutic agent for hypercholesterolemia or cardiovascular and metabolic diseases.

[0013] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those with ordinary knowledge in the technical field to which the present invention pertains from the following description.

Means for Solving the Problems

[0014] In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for reducing blood cholesterol, comprising, as an active ingredient, a binding inhibitor between CAP1 (Adenylyl cyclase-associated Protein 1) consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 (proprotein convertase subtilisin / kexin type-9) consisting of the amino acid sequence of SEQ ID NO: 2.

[0015] Further, the present invention provides a method for reducing blood cholesterol, comprising the step of administering to an individual a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2.

[0016] Furthermore, the present invention provides the use of a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2 for reducing blood cholesterol.

[0017] Moreover, the present invention provides the use of a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2 for producing a drug used for preventing or treating blood cholesterol reduction or hypercholesterolemia.

[0018] In one embodiment of the present invention, the binding inhibitor may be one or more selected from the group consisting of a protein, a peptide, a peptidomimetic, a substrate analog, an aptamer, and an antibody that specifically binds to CAP1 or PCSK9, but is not limited thereto.

[0019] In another embodiment of the present invention, the binding inhibitor may be a fusion protein comprising a CAP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof; and an Fc fragment of an immunoglobulin heavy chain.

[0020] In yet another embodiment of the present invention, the fusion protein may consist of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, but is not limited thereto.

[0021] In yet another embodiment of the present invention, the binding inhibitor may bind to one or more domains selected from the group consisting of the SH3 (Src homology 3) binding domain of CAP1 and the cysteine rich domain (CRD) of PCSK9, but is not limited thereto.

[0022] In yet another embodiment of the present invention, the SH3 binding domain of CAP1 may include the amino acid sequence of SEQ ID NO: 10, but is not limited thereto.

[0023] In yet another embodiment of the present invention, the cysteine rich domain of PCSK9 may consist of the amino acid sequence of SEQ ID NO: 11, but is not limited thereto.

[0024] In yet another embodiment of the present invention, the cysteine rich domain of PCSK9 may include the M1 domain consisting of the amino acid sequence of SEQ ID NO: 12, but is not limited thereto.

[0025] In yet another embodiment of the present invention, the cysteine rich domain of PCSK9 may include the M3 domain consisting of the amino acid sequence of SEQ ID NO: 13, but is not limited thereto.

[0026] In still another embodiment of the present invention, the binding inhibitor may specifically bind to a site containing aspartic acid at position 34B present in the SH3 binding domain of CAP1, but is not limited thereto.

[0027] In still another embodiment of the present invention, the binding inhibitor may specifically bind to a site containing lysine at position 494 present in the M1 domain of PCSK9, but is not limited thereto.

[0028] In still another embodiment of the present invention, the binding inhibitor may specifically bind to a site containing arginine at position 659 present in the M3 domain of PCSK9, but is not limited thereto.

[0029] Furthermore, the present invention provides a pharmaceutical composition for reducing blood cholesterol, comprising, as an active ingredient, an inhibitor of the expression of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0030] Furthermore, the present invention provides a method for reducing blood cholesterol, comprising the step of administering to an individual an inhibitor of the expression of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0031] In addition, the present invention provides the use of an inhibitor of the expression of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 for reducing blood cholesterol.

[0032] Furthermore, the present invention provides the use of an inhibitor of the expression of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 for producing a medicament used for the prevention or treatment of blood cholesterol reduction or hypercholesterolemia.

[0033] In one embodiment of the present invention, the expression inhibitor may be one or more selected from the group consisting of antisense nucleotides, siRNAs, shRNAs, miRNAs, ribozymes, and PNAs that can bind complementarily to the mRNA of the CAP1 gene, but is not limited thereto.

[0034] In another embodiment of the present invention, the expression inhibitor may be an siRNA consisting of the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

[0035] In still another embodiment of the present invention, the expression inhibitor may be an shRNA consisting of the nucleotide sequence of SEQ ID NO: 9, but is not limited thereto.

[0036] In still another embodiment of the present invention, the composition may inhibit the degradation of LDL (Low-density lipoprotein) receptors.

[0037] In still another embodiment of the present invention, the cholesterol may be LDL cholesterol, but is not limited thereto.

[0038] Further, the present invention provides a health functional food composition for reducing blood cholesterol, comprising (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; or (iii) a mixture of (i) and (ii).

[0039] Further, the present invention provides a pharmaceutical composition for preventing or treating cardiovascular and metabolic diseases or a health functional food composition for preventing or improving cardiovascular and metabolic diseases, comprising (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; or (iii) a mixture of (i) and (ii).

[0040] Furthermore, the present invention provides a method for preventing or treating a cardiometabolic disease, comprising administering to an individual a composition comprising (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; or (iii) a mixture of (i) and (ii).

[0041] In addition, the present invention provides the use of a composition comprising (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; or (iii) a mixture of (i) and (ii) for preventing or treating a cardiometabolic disease.

[0042] Furthermore, the present invention provides the use of a composition comprising (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; or (iii) a mixture of (i) and (ii) for producing a medicament used for preventing or treating a cardiometabolic disease.

[0043] In one embodiment of the present invention, the binding inhibitor may be one or more selected from the group consisting of a protein, a peptide, a peptidomimetic, a substrate analog, an aptamer, and an antibody that specifically binds to CAP1 or PCSK9, but is not limited thereto.

[0044] In another embodiment of the present invention, the binding inhibitor may be a fusion protein comprising a CAP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof; and an Fc fragment of an immunoglobulin heavy chain.

[0045] In still another embodiment of the present invention, the fusion protein may consist of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, but is not limited thereto.

[0046] In still another embodiment of the present invention, the expression inhibitor may be one or more selected from the group consisting of antisense nucleotides, siRNAs, shRNAs, miRNAs, ribozymes, and PNAs that can bind complementarily to the mRNA of the CAP1 gene, but is not limited thereto.

[0047] In still another embodiment of the present invention, the expression inhibitor may be an siRNA consisting of the nucleotide sequence of SEQ ID NO: 8, but is not limited thereto.

[0048] In still another embodiment of the present invention, the expression inhibitor may be an shRNA consisting of the nucleotide sequence of SEQ ID NO: 9, but is not limited thereto.

[0049] In still another embodiment of the present invention, the cardiometabolic disease may be a disease selected from the group consisting of diabetes, obesity, dyslipidemia, fatty liver, hypertension, gout, stroke, arteriosclerosis, myocardial infarction, angina pectoris, peripheral vascular disease, and combinations thereof, but is not limited thereto.

[0050] Further, the present invention provides a pharmaceutical composition for anti-inflammatory use, comprising, as an active ingredient, one or more selected from the group consisting of (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and resistin consisting of the amino acid sequence of SEQ ID NO: 3; and (iii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0051] In one embodiment of the present invention, the binding inhibitor of (i) may be one or more selected from the group consisting of proteins, peptides, peptidomimetics, substrate analogs, aptamers, and antibodies that specifically bind to PCSK9, but is not limited thereto.

[0052] In another embodiment of the present invention, the binding inhibitor of (ii) may be one or more selected from the group consisting of a protein, peptide, peptidomimetic, substrate analog, aptamer, and antibody that specifically binds to resistin, but is not limited thereto.

[0053] In yet another embodiment of the present invention, the binding inhibitor of (i) or (ii) may be a fusion protein comprising the CAP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof; and the Fc fragment of an immunoglobulin heavy chain.

[0054] In yet another embodiment of the present invention, the fusion protein may consist of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, but is not limited thereto.

[0055] In yet another embodiment of the present invention, the expression inhibitor of (iii) may be one or more selected from the group consisting of an antisense nucleotide, siRNA, shRNA, miRNA, ribozyme, and PNA that can complementarily bind to the mRNA of the CAP1 gene, but is not limited thereto.

[0056] In yet another embodiment of the present invention, the composition can suppress the activity of NF-κB.

[0057] The present invention also provides a diagnostic composition for hypercholesterolemia or cardiometabolic diseases, comprising a preparation for measuring the binding level between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2 or resistin consisting of the amino acid sequence of SEQ ID NO: 3.

[0058] The present invention also provides a diagnostic method for hypercholesterolemia or cardiometabolic diseases or a method for providing information for the diagnosis thereof, comprising the step of measuring the binding level between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2 or resistin consisting of the amino acid sequence of SEQ ID NO: 3 in a sample from a patient.

[0059] In one embodiment of the present invention, the sample of the patient may be selected from the group consisting of liver tissue, hepatocytes, blood, serum, plasma, saliva, sputum, and urine, but is not limited thereto.

[0060] The present invention also provides a method for screening a therapeutic agent for hypercholesterolemia or cardiometabolic diseases, comprising: (a) treating a test substance with a sample containing (i) a CAP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof; and (ii) a PCSK9 protein consisting of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, or a resistin protein consisting of the amino acid sequence of SEQ ID NO: 3 or a fragment thereof; (b) measuring the binding level between the CAP1, and the PCSK9 protein or a fragment thereof, or the resistin protein or a fragment thereof; and (c) selecting a test substance in which the binding level is decreased as compared with a control group sample.

[0061] In one embodiment of the present invention, the measurement of the binding level in step (b) may be performed using any one selected from the group consisting of Yeast two-hybrid, Surface Plasmon Resonance (SPR), immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, Western Blotting, and flow cytometry (FACS), but is not limited thereto.

Advantages of the Invention

[0062] According to the present invention, CAP1 which directly binds to PCSK9 and regulates the life cycle of LDL receptor inhibits the binding of PCSK9 or resistin to CAP1, or suppresses the expression of CAP1 gene, thereby regulating cholesterol levels. Therefore, a binding inhibitor between CAP1 and PCSK9 or resistin according to the present invention, an expression inhibitor of CAP1 gene, etc. can reduce the blood cholesterol level, and thus can be usefully used as a composition for treating various cardiovascular and metabolic diseases associated with or caused by abnormal blood cholesterol levels, and can also exhibit an anti-inflammatory effect through suppression of NF-κB activation.

Brief Description of Drawings

[0063]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0064] By identifying CAP1 as a new binding partner of PCSK9, the inventors of the present invention have expanded the understanding of the endocytosis of LDL receptor by PCSK9.

[0065] First, in one embodiment of the present invention, it was shown that the SH3BD (Src Homology 3 Binding Domain) of CAP1 directly binds to the CRD (cysteine rich domain) of PCSK9 (see Example 1).

[0066] In another embodiment of the present invention, it was confirmed that two loss-of-function polymorphisms discovered in human PCSK9 are defective in their interaction with CAP1 (see Example 2).

[0067] In yet another embodiment of the present invention, it was confirmed that not only siRNA against CAP1 in hepatocytes but also the degradation of PCSK9-mediated LDL receptor is prevented in heterozygous CAP1 knockout mice with low plasma LDL cholesterol levels (see Example 3).

[0068] In yet another embodiment of the present invention, it was demonstrated that CAP1 binds to caveolin-1 and then leads the PCSK9-LDL receptor complex to caveola-dependent endocytosis and lysosomal degradation (see Example 4).

[0069] In yet another embodiment of the present invention, mFc-CAP1 was produced as a competitive inhibitor of CAP1 (see Example 5), and this was treated on peripheral blood mononuclear cells and hepatocytes to confirm the protective effect on LDL receptor and the inhibitory effect on NF-κB related to the regulation of almost all inflammatory reactions in the body (see Examples 6 and 7).

[0070] In yet another embodiment of the present invention, it was confirmed that when CAP1 was knocked down to suppress its expression, the absorption of LDL cholesterol was suppressed (see Example 8).

[0071] Accordingly, the present invention provides a pharmaceutical composition for reducing blood cholesterol, comprising, as an active ingredient, a binding inhibitor between CAP1 (Adenylyl cyclase-associated Protein 1) consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 (proprotein convertase subtilisin / kexin type-9) consisting of the amino acid sequence of SEQ ID NO: 2.

[0072] As used herein, the term "CAP1" means "Adenylyl cyclase-associated Protein 1" and can be divided into three domains in terms of structure and function. First, the highly conserved carboxyl-terminal domain binds to monomeric actin and is essential for general cell morphology. Second, the amino-terminal domain of CAP1 interacts with adenylyl cyclase in yeast. Third, the centrally located proline-rich domain interacts with the SH3 (Src homology 3) domain of specific proteins. The CAP1 according to the present invention may, for example, contain the amino acid sequence represented by SEQ ID NO: 1, consist of the amino acid sequence represented by SEQ ID NO: 1, or consist of an amino acid sequence having a sequence homology of 80% or more, more preferably 90% or more, and even more preferably 95% or more with the amino acid sequence of SEQ ID NO: 1. For example, it may include amino acid sequences having sequence homologies of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% with the amino acid sequence. The "%" of sequence homology to an amino acid sequence is confirmed by comparing the comparison region with two optimally aligned sequences, and a part of the amino acid sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions) for the optimal alignment of the two sequences.

[0073] As used herein, the term "PCSK9" means "proprotein convertase subtilisin / kexin type-9". The human PCSK9 gene is located on chromosome 1p32.3 and has a length of 25,378 bp. It contains 12 exons encoding 692 amino acids. The PCSK9 protein contains a signal peptide, a prodomain, a catalytic domain, and a C-terminal cysteine-histidine-rich domain composed of three modules (M1, M2, and M3). The PCSK9 according to the present invention may, for example, comprise the amino acid sequence represented by SEQ ID NO: 2, consist of the amino acid sequence represented by SEQ ID NO: 2, or consist of an amino acid sequence having a sequence homology of 80% or more, more preferably 90% or more, still more preferably 95% or more with the amino acid sequence of SEQ ID NO: 2. For example, it may include amino acid sequences having sequence homologies of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% with the amino acid sequence. The meaning of "% sequence homology" for an amino acid sequence is as described above.

[0074] In the present invention, the binding inhibitor may be one or more selected from the group consisting of a protein, a peptide, a peptidomimetic, a substrate analog, an aptamer, and an antibody that specifically binds to CAP1 or PCSK9. However, all substances that bind to CAP1 or PCSK9 and can interfere with their interaction are included in the scope of the present invention.

[0075] As used herein, the term "Peptide mimetics" refers to those that bind to the binding domain of CAP1 or PCSK9 and inhibit the binding between CAP1 and PCSK9. The peptide mimetic may be a peptide or a non-peptide, and may be composed of amino acids linked by non-peptide bonds such as psi bonds. Further, it can be a conformationally constrained peptide, a cyclic mimetic, a cyclic mimetic containing at least one exocyclic domain, a binding moiety (binding amino acid) and an active site. The peptide mimetic is structured similarly to the secondary structure characteristics of the CAP1 or PCSK9 protein, can mimic the inhibitory characteristics of large molecules such as antibodies or water-soluble receptors, and can be a novel small molecule that can act with an effect equivalent to that of a natural antagonist.

[0076] As used herein, the term "antibody" means a proteinaceous molecule that can specifically bind to an antigenic site of a protein or peptide molecule, and such an antibody can be produced by cloning each gene into an expression vector by a conventional method to obtain the protein encoded by the marker gene and then producing it from the obtained protein by a conventional method.

[0077] As used herein, the term "aptamer" means a nucleic acid molecule having binding activity to a predetermined target molecule. The aptamer may be RNA, DNA, a modified nucleic acid, or a mixture thereof, and may be in a linear or cyclic form. Generally, the shorter the nucleotide sequence constituting the aptamer, the easier it is for chemical synthesis and mass production, and it has the advantage of being excellent in terms of cost, is easy to chemically modify, is excellent in in vivo stability, and is known to have low toxicity.

[0078] In the present invention, the binding inhibitor may be a fusion protein comprising a CAP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof; and an Fc fragment of an immunoglobulin heavy chain. The CAP1 protein may be a fragment capable of specifically binding to PCSK9, for example, a fragment capable of specifically binding to a cysteine-rich domain corresponding to the amino acid portion from the 421st amino acid to the 629th amino acid of PCSK9 represented by SEQ ID NO: 2. The fragment may be a polypeptide comprising all or part of the SH3 binding domain of the CAP1 protein, and there is no limitation on the length of the polypeptide.

[0079] The Fc fragment may be derived from an immunoglobulin heavy chain of mammals including humans, such as monkeys, orangutans, chimpanzees, mice, dogs, cats, cows, pigs, horses, etc. Preferably, it may be derived from an immunoglobulin heavy chain of humans or mice, but is not limited thereto. The sequence of the Fc fragment can be appropriately modified / altered within the limit that a person with ordinary knowledge in the technical field of the present invention can achieve the purpose of inhibiting the binding of CAP1 in vivo to PCSK9 or resistin, binding to PCSK9 or resistin instead of the in vivo CAP1, and inhibiting the degradation of the LDL receptor regulated thereby.

[0080] Also, the Fc fragment can be bound to the N-terminal portion or preferably the C-terminal portion of the CAP1 protein, and can be directly bound or indirectly linked through a peptide linker or hinge widely known in the technical field of the present invention.

[0081] In the present invention, the fusion protein may be a CAP1 protein fused with an Fc fragment of a human immunoglobulin heavy chain or an Fc fragment of a mouse immunoglobulin heavy chain, respectively. At this time, the CAP1 protein fused with the Fc fragment of the human immunoglobulin heavy chain may consist of the amino acid sequence of SEQ ID NO: 4 or may be encoded by the nucleotide sequence of SEQ ID NO: 5. Further, the CAP1 protein fused with the Fc fragment of the mouse immunoglobulin heavy chain may consist of the amino acid sequence of SEQ ID NO: 6 or may be encoded by the nucleotide sequence of SEQ ID NO: 7. The fusion protein also includes functional equivalents of the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 6 within the scope of the present invention. The functional equivalent means a polypeptide having at least 60% or more, preferably 70% or more, more preferably 80% or more, and most preferably 90% or more sequence homology with the amino acid sequence as a result of addition, substitution, or deletion of amino acids, and having substantially the same activity as the amino acid sequence represented by SEQ ID NO: 4 or SEQ ID NO: 6. It means a polypeptide that can specifically bind to PCSK9 and is not limited to this as long as it has an amino acid sequence that can specifically bind to PCSK9.

[0082] The inventors of the present invention produced a fusion protein (Fc-CAP1) in which an Fc fragment of an immunoglobulin heavy chain was conjugated to a human CAP1 protein, directly observed the protective effect on the LDL receptor, and thereby confirmed that the Fc-CAP1 can reduce the blood LDL cholesterol level.

[0083] In the present invention, the binding inhibitor may bind to one or more domains selected from the group consisting of the SH3 (Src homology 3) binding domain of CAP1 and the cysteine rich domain (CRD) of PCSK9.

[0084] At this time, the SH3 binding domain of CAP1 with which the binding inhibitor interacts may consist of the amino acid sequence of SEQ ID NO: 10, but is not limited thereto. For example, the amino acid sequence may specifically bind to an amino acid site having a length of 3 to 250, 3 to 200, 3 to 150, 3 to 100, 3 to 50, 3 to 25, 3 to 10, 3 to 7, or 3 to 5 that includes Asp34B of the SH3 binding domain.

[0085] In the present invention, the binding inhibitor may specifically bind to a site containing Asp34B present in the SH3 binding domain of CAP1. For example, it may specifically bind to a site containing Asp34B within the SH3 binding domain of CAP1 and inhibit the binding to PCSK9.

[0086] Also, in the present invention, the CRD of the PCSK9 may consist of the amino acid sequence of SEQ ID NO: 11. The CRD includes the M1 domain consisting of the amino acid sequence of SEQ ID NO: 12 or the M3 domain consisting of the amino acid sequence of SEQ ID NO: 13.

[0087] Also, in the present invention, the binding inhibitor may specifically bind to a site containing lysine 494 present in the M1 domain of PCSK9. For example, it may specifically bind to a site containing the 494th amino acid of the amino acid sequence represented by SEQ ID NO: 2 and inhibit the binding to PCSK9.

[0088] At this time, the M1 domain of PCSK9 to which the binding inhibitor binds may include the amino acid sequence of SEQ ID NO: 12, but is not limited thereto. For example, it may specifically bind to an amino acid site having a length of 3 to 70, 3 to 60, 3 to 50, 3 to 40, 3 to 30, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 7, or 3 to 5 that includes the 42nd amino acid of the amino acid sequence (or the 494th amino acid of the amino acid sequence represented by SEQ ID NO: 2).

[0089] In the present invention, the binding inhibitor may specifically bind to a site containing arginine at position 659 present in the M3 domain of PCSK9. For example, it may specifically bind to a site containing the 659th amino acid of the amino acid sequence represented by SEQ ID NO: 2 and inhibit the binding to PCSK9.

[0090] At this time, the M3 domain of PCSK9 to which the binding inhibitor binds may include the amino acid sequence of SEQ ID NO: 13, but is not limited thereto. For example, it may specifically bind to an amino acid site having a length of 3 to 85, 3 to 75, 3 to 65, 3 to 55, 3 to 45, 3 to 35, 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 7 or 3 to 5, including the 56th amino acid of the amino acid sequence (or the 659th amino acid of the amino acid sequence represented by SEQ ID NO: 2).

[0091] In another aspect of the present invention, the present invention provides a pharmaceutical composition for reducing blood cholesterol, comprising, as an active ingredient, an inhibitor of the expression of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0092] In still another aspect of the present invention, the present invention provides a health functional food composition for reducing blood cholesterol, comprising (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) an inhibitor of the expression of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; or (iii) a mixture of (i) and (ii).

[0093] In the present invention, the inhibitor of the expression of the CAP1 gene may be one or more selected from the group consisting of antisense nucleotides, siRNA, shRNA, miRNA, ribozymes, and PNA that can complementarily bind to the mRNA of the CAP1 gene, but is not limited thereto.

[0094] In the present invention, "expression suppression" means including transcriptional suppression and translational suppression of the CAP1 gene. Also, it includes not only those in which the gene expression has completely stopped but also those in which the expression has decreased.

[0095] As used herein, the terms "siRNA, shRNA, and miRNA" mean nucleic acid molecules that mainly bind to mRNA transcribed from a target gene to inhibit the translation of the mRNA in order to mediate RNA interference or gene silencing. Since the miRNA, siRNA, and shRNA can suppress the expression of a target gene at the translational level, they can be used in an efficient gene knockdown method or gene therapy method.

[0096] As used herein, the term "antisense oligonucleotide" means DNA or RNA or derivatives thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, but can exhibit the effect of binding to the complementary sequence in the mRNA to inhibit the translation of the mRNA into protein.

[0097] As used herein, the term "ribozyme" can suppress the protein expression of a target gene by recognizing a specific nucleotide sequence within a target RNA molecule and cleaving it site-specifically.

[0098] As used herein, the term "PNA" means a nucleic acid mimic, for example, a DNA mimic, where the deoxyribose phosphate backbone is replaced with a pseudopeptide backbone and only the original four nucleobases are maintained. The neutral backbone of PNA is known to provide hybrids specific for DNA and RNA under conditions of low ionic strength, and can be used as an antisense or antigenic agent for inducing transcriptional or translational suppression or replication suppression for sequence-specific regulation of gene expression.

[0099] In the present invention, the expression inhibitor may be siRNA consisting of the nucleotide sequence of SEQ ID NO: 8, shRNA consisting of the nucleotide sequence of SEQ ID NO: 9, or a mixture thereof. However, the siRNA sequence or shRNA sequence can be appropriately modified / altered and used within the limit that a person having ordinary knowledge in the technical field of the present invention can achieve the purpose of suppressing (or knocking down) the expression of the CAP1 gene.

[0100] In the present invention, the cholesterol may be LDL cholesterol, but is not limited thereto. For example, it may be total cholesterol including LDL cholesterol.

[0101] In the present invention, the composition may be one that suppresses the degradation of the LDL (Low-density lipoprotein) receptor. Suppressing the expression or activity of CAP1 suppresses the degradation of the LDL receptor by PCSK9, and as a result, has the effect of reducing the blood LDL cholesterol level.

[0102] In another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cardiovascular and metabolic diseases or a health functional food composition for preventing or improving cardiovascular and metabolic diseases, comprising (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1; or (iii) a mixture of (i) and (ii).

[0103] In the present invention, the binding inhibitor may be one or more selected from the group consisting of a protein, peptide, peptidomimetic, substrate analog, aptamer, and antibody that specifically binds to CAP1 or PCSK9, but is not limited thereto.

[0104] In the present invention, the binding inhibitor may be a fusion protein comprising a CAP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof; and an Fc fragment of an immunoglobulin heavy chain. Matters regarding the fusion protein are as described above.

[0105] In the present invention, the expression inhibitor may be one or more selected from the group consisting of antisense nucleotides, siRNAs, shRNAs, miRNAs, ribozymes, and PNAs that can bind complementarily to the mRNA of the CAP1 gene, but is not limited thereto.

[0106] In the present invention, the expression inhibitor may be an siRNA consisting of the nucleotide sequence of SEQ ID NO: 8, an shRNA consisting of the nucleotide sequence of SEQ ID NO: 9, or a mixture thereof, but is not limited thereto.

[0107] In the present invention, the cardiovascular and metabolic disease may be a disease selected from the group consisting of diabetes, obesity, dyslipidemia, fatty liver, hypertension, gout, stroke, arteriosclerosis, myocardial infarction, angina pectoris, peripheral vascular disease, and combinations thereof, but is not limited thereto as long as it is an abnormality in blood cholesterol levels or a disease caused thereby.

[0108] In yet another aspect of the present invention, the present invention provides a pharmaceutical composition for anti-inflammatory use, comprising as an active ingredient one or more selected from the group consisting of: (i) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2; (ii) a binding inhibitor between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and resistin consisting of the amino acid sequence of SEQ ID NO: 3; and (iii) an expression inhibitor of the CAP1 gene consisting of the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

[0109] In the present invention, the binding inhibitor of (i) may be one or more selected from the group consisting of proteins, peptides, peptidomimetics, substrate analogs, aptamers, and antibodies that specifically bind to PCSK9, and the binding inhibitor of (ii) may be one or more selected from the group consisting of proteins, peptides, peptidomimetics, substrate analogs, aptamers, and antibodies that specifically bind to resistin, but is not limited thereto.

[0110] In the present invention, the binding inhibitor of (i) or (ii) may be a CAP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof; and a fusion protein containing the Fc fragment of an immunoglobulin heavy chain. The fusion protein may consist of the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, but is not limited thereto.

[0111] In the present invention, the expression inhibitor of (iii) may be one or more selected from the group consisting of antisense nucleotides, siRNA, shRNA, miRNA, ribozymes, and PNA that can bind complementarily to the mRNA of the CAP1 gene, but is not limited thereto.

[0112] In the present invention, the composition can suppress the activity of NF-κB.

[0113] On the other hand, the pharmaceutical composition according to the present invention may further contain, in addition to the active ingredient, appropriate carriers, excipients, and / or diluents usually used for manufacturing the pharmaceutical composition. Also, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions by ordinary methods and used.

[0114] Examples of the carriers, excipients, and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating the composition, it can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants usually used.

[0115] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined by factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route and excretion rate, the treatment period, the drugs used concomitantly, and other factors well known in the medical field.

[0116] It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects considering all the above-mentioned factors, and this can be determined by those skilled in the art. Specifically, the effective amount of the pharmaceutical composition according to the present invention can vary depending on the patient's age, gender, condition, weight, absorption degree, inactivation rate and excretion rate of the active ingredient in the body, the type of disease, and the drugs used concomitantly.

[0117] The pharmaceutical composition of the present invention can be administered to an individual by various routes. For example, it can be administered by oral administration, intranasal administration, transbronchial administration, arterial injection, intravenous injection, subcutaneous injection, intramuscular injection or intraperitoneal injection. The daily dosage can be administered once a day or divided into several times a day.

[0118] As used herein, the term "individual" means a subject in need of disease prevention, treatment, treatment enhancement or resistance suppression. For example, the individual can be a human or a mammal including non-human primates, mouse, dog, cat, horse, sheep and cow.

[0119] In the present invention, "prevention" means all actions that suppress or delay the onset of the target disease, "treatment" means all actions that improve or beneficially change the target disease and the metabolic disorder symptoms caused thereby by the administration of the pharmaceutical composition according to the present invention, and "improvement" means all actions that reduce the parameters related to the target disease, such as the degree of symptoms, by the administration of the composition according to the present invention.

[0120] In the present invention, the composition according to the present invention can be manufactured as a food composition. The food composition can add the active ingredient directly to the food, or can be used together with other foods or food ingredients, and can be appropriately used by ordinary methods. The mixing amount of the active ingredient can be preferably determined according to its purpose of use (for prevention or improvement).

[0121] Except for containing the active ingredient as an essential component at the indicated ratio, other components of the food composition are not particularly limited, and like ordinary beverages, various flavoring agents or natural carbohydrates can be contained as additional components. Examples of the above-mentioned natural carbohydrates are ordinary sugars such as monosaccharides, for example, glucose, fructose, etc.; disaccharides, for example, maltose, sucrose, etc.; and polysaccharides, for example, dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents other than those mentioned above, natural flavoring agents (such as thaumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (such as saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrates can be appropriately determined by the selection of those skilled in the art.

[0122] In addition to the above, the food composition of the present invention can contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and enhancers (such as cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Such components can be used independently or in combination. Also, the ratio of such additives can be appropriately selected by those skilled in the art.

[0123] The food composition of the present invention may include functional foods for health. As used herein, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials and ingredients having functional properties useful to the human body. Here, functionality means obtaining effects useful for health purposes such as regulating nutrients with respect to the structure and function of the human body or physiological actions. The health functional food of the present invention can be manufactured by methods commonly used in the art, and during the manufacture, raw materials and ingredients commonly added in the art can be added for manufacture. Also, different from general drugs, it has the advantage of having no side effects that may occur during long-term use of drugs using food as a raw material, and is excellent in portability. The health functional food of the present invention can be ingested as an adjuvant for enhancing the anti-metabolic disease effect.

[0124] The health functional food of the present invention contains a binding inhibitor between CAP1 and PCSK9 or resistin or an expression inhibitor of the CAP1 gene, and may further contain an appropriate food adjuvant.

[0125] In still another aspect of the present invention, the present invention provides a diagnostic composition for hypercholesterolemia or cardiometabolic disease, comprising a preparation for measuring the binding level between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2 or resistin consisting of the amino acid sequence of SEQ ID NO: 4. In this specification, when the binding between CAP1 and PCSK9 increases, the degradation of the LDL receptor increases, and the concentration of cholesterol in the blood increases. Conversely, when the binding between CAP1 and resistin is inhibited by Fc-CAP1, the degradation of the LDL receptor decreases, and the concentration of cholesterol in the blood decreases. When the binding level between CAP1 and PCSK9 or the binding level between CAP1 and resistin is measured and a higher binding level is shown compared to the normal control group, it can be diagnosed that there is hypercholesterolemia or cardiometabolic disease.

[0126] In yet another aspect of the present invention, the present invention provides a method for diagnosing hypercholesterolemia or cardiometabolic disease or providing information for diagnosis, comprising measuring the binding level between CAP1 consisting of the amino acid sequence of SEQ ID NO: 1 and PCSK9 consisting of the amino acid sequence of SEQ ID NO: 2 or resistin consisting of the amino acid sequence of SEQ ID NO: 3 in a sample of a patient.

[0127] In the present invention, the sample of the patient may be selected from the group consisting of liver tissue, hepatocytes, blood, serum, plasma, saliva, sputum and urine, but is not limited thereto.

[0128] In yet another aspect of the present invention, the present invention provides a method for screening a therapeutic agent for hypercholesterolemia or cardiometabolic disease, comprising: (a) treating a sample containing CAP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a fragment thereof; and PCSK9 protein consisting of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, or resistin protein consisting of the amino acid sequence of SEQ ID NO: 3 or a fragment thereof, with a test substance; (b) measuring the binding level between the CAP1 and the PCSK9 protein or a fragment thereof, or the resistin protein or a fragment thereof; and (c) selecting a test substance in which the binding level is decreased as compared with a control group sample.

[0129] In the present invention, the measurement of the binding level in step (b) may be performed using any one selected from the group consisting of the yeast two-hybrid method, surface plasmon resonance (SPR), immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, Western blotting and flow cytometry (FACS). However, as long as the binding level between CAP1 and PCSK9 can be measured, it is not limited thereto.

[0130] The terms and words used in this specification and claims should not be construed as being limited to their ordinary or preconceived meanings. Based on the principle that the inventor can appropriately define the concepts of the terms in order to explain his invention in the best possible way, they must be construed as meanings and concepts that conform to the technical idea of the present invention.

[0131] Hereinafter, preferred embodiments will be presented to assist in understanding the present invention. However, the following embodiments are provided to more easily understand the present invention, and the content of the present invention is not limited by the following embodiments.

Embodiment

[0132] [Example 1. CAP1 binds directly to the CRD (cysteine rich domain) of PCSK9] The physical interaction between CAP1 and PCSK9 was confirmed using immunoprecipitation in mouse liver tissue (Figure 1a). For far-western blot analysis, purified mFc-CAP1 or His-PCSK9 under non-reducing conditions was used as prey, and His-PCSK9 or mFc-CAP1 was used as bait for each (Figure 1b). To visualize the above interaction in living cells, a bimolecular fluorescence complementation assay based on the complementarity between two non-fluorescent fragments of fluorescent proteins that come together by protein-protein interactions fused to each fragment was performed. Through this, the direct binding between hCAP1 and hPCSK9 was clearly visualized. As shown in Figure 1c, when CAP1 and PCSK9 were fused to their respective fragments (pVC155 and pVN173) and both were expressed, green fluorescence was observed (left panel). However, when human CAP1 or PCSK9 was expressed alone, no fluorescence was detected (middle and right panels respectively).

[0133] Moreover, when recombinant PCSK9 was treated with HepG2 cells through immunofluorescent staining, it was demonstrated that PCSK9 was localized with CAP1 and LDL receptor in the cell membrane and cytosol (Figure 1d). Finally, as a result of direct binding analysis using a surface plasmon resonance-based system, it was confirmed that the response unit between CAP1 and PCSK9 increased in a dose-dependent manner of PCSK9 (Figure 1e).

[0134] Furthermore, the inventors tested whether the CRD of PCSK9 binds to the SH3BD (Src Homology 3 Binding Domain) of CAP1. For this purpose, in vitro co-immunoprecipitation analysis was performed using wtPCSK9-Flag and the following CAP1 mutants: adenylyl cyclase binding domain deletion (ΔACBD) mutant, actin binding domain deletion (ΔActinBD) mutant, and SH3BD and ActinBD deletion (ΔSH3BD ΔActinBD) mutant.

[0135] As a result, as shown in Figure 1f, PCSK9 interacted with wtCAP1, ΔACBD mutant, and ΔActinBD mutant, but did not interact with the ΔSH3BD ΔActinBD mutant, suggesting that CAP1 binds to PCSK9 through SH3BD. Also, as shown in Figure 1g, it was confirmed that the SH3BD of CAP1 is sufficient for interaction with PCSK9. CAP1 bound to wtPCSK9, but not to CRD-deleted PCSK9 (Figure 1h).

[0136] Furthermore, as shown in Figure 1i, through 3D molecular modeling analysis of the complex using protein-protein docking simulation and binding energy score analysis, it was confirmed that the interaction between Asp34B present in the SH3BD of CAP1 and Lys494 present in the M1 domain of PCSK9-CRD and Arg659 present in the M3 domain is important.

[0137] [Example 2. PCSK9 loss-of-function mutants cannot interact with CAP1] The inventors generated eight PCSK9 point mutants by site-directed mutagenesis of PCSK9-CRD, including well-known loss-of-function and gain-of-function mutations discovered in human gene studies (Figure 2a; arrows indicate the sites where point mutations were induced). The Q544E and H683fs mutants of PCSK9 were not expressed in HepG2 cells, suggesting that these mutants are related to protein expression or stability.

[0138] In contrast, as shown in Figure 2b, the S668R and G670E mutants of PCSK9 CRD were well-expressed, but it was confirmed by immunoprecipitation that their binding ability to CAP1 was severely damaged. Such results suggest that the CRD of PCSK9 is important for binding to CAP1 and is important for regulating the LDL receptor protein, i.e., the level of LDL cholesterol.

[0139] To further investigate the additional characteristics of the direct interaction between PCSK9-CRD mutations and CAP1, the inventors measured the binding affinity of PCSK9 WT, PCSK9 A514T, PCSK9 G670E, and PCSK9 S668RG670E to CAP1 using the BLItz system (Molecular Devices, LLC., USA). As a result, as shown in Figure 2c, PCSK9 A514T showed a stronger interaction compared to WT (wild type), while the G670E and G670ES668R mutants showed a weaker interaction.

[0140] [Example 3. CAP1 induces PCSK9-mediated degradation of LDL receptor and increases the level of LDL cholesterol] [(3.1. Study on the effect of suppressing CAP1 expression using siRNA)] Treatment with PCSK9 in HepG2 cells promotes the degradation of LDL receptors in a dose-dependent manner. To investigate the effect of CAP1 expression suppression on LDL receptor degradation, the present inventors suppressed the expression of the CAP1 gene using siRNA. The siRNA sequences are as follows:

[0141] CAP1 siRNA, 5′-AAACCGAGTCCTCAAAGAGTA-3′ (SEQ ID NO: 8).

[0142] As a result, as shown in Fig. 3a, when CAP1 was depleted by siRNA (siCAP1), the degradation of LDL receptors by exogenous PCSK9 was significantly reduced. Also, the results shown in Fig. 3b demonstrate that after treating His-rhPCSK9 in the cytoplasmic matrix of cells treated with CAP1 siRNA for 30 or 60 minutes, both LDL receptors and exogenous His-tagged PCSK9 were detected less.

[0143] The present inventors rescued CAP1-deficient cells (siCAP1) having wtCAP1 or each CAP1 mutant by overexpression and investigated PCSK9-mediated LDL receptor degradation. As a result, as shown in Fig. 3c, only wtCAP1 and the ΔactinBD mutant restored the attenuated PCSK9-mediated LDL receptor degradation, suggesting that SH3BD and ACBD are important for exogenous PCSK9-mediated LDL receptor degradation.

[0144] (3.2. Study on the effect of CAP1 on LDL receptor degradation using CAP1 knockout mice) To investigate the role of CAP1 in vivo, CAP1 knockout mice targeting exon 3 of CAP1 were generated using TALEN (Transcription activator-like effector nuclease). Since homozygous knockout mice died on embryonic day 16.5, heterozygous knockout mice (CAP1 + / - mice) were used. CAP1 + / -The organs of the mouse are indistinguishable from those of wild-type mice until approximately 16 weeks of age, but the mRNA and protein levels of CAP1 + / - were shown to be significantly decreased in various organs of the mouse (Figure 3d). Accordingly, the present inventors compared the expression levels of LDL receptor and PCSK9 in the liver of CAP1 + / - mice with or without a high-fat diet and CAP1 + / + mice. Although no significant difference was confirmed in the investigation of mRNA expression of LDL receptor through rt-PCR, the protein level of LDL receptor was significantly higher in CAP1 + / + mice than in CAP1 + / - mice (Figure 3e). From the above results, it was confirmed that CAP1 + / - mice had even lower total cholesterol and LDL cholesterol values compared to wild-type mice (Figure 3f). There was no significant difference in the levels of plasma triglyceride (TG) and high-density lipoprotein (HDL) cholesterol.

[0145] Next, the present inventors fractionated plasma lipoproteins by fast protein liquid chromatography (FPLC) and confirmed that the levels of LDL cholesterol and VLDL cholesterol decreased and HDL cholesterol shifted to a large-buoyant form in CAP1 + / + mice compared to CAP1 + / - mice fed a high-fat diet (Figure 3g).

[0146] Subsequently, the present inventors used CAP1 + / - and CAP1 + / +After overexpressing PCSK9 using adeno-associated virus in mice, the expression levels of LDL receptor and the levels of LDL cholesterol were measured. As a result, as shown in FIGS. 3h to 3j, in CAP1 heterozygous knockout mice, the decrease or degradation of LDL receptor protein due to transduction of PCSK9 was prevented, showing an improved cholesterol profile compared to wild-type animals. Such results indicate that the CAP1 protein is essential for the degradation of LDL receptor protein by PCSK9.

[0147] [Example 4. CAP1 induces caveola-dependent endocytosis of the PCSK9-LDL receptor complex and causes degradation of the LDL receptor] The PCSK9-mediated degradation of the LDL receptor was exclusively blocked by the lysosomal protease inhibitor (E-64d), but not by the inhibitors of proteasome (lactacystin) or autophagy (bafilomycin), suggesting that the LDL receptor is degraded by the lysosomal pathway as previously reported. Such findings were also explained by tracking the LDL receptor with PCSK9-Cy3, the endosome marker EEA1 (early endosome antigen 1), and the lysosome marker LAMP2 (lysosome-associated membrane protein 2) in HepG2 cells treated with PCSK9 conjugated with Cy3 dye (PCSK9-Cy3) at various time points. That is, within 30 minutes after PCSK9-Cy3 treatment, EEA1 was co-localized with PCSK9 and the LDL receptor (FIG. 4a). Then, LAMP2, which was co-localized with PCSK9 and the LDL receptor, appeared within 60 minutes (FIG. 4b). This increased until 240 minutes when PCSK9 and the LDL receptor disappeared (FIGS. 4a and 4c). Such lysosome formation without initial endosome formation was blocked by CAP1 depletion (FIG. 4d).

[0148] In the raft separation experiment, endogenous PCSK9 before exogenous PCSK9 treatment was more distributed in the non-raft fraction [raft (36.8%) / non-raft (63.2%)]. Within 30 minutes after exogenous PCSK9 treatment, it was mainly included in the lipid raft fraction [raft (54.3%) / non-raft (45.7%)], which is closely related to the formation of caveolae (Figure 4e and Figure 4f). The expression of LDL receptor in the membrane fraction decreased 60 minutes after PCSK9 treatment. However, despite treating CAP1-deficient cells with PCSK9, no significant changes appeared in the expression patterns of PCSK9 and LDL receptor (Figure 4e and Figure 4f).

[0149] The initial endosome formation by PCSK9 is known to be mediated by the clathrin pathway. However, PCSK9 treatment increased the number of endosomes localized with not only clathrin but also caveolin (Figure 4g). Interestingly, in relation to the enhanced endosome formation, knockdown of CAP1 after PCSK9 treatment decreased caveolin-endosomes but not clathrin-endosomes, and such results suggest that CAP1 induces caveolin-mediated endocytosis rather than clathrin-mediated endocytosis of the PCSK9-LDL receptor complex (Figure 4h and Figure 4i).

[0150] Therefore, caveolin or clathrin was knocked down to compare caveolin- and clathrin-mediated LDL receptor endocytosis by PCSK9 treatment, respectively. As a result, as shown in Figure 4j, the LDL receptor was not degraded in caveolin-deficient cells despite PCSK9 treatment. In comparison, as shown in Figure 4k, it was confirmed that the LDL receptor was degraded in a dose-dependent manner by PCSK9 treatment in clathrin-deficient cells, and such results suggest that PCSK9-mediated LDL receptor degradation is clathrin-independent.

[0151] In the absence of caveolin, LAMP2 could not be formed and the LDL receptor was not degraded by PCSK9. In contrast, in the absence of clathrin, LAMP2 appeared and the LDL receptor was degraded (Figure 4l). Also, knockdown of CAP1 could not produce LAMP2, suggesting that CAP1 is closely related to caveolin-mediated degradation of the LDL receptor by PCSK9 (Figure 4l).

[0152] Next, the inventors evaluated the effect of CAP1 deficiency on the endocytosis of EGF or albumin. EGF and its receptor complex are mainly internalized by clathrin-dependent endocytosis, whereas albumin uptake is known to depend on caveolae. As a result, as shown in Figure 4m, caveolin-dependent albumin endocytosis was significantly decreased by CAP1 deficiency, while clathrin-dependent EGF receptor endocytosis was not affected at all.

[0153] Such observations suggest that CAP1 can be involved not only in the endocytosis of the PCSK9-LDL receptor complex but also in general caveolin-dependent endocytosis. Also, as shown in Figure 4n, the degradation of the LDL receptor mediated by endogenously overexpressed PCSK9 was attenuated by siRNA against CAP1 or caveolin. Electron microscopy analysis also showed that only the formation of non-clathrin caveosomes was significantly weakened by CAP1 siRNA after PCSK9 treatment (Figure 4o). The mechanism by which CAP1 induces the PCSK9-LDL receptor complex into caveosomes is based on the binding of the AC-domain of CAP1 to caveolin-1. In an immunoprecipitation experiment, PCSK9 containing the LDL receptor could form a complex with wtCAP1 and caveolin-1, but it was impossible in the presence of mutant CAP1 such as ΔSH3BD ΔactinBD or ΔACBD CAP1 (Figure 4p). Such results prove that the SH3BD of CAP1 is essential for the binding to the CRD of PCSK9.

[0154] In addition, the ActinBD of CAP1 is required for binding to caveolin. As shown in Fig. 4q, immunoprecipitation analysis of liver lysates from wild-type mice showed that caveolin binds to the LDL receptor, CAP1, and PCSK9. Furthermore, the LDL receptor-PCSK9-CAP1 complex was localized with caveolin in the mouse liver. One hour after treatment with LDL cholesterol, LDL receptor endocytosis, which was stained with the early endosome marker Rab5, was significantly decreased by both clathrin siRNA and caveolin siRNA. When PCSK9 was blocked in the siRNA-depleted LDL receptor degradation pathway, caveolin-mediated LDL receptor endocytosis was significantly decreased, whereas there was no significant change in clathrin-mediated endocytosis. Moreover, 4 hours after LDL cholesterol treatment, when Rab11, which is related to the recycling mechanism, was co-stained with the LDL receptor, the amount of recycled LDL receptor was significantly decreased when treated with clathrin siRNA compared with when treated with caveolin siRNA (Fig. 4r).

[0155] Summarizing the results demonstrated through Examples 1 to 4 above, as shown in the schematic diagram of Fig. 4s, the LDL receptor binds to LDL cholesterol, enters the cell by clathrin-dependent endocytosis, and then, the acidic pH of the endosome induces allosteric dissociation, and it is regenerated on the cell surface. PCSK9 also promotes clathrin-dependent endocytosis but does not cause lysosomal degradation of the LDL receptor. However, when the complex of the LDL receptor and PCSK9 interacts with CAP1, since CAP1 can bind to caveolin-1 through the actin-binding domain, these proteins enter the cell by caveolin-dependent endocytosis. Subsequently, the caveolin-coated endosome containing the LDL receptor-PCSK9-CAP1 complex is induced in the lysosome for degradation. That is, CAP1 was the first to clarify that it is an essential molecule that mediates caveolin-dependent endocytosis and lysosomal degradation of the LDL receptor as a binding partner of PCSK9.

[0156] [Example 5. Production of the competitive inhibitor Fc-CAP1 of CAP1] hFc-CAP1 according to the present invention was prepared by commissioning a protein synthesizer according to the amino acid sequence of SEQ ID NO: 4. For the expression of mFc-CAP1, the pCEP4 expression vector was used.

[0157] The culture of Expi293 cells used for protein purification was used with a partial modification of the culture method of the manufacturer (Thermo Scientific, USA). More specifically, 0.5X antibiotic-antimycotic (Gibco, 15240-062, USA) was added to FreeStyle medium and cultured in a shaker maintained at a temperature of 37 °C, a CO2 partial pressure of 7%, and 140 RPM. When transfecting the mFc-CAP1 plasmid, 1 × 10 6300 ml was cultured with the cell count. The next day, a transfection mixture [30 ml of 150 mM NaCl + 600 μg (2 μg / ml) of mFc-CAP1 plasmid + 1200 μg of PEI] was prepared, cultured at room temperature for 30 minutes, and then added to the cells in a drop wise manner. On the 7th day from the day of transfection, the cells and the medium were centrifuged at 3,000 rpm for 10 minutes, and the supernatant was collected. The collected supernatant was concentrated and then filtered using a column and mFc beads (CaptureSelect TM IgG-Fc, ms), and sequentially separated from the beads using 0.1 M glycine (pH 2.8). Thereafter, the degree of protein expression was confirmed by western blot, and the buffer was dialyzed with PBS using a column (Zeba TM Spin Desalting Columns).

[0158] The mFc-CAP1 separated and purified as described above was used as a binding inhibitor (or suppressor) between CAP1 and resistin or CAP1 and PCSK9 in Examples 6 to 8 below.

[0159] [Example 6. mFc-CAP1 suppresses the activation of the NF-κB p65 subunit in peripheral blood mononuclear cells] The human monocytic cell line THP-1 cells were cultured in RPMI medium containing 1X antibiotic-antimycotic (Gibco, 15240-062, USA) and 10% FBS (fetal bovine serum) in an incubator maintained at a temperature of 37°C and a CO2 partial pressure of 5% according to the culture method of ATCC (American Type Culture Collection, USA). Thereafter, after diluting the THP-1 cells in RPMI medium, 1×10 6 cells were uniformly added per well and cultured in an incubator at 37°C and 5% CO2 for 24 hours.

[0160] To maximize the effect on THP-1 cells, the cells were cultured in 0.1% FBS RPMI at 37°C and 5% CO2 for 16 hours to induce cell starvation. Then, 1 μg / ml of recombinant human (rh) PCSK9 or 50 ng / ml of recombinant human resistin (rhResistin) was mixed with mFc-CAP1 at each concentration (0.1, 0.5, 2 μg / ml) and pre-cultured for 30 minutes, and then this was treated with THP-1 cells for 30 minutes. Thereafter, proteins were separated from THP-1 cells using lysis buffer (CST, #9803) and confirmed by Western blot.

[0161] As a result, as shown in Figure 5a, treatment with resistin in THP-1 phosphorylates the S276 site of the p65 subunit, inducing the activation of NF-κ, but in the group co-treated with mFc-CAP1, it was confirmed that the phosphorylation of p65 was suppressed in a concentration-dependent manner. In the case of PCSK9, also, as shown in Figure 5b, treatment with PCSK9 in THP-1 phosphorylates the S276 site of the p65 subunit, and it was confirmed that when co-treated with mFc-CAP1, the phosphorylation of p65 was suppressed in a concentration-dependent manner. The above results mean that the induction of inflammation by the activation of NF-κB by PCSK9 is effectively inhibited by mFc-CAP1.

[0162] [Example 7. In hepatocytes, mFc-CAP1 suppresses the degradation of LDL receptor and the activation of NF-κB, and promotes the activation of the AMPK pathway] (7.1. Culture of HepG2 human liver cancer cells) HepG2 cells were cultured in a incubator maintained at 37 °C and 5% CO2 partial pressure with DMEM (Dulbecco's Modified Eagle's medium, high glucose) medium containing 1X antibacterial - antifungal agent (Gibco) and 10% FBS according to the culture method of ATCC. Thereafter, after diluting HepG2 cells in DMEM medium, 5×10 5 cells were evenly added per well and cultured in an incubator at 37 °C and 5% CO2 environment for 24 hours.

[0163] (Confirmation of the protective effect of 7.2.mFc - CAP1 on PCSK9 - mediated LDL receptor and the activation effect on AMPK pathway) HepG2 cells were cultured in a 37 °C and 5% CO2 incubator with basic (basal) DMEM for 6 hours. Thereafter, the cells were simultaneously treated with 2 μg / ml recombinant human PCSK9 and mFc - CAP1 at each concentration (0.1, 1 μg / ml or 0.05, 0.15, 0.5 μg / ml) for 4 hours. Thereafter, proteins were separated from HepG2 cells using cell lysis buffer (CST, #9803), electrophoresed, transferred to a PVDF membrane (polyvinylidene fluoride membrane; Millipore, USA), and then reacted with each antibody.

[0164] As a result, as shown in FIGS. 6a and 6b, when HepG2 cells were treated with PCSK9 alone, the degradation of LDL receptor was promoted by PCSK9, and when mFc - CAP1 was treated simultaneously, it was confirmed that the protective effect on LDL receptor appeared in a concentration - dependent manner. Also, when treated with mFc - CAP1, AMPK was effectively phosphorylated, and thereby, it was confirmed that the phosphorylation of ACC was suppressed.

[0165] The above results directly show that mFc - CAP1 according to the present invention can successfully suppress LDL receptor degradation through AMPK activation, and thus has a protective effect on LDL receptor for reducing the blood LDL cholesterol value.

[0166] (Confirmation of the protective effect of mFc-CAP1 on resistin-mediated LDL receptor and NF-κ inhibition effect) HepG2 cells were treated with 50 ng / ml of rh resistin and mFc-CAP1 at each concentration (0.1, 1 μg / ml or 0.05, 0.15, 0.5 μg / ml), and cultured in a 37 °C, 5% CO2 incubator for 12 - 16 hours. Thereafter, proteins were obtained by the same method as in Example 7.2 above, electrophoresed, and then reacted with specific antibodies respectively.

[0167] As a result, as shown in FIGS. 6c and 6d, when mFc-CAP1 and rh resistin were co-treated overnight, it was shown that the degradation level of the LDL receptor by rh resistin was relatively mild. Also, p-p65 activated by resistin decreased by the treatment of mFc-CAP1, while it was confirmed that pAMPK was activated by the mFc-CAP1 treatment.

[0168] The above results confirmed that mFc-CAP1 according to the present invention can suppress resistin-induced LDL receptor degradation, which is due to the suppression of phosphorylation of p65 and the activation of AMPK, and proved that the competitive inhibitor of CAP1 has the protective effect of the LDL receptor for reducing the value of blood LDL cholesterol.

[0169] (Confirmation of the protective effect of mFc-CAP1 on resistin-mediated LDL receptor and the activation effect of the AMPK pathway) After changing the cultured HepG2 cells to basic DMEM medium, they were cultured in a 37 °C, 5% CO2 incubator for 6 hours. After 6 hours, AICAR (5-aminoimidazole-4-carboxamide riboside), an AMPK activator, was pretreated for 1 hour, and then 50 ng / ml of rh resistin and mFc-CAP1 at each concentration (0.05, 0.5 μg / ml) were co-treated for 4 hours.

[0170] As a result, as shown in Fig. 6e, the activation of AMPK by AICAR was suppressed by resistin, and it was confirmed that this was further activated by the treatment with mFc-CAP1.

[0171] Also, after changing the culture medium of HepG2 cells during culture to basic DMEM medium, the cells were cultured in a 37°C, 5% CO2 incubator for 6 hours, and when treated simultaneously with 50 ng / ml of rh resistin and mFc-CAP1 at each concentration (0.1, 1 μg / ml or 0.05, 0.15, 0.5 μg / ml), as shown in Fig. 6f, it was confirmed that the degradation of the LDL receptor promoted by the treatment with rh resistin alone was suppressed by the simultaneous treatment with mFc-CAP1.

[0172] [Example 8. Knock-down of CAP1 in human umbilical vein endothelial cells (HUVEC) inhibits the absorption of LDL cholesterol] To directly confirm the LDL cholesterol absorption inhibitory effect of mFc-CAP1 according to the present invention, the inventors suppressed the expression of the CAP1 gene in human umbilical vein endothelial cells using shRNA. The following shCAP1 of SEQ ID NO: 9 designed to target the CAP1 gene was produced by cloning at the HpaI and XhoI restriction enzyme sites of the pLL3.7 lentiviral vector.

[0173] CAP1 shRNA, 5′-AGATGTGGATAAGAAGCAT-3′ (SEQ ID NO: 9).

[0174] Blood LDL cholesterol is known to induce arteriosclerosis by passing through the endothelial cells of arterial blood vessels. Based on this, the inventors confirmed whether there would be a change in the absorption of LDL cholesterol when the expression of CAP1 was suppressed using shCAP1. As a result, as shown in Figure 7, when PCSK9 was treated in human umbilical vein endothelial cells (HUVEC), LDL cholesterol flowed into the blood vessel wall through it. At this time, it was shown that in the vascular endothelial cells with CAP1 knocked down, LDL cholesterol did not enter the endothelial cells. The above results suggest that CAP1 plays an important role in the intracellular absorption of LDL cholesterol that can induce arteriosclerosis, and therefore, various cardiovascular and metabolic diseases including arteriosclerosis can be treated by inhibiting the binding or suppressing the expression of CAP1.

[0175] The above description of the present invention is for illustrative purposes, and those with ordinary knowledge in the technical field to which the present invention pertains can easily understand that it can be easily deformed into other specific forms without changing the technical idea and essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all aspects and not restrictive.

Industrial Applicability

[0176] According to the present invention, CAP1, which directly binds to PCSK9 and regulates the life cycle of the LDL receptor, can inhibit the binding of CAP1 to PCSK9 or resistin, or suppress the expression of the CAP1 gene, thereby regulating cholesterol levels. Therefore, a binding inhibitor between CAP1 and PCSK9 or resistin according to the present invention, or an expression inhibitor of the CAP1 gene, etc., can reduce the level of blood cholesterol, and thus can be usefully used as a composition for treating various cardiovascular and metabolic diseases related to or caused by abnormal levels of blood cholesterol. In addition, it is expected to have great industrial utility value from the point of having an anti-inflammatory effect.

Claims

1. A composition for reducing blood LDL cholesterol, comprising an inhibitor of the binding between CAP1 (adenylyl cyclase-associated protein 1) and PCSK9 (proprotein convertase subtilisin / kexin type-9), wherein the binding inhibitor is a fusion protein comprising a CAP1 protein having the amino acid sequence of SEQ ID NO: 1, or a fragment comprising its SH3 binding domain, A composition for reducing blood LDL cholesterol.

2. The composition according to claim 1, wherein the binding inhibitor specifically binds to a site containing aspartic acid at position 34B present in the SH3 binding domain of CAP1 (aspartic acid).

3. The composition according to claim 1, wherein the binding inhibitor specifically binds to a site containing lysine at position 494 present in the M1 domain of PCSK9 or arginine at position 659 present in the M3 domain of PCSK9.

4. The composition according to claim 1, which inhibits the degradation of LDL (Low-density lipoprotein) receptors.

5. A pharmaceutical composition for preventing or treating cardiometabolic diseases, comprising an inhibitor of the binding between CAP1 (adenylyl cyclase-associated protein 1) and PCSK9 (proprotein convertase subtilisin / kexin type-9), wherein the binding inhibitor is a fusion protein comprising a CAP1 protein having the amino acid sequence of SEQ ID NO: 1, or a fragment comprising its SH3 binding domain, wherein the cardiometabolic disease is a disease selected from the group consisting of diabetes, obesity, dyslipidemia, fatty liver, hypertension, gout, stroke, arteriosclerosis, myocardial infarction, angina pectoris, peripheral vascular disease, and combinations thereof, A pharmaceutical composition for preventing or treating cardiometabolic diseases.

6. An anti-inflammatory pharmaceutical composition, comprising an inhibitor of the binding between CAP1 (adenylyl cyclase-associated protein 1) and PCSK9 (proprotein convertase subtilisin / kexin type-9), wherein the binding inhibitor is a fusion protein comprising a CAP1 protein having the amino acid sequence of SEQ ID NO: 1, or a fragment comprising its SH3 binding domain, An anti-inflammatory pharmaceutical composition, wherein the inflammation is inflammation resulting from the activation of NF-κB.

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