Use of polypeptide blocking lipid synthesis in treatment of tumor and lipid metabolism abnormality diseases

Abstract

Provided is a polypeptide. The polypeptide has the effects of inhibiting Insig1 / 2 phosphorylation and inhibiting lipid synthesis. Also provided are a nucleic acid molecule encoding the polypeptide, a corresponding expression vector and host cell, and a method for producing the polypeptide. The polypeptide can bind to PCK1 pS90 with high affinity, and can be used for detection of PCK1 pS90 and targeted treatment of tumors.

Description

Application of a peptide that blocks lipid synthesis in the treatment of tumors and lipid metabolism disorders Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a polypeptide that blocks lipid synthesis in the treatment of tumors and lipid metabolism disorders. Background Technology

[0002] Lipid synthesis is a complex and tightly regulated biochemical reaction in a normal body. De novo fatty acid synthesis is a metabolic pathway in which the body synthesizes fatty acids from excess carbohydrates under conditions of sufficient energy supply, ultimately storing them as triglycerides for energy. Under conditions of insufficient energy supply, triglycerides can generate ATP through fatty acid β-oxidation for energy. Disorders in both lipid synthesis and catabolism can lead to a series of metabolic diseases, such as obesity, non-alcoholic fatty liver disease, metabolic syndrome, and cancer. Current research has shown that lipid synthesis plays a crucial role in the replication of various viruses and the process of tumor growth. Blocking lipid synthesis in tumors can inhibit tumor growth and promote tumor cell death. Furthermore, orlistat, as an inhibitor of fatty acid synthase (FASN), has been proven effective in the treatment of obesity in clinical practice. Therefore, targeting lipid synthesis pathways can provide new insights for the treatment of diseases caused by abnormal lipid metabolism.

[0003] Phosphoenolpyruvate carboxylase (PCK) is the rate-limiting enzyme in the gluconeogenesis pathway, involved in maintaining blood glucose levels. There are two subtypes of PCK, PCK1 and PCK2, which play important roles in the biological processes of diseases such as carbohydrate metabolism, lipid metabolism, aging, and tumors.

[0004] The SREBP1 protein plays a crucial role in the disease progression caused by abnormal lipid synthesis and metabolism. Under normal circumstances, a complex composed of INSIG protein, SREBP cleavage activator protein (SCAP), and sterol in the endoplasmic reticulum can inhibit SREBP. The interaction between INSIG and SCAP, regulated by sterol levels, is essential for the dissociation of the SCAP-SREBP complex from the endoplasmic reticulum and the activation of SREBP1.

[0005] In tumors, existing research has shown that under the stimulation of insulin-like growth factor, overactivated AKT can phosphorylate PCK1 protein. Phosphorylated PCK1 can promote the translocation of the SCAP-SREBP complex from the endoplasmic reticulum to the Golgi apparatus, activate the transcription of SREBP1 protein and downstream lipid-related genes, thereby promoting cellular lipid synthesis and providing conditions for tumor proliferation.

[0006] Therefore, there is currently a lack of methods in this field to effectively treat lipid metabolism disorders by targeting and inhibiting lipid synthesis pathways.

[0007] Therefore, developing a new method to accurately and efficiently treat lipid metabolism disorders by targeting lipid synthesis pathways is of great significance to this field. Summary of the Invention

[0008] This invention provides a novel method for accurately and efficiently treating tumors by targeting lipid synthesis pathways.

[0009] In a first aspect of the invention, a polypeptide is provided, said polypeptide being selected from the group consisting of:

[0010] (P1) comprises polypeptide A, which has the amino acid sequence shown in SEQ ID NO:1;

[0011] (P2) A polypeptide B that has the same function as polypeptide A, which is formed by adding, deleting, modifying and / or substituting at least one amino acid, or cyclizing it.

[0012] (P3) The combination of (P1) and (P2) above.

[0013] In another preferred embodiment, the polypeptide B retains the affinity of the polypeptide A.

[0014] In another preferred embodiment, the polypeptide B is obtained by substituting at least one amino acid into polypeptide A, and the polypeptide B has the same function as the polypeptide A.

[0015] In another preferred embodiment, the number of substituted amino acids is 1 to 8, for example 1, 2, 3, 4, 5, 6, 7, or 8.

[0016] In another preferred embodiment, the polypeptide B is selected from the group consisting of:

[0017] (PB1) includes the amino acid sequence shown in SEQ ID NO:1, and has the following mutations in the amino acid sequence: glycine at position 10 is mutated to alanine (G10A), glutamic acid at position 11 is mutated to alanine (E11A), and proline at position 12 is mutated to alanine (P12A).

[0018] (PB2) includes the amino acid sequence shown in SEQ ID NO:1, and has the following mutations in the amino acid sequence: aspartic acid at position 6 is mutated to tryptophan (D6W) and serine at position 7 is mutated to threonine (S7T).

[0019] (PB3) comprises the amino acid sequence shown in SEQ ID NO:1, and has the following mutations in the amino acid sequence: aspartic acid at position 6 is mutated to tryptophan (D6W), serine at position 7 is mutated to threonine (S7T), histidine at position 8 is mutated to lysine (H8K), leucine at position 9 is mutated to glycine (L9G), and glycine at position 10 is mutated to proline (G10P).

[0020] (PB4) includes the amino acid sequence shown in SEQ ID NO:1, and has the following mutations in the amino acid sequence: isoleucine at position 5 is mutated to tryptophan (I5W), aspartic acid at position 6 is mutated to methionine (D6M), and proline at position 12 is mutated to valine (P12V).

[0021] (PB5) comprises the amino acid sequence shown in SEQ ID NO:1, and has the following mutations in the amino acid sequence: cysteine ​​at position 4 is mutated to methionine (C4M), isoleucine at position 5 is mutated to serine (I5S), aspartic acid at position 6 is mutated to histidine (D6H), glycine at position 10 is mutated to cysteine ​​(G10C), glutamic acid at position 11 is mutated to glutamine (E11Q), proline at position 12 is mutated to glycine (P12G), and histidine at position 13 is mutated to tryptophan (H13W).

[0022] (PB6) includes the amino acid sequence shown in SEQ ID NO:1, and has the following mutations in the amino acid sequence: cysteine ​​at position 4 is mutated to valine (C4V), isoleucine at position 5 is mutated to cysteine ​​(I5C), aspartic acid at position 6 is mutated to histidine (D6H), glutamic acid at position 11 is mutated to asparagine (E11N), proline at position 12 is mutated to isoleucine (P12T), and histidine at position 13 is mutated to leucine (H13L);

[0023] (PB7) comprises the amino acid sequence shown in SEQ ID NO:1, and has the following mutations in the amino acid sequence: isoleucine at position 5 is mutated to methionine (I5M), aspartic acid at position 6 is mutated to methionine (D6M), glycine at position 10 is mutated to isoleucine (G10I), glutamic acid at position 11 is mutated to glycine (E11G), proline at position 12 is mutated to alanine (P12A), and histidine at position 13 is mutated to serine (H13S).

[0024] Or a combination thereof.

[0025] In another preferred embodiment, the polypeptide B is selected from the group consisting of:

[0026] (B1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO:4;

[0027] (B2) A polypeptide comprising the amino acid sequence shown in SEQ ID NO:5;

[0028] (B3) includes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:6;

[0029] (B4) A polypeptide comprising the amino acid sequence shown in SEQ ID NO:7;

[0030] (B5) includes a polypeptide as shown in the amino acid sequence of SEQ ID NO:8;

[0031] (B6) includes a polypeptide comprising the amino acid sequence shown in SEQ ID NO:9;

[0032] (B7) A polypeptide comprising the amino acid sequence shown in SEQ ID NO:10;

[0033] Or a combination thereof.

[0034] In another preferred embodiment, the polypeptide comprises the polypeptide shown in SEQ ID NO:4.

[0035] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5, more preferably 1-2, and even more preferably 1.

[0036] In another preferred embodiment, the N-terminus of the polypeptide also contains a fusion element.

[0037] In another preferred embodiment, the fusion element is a transmembrane peptide.

[0038] In another preferred embodiment, the sequence of the membrane-penetrating peptide is SEQ ID NO:3.

[0039] In a second aspect of the invention, a nucleic acid molecule is provided that encodes the polypeptide described in the first aspect of the invention.

[0040] In another preferred embodiment, the nucleic acid molecule includes DNA or RNA.

[0041] In another preferred embodiment, the nucleic acid molecule comprises a sequence as shown in SEQ ID NO:2.

[0042] In a third aspect of the invention, an expression vector is provided, the expression vector containing the nucleic acid molecule described in the second aspect of the invention.

[0043] In a fourth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the third aspect of the invention, or having the nucleic acid molecule described in the second aspect of the invention integrated into its genome.

[0044] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0045] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0046] In a fifth aspect of the invention, the use of the polypeptide described in the first aspect of the invention, or the nucleic acid molecule described in the second aspect of the invention, or the expression vector described in the third aspect of the invention, is provided for the preparation of formulations or compositions, said formulations or compositions being used for:

[0047] (a) Competitively binds to phosphorylated PCK1 S90 (pS90) protein;

[0048] (b) Prevention and / or treatment of tumors.

[0049] In another preferred embodiment, the formulation or composition is also used for (c) prevention and / or treatment of diseases caused by abnormal lipid metabolism.

[0050] In another preferred embodiment, the lipid metabolism disorder is selected from the group consisting of: obesity, non-alcoholic fatty liver disease, hyperlipidemia, hypertension, coronary heart disease, diabetes, or a combination thereof.

[0051] In another preferred embodiment, the lipid metabolism disorder is a metabolic syndrome.

[0052] In another preferred embodiment, the polypeptide is a truncated version of the INSIG protein.

[0053] In another preferred embodiment, the tumor is a PCK1 S90 phosphorylated tumor.

[0054] In another preferred embodiment, the tumor is selected from the group consisting of: gastrointestinal cancer, cancer of the central or peripheral nervous system, cancer of the endocrine or neuroendocrine system or cancer of the hematopoietic system, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, or combinations thereof.

[0055] In another preferred embodiment, the formulation or composition is used for:

[0056] (i) Block phosphorylation of Insig1 / 2 protein;

[0057] (ii) Inhibit the activation of SREBP protein;

[0058] (iii) Inhibits lipid synthesis in cells;

[0059] (iv) Inhibit the growth of tumor cells.

[0060] In another preferred embodiment, the formulation is a laboratory formulation.

[0061] In another preferred embodiment, the composition is a pharmaceutical composition.

[0062] In a sixth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0063] (a1) The polypeptide described in the first aspect of the present invention, or the nucleic acid molecule described in the second aspect of the present invention, or the expression vector described in the third aspect of the present invention;

[0064] (b) Pharmaceutically acceptable carriers.

[0065] In another preferred embodiment, the pharmaceutical composition further comprises:

[0066] (a2) Other drugs used to inhibit Insig1 / 2 phosphorylation or to treat tumors.

[0067] In another preferred embodiment, the pharmaceutical composition is an injectable formulation.

[0068] In another preferred embodiment, (a1) is encapsulated in a pharmaceutically acceptable carrier.

[0069] In another preferred embodiment, the pharmaceutically acceptable carrier is a lipid particle.

[0070] In another preferred embodiment, the pharmaceutically acceptable carrier is lipid nanoparticles (LNP).

[0071] In another preferred embodiment, the pharmaceutical composition is lipid particles.

[0072] In another preferred embodiment, the pharmaceutical composition is lipid nanoparticles.

[0073] In another preferred embodiment, the lipid nanoparticles are encapsulated with components selected from the group consisting of: polypeptides according to the first aspect of the invention, nucleic acid molecules according to the second aspect of the invention, expression vectors according to the third aspect of the invention, or combinations thereof.

[0074] In another preferred embodiment, the lipid nanoparticles further contain an element that targets tumor cells.

[0075] In another preferred embodiment, the lipid nanoparticles include: ionizable lipids, lecithin, phospholipids, and PEG esters.

[0076] In another preferred embodiment, the lipid nanoparticles are ionizable lipids.

[0077] In another preferred embodiment, the encapsulation efficiency is ≥80%, and the average particle size of the encapsulated stock solution is 50-150 nm.

[0078] In another preferred embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0079] In another preferred embodiment, the pharmaceutically acceptable excipient is selected from the group consisting of diluents, excipients, surfactants, lubricants, disintegrants, or combinations thereof.

[0080] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, lyophilized formulations, nebulized inhalers, oral formulations, and topical formulations.

[0081] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of oral, intravenous, and intraperitoneal injection.

[0082] In another preferred embodiment, the pharmaceutical composition is administered by injection, i.e., intravenous, intratumoral, intramuscular, subcutaneous, or intraperitoneal injection.

[0083] In another preferred embodiment, the pharmaceutical composition is administered transdermally, such as by topical application or electrode delivery.

[0084] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for the prevention and / or treatment of cancer or tumors.

[0085] In a seventh aspect of the invention, the use of the pharmaceutical composition described in the sixth aspect of the invention is provided for the preparation of a medicament for the prevention and / or treatment of cancer or tumors.

[0086] In another preferred embodiment, the drug is also used to prevent and / or treat lipid metabolism disorders.

[0087] In another preferred embodiment, the lipid metabolism disorder is selected from the group consisting of: obesity, non-alcoholic fatty liver disease, hyperlipidemia, hypertension, coronary heart disease, diabetes, or a combination thereof.

[0088] In another preferred embodiment, the lipid metabolism disorder is a metabolic syndrome.

[0089] In another preferred embodiment, the cancer or tumor is a PCK1 pS90 phosphorylated cancer or tumor.

[0090] In another preferred embodiment, the cancer or tumor is selected from the group consisting of: gastrointestinal cancer, cancer of the central or peripheral nervous system, cancer of the endocrine or neuroendocrine system or cancer of the hematopoietic system, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, leukemia, or combinations thereof.

[0091] In an eighth aspect of the invention, a reagent combination is provided, the reagent combination comprising:

[0092] (a) the polypeptide described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, or the expression vector described in the third aspect of the present invention; and

[0093] (b) Detection reagent for detecting PCK1 S90 phosphorylation.

[0094] In another preferred embodiment, the reagent combination further includes: (c) a detection-acceptable carrier.

[0095] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0096] In another preferred embodiment, the reagent combination is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0097] In another preferred embodiment, the reagent combination is used for in vivo or in vitro detection.

[0098] In another preferred embodiment, the dosage form of the reagent combination is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, inhaler).

[0099] In another preferred embodiment, the detection reagent is a PCK1 S90 phosphorylated antibody or a PCK1 non-phosphorylated antibody.

[0100] In another preferred embodiment, the detection reagent is a PCK1 S90 phosphorylated antibody.

[0101] In another preferred embodiment, the expression vector is a tumor-targeting expression vector.

[0102] In another preferred embodiment, the expression vector is a lipid nanoparticle (LNP) containing the nucleic acid molecule described in the second aspect of the present invention.

[0103] In another preferred embodiment, the reagent combination is used to detect the phosphorylation level of PCK1 S90.

[0104] In a ninth aspect of the invention, a reagent kit is provided, the reagent kit comprising the reagent combination as described in the eighth aspect of the invention.

[0105] In a tenth aspect of the invention, a method for inhibiting Insig1 / 2 phosphorylation in vitro in the presence of PCK1 pS90 is provided, comprising the steps of:

[0106] (s1) PCK1 pS90 is contacted with the polypeptide described in the first aspect of the present invention to inhibit Insig1 / 2 phosphorylation.

[0107] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0108] In another preferred embodiment, the method is an in vitro method.

[0109] In an eleventh aspect of the present invention, a method for generating the polypeptide described in the first aspect of the present invention is provided, comprising the steps of:

[0110] (a) Culturing the host cells described in the fourth aspect of the invention under conditions suitable for polypeptide production, thereby obtaining a culture containing said polypeptide; and

[0111] (b) Isolate or recover the polypeptide from the culture.

[0112] In a twelfth aspect of the invention, a method for treating and / or alleviating tumors is provided, the method comprising: administering to a desired subject a polypeptide as described in the first aspect of the invention, or a nucleic acid molecule as described in the second aspect, or a pharmaceutical composition as described in the sixth aspect of the invention.

[0113] In another preferred embodiment, the method includes mammals, such as humans.

[0114] In another preferred embodiment, the cancer or tumor is a PCK1 S90 phosphorylated cancer or tumor.

[0115] In another preferred embodiment, the cancer or tumor is selected from the group consisting of: gastrointestinal cancer, cancer of the central or peripheral nervous system, cancer of the endocrine or neuroendocrine system or cancer of the hematopoietic system, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, leukemia, or combinations thereof.

[0116] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0117] Figure 1 shows that the Insig1 / 2 blocking peptide inhibits IGF-mediated SREBP1 activation in Huh7 hepatocellular carcinoma cells.

[0118] Figure 2 shows that the Insig1 / 2 blocking peptide inhibits IGF-mediated phosphorylation of the Insig1 / 2 protein in Huh7 hepatocellular carcinoma cells.

[0119] Figure 3 shows that the Insig1 / 2 blocking peptide can bind to the PCK1 pS90 protein.

[0120] Figure 4 shows that the Insig1 / 2 blocking peptide mutant can bind to the PCK1 pS90 protein.

[0121] Figure 5 shows that the Insig1 / 2 blocking peptide can inhibit the growth of liver cancer in nude mice.

[0122] Figure 6 shows that the Insig1 / 2 blocking peptide can inhibit the phosphorylation of Insig protein in tumor tissue. Detailed Implementation

[0123] Through extensive and in-depth research and screening, the inventors have developed a polypeptide (Insig1 / 2 blocking peptide) that binds with high affinity to phosphorylated PCK1 protein (PCK1 pS90). This blocks the binding of PCK1 pS90 to Insig1 / 2, thereby inhibiting Insig1 / 2 phosphorylation, suppressing lipid synthesis, and inhibiting tumor cell growth. Experiments show that the polypeptide of this invention has high affinity for PCK1 pS90 protein and can effectively inhibit the growth of various tumor cells, with the inhibition rate showing a concentration-dependent relationship. Based on this, the present invention was completed.

[0124] the term

[0125] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0126] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0127] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0128] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0129] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0130] As used in this article, “PCK1 S90”, “PCK1 pS90” and “phosphorylated PCK1” are used interchangeably and all refer to PCK1 protein phosphorylated at position S90.

[0131] As used herein, “blocking peptide of the present invention”, “blocking polypeptide of the present invention”, “inhibitory polypeptide of the present invention”, “Insig1 / 2 blocking peptide of the present invention”, “PCK1 peptide of the present invention” and “polypeptide of the present invention” are used interchangeably and all refer to polypeptides that can bind to PCK1 or phosphorylated PCK1 (PCK1 pS90) with high affinity.

[0132] PCK1 protein, its phosphorylation and its role in Insig1 / 2

[0133] Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme in the first step of the gluconeogenesis pathway, catalyzing the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP).

[0134] Previous studies have found that PCK1 participates in the regulation of important metabolic processes such as the pentose phosphate pathway, hexosamine biosynthesis, and glycerol synthesis. Under the stimulation of insulin-like growth factor, the AKT signaling pathway in tumor cells is activated, promoting AKT phosphorylation of the PCK1 protein at its S90 site. S90 phosphorylated PCK1 protein can bind to Insig1 / 2 protein, promoting Insig1 / 2 phosphorylation, which further promotes the translocation of the SCAP-SREBP complex from the endoplasmic reticulum to the Golgi apparatus, activating the transcription of SREBP1 protein and downstream lipid-related genes, and promoting lipid synthesis in tumor cells.

[0135] Insig1 / 2 protein, its phosphorylation and its role in lipid synthesis

[0136] Insulin-inducible genes (INSIG) are proteins anchored in the endoplasmic reticulum (ER). In mammals, there are two types of INSIG proteins: INSIG-1 and INSIG-2. Both INSIG proteins consist of six transmembrane helices, which are mostly embedded in the ER membrane. They share the same function in regulating SREBP processing. The SCAP-SREBP complex plays a central role in the feedback regulation of SREBP processing by being retained in the ER.

[0137] Under normal circumstances, a complex composed of INSIG protein, SREBP cleavage activator protein (SCAP), and sterols in the endoplasmic reticulum (ER) inhibits SREBP. The interaction between INSIG and SCAP, regulated by sterol levels, is crucial for the dissociation of the SCAP-SREBP complex from the ER and the activation of SREBP. SREBP protein plays an important role in the progression of many diseases caused by abnormal lipid synthesis and metabolism, such as hepatic steatosis, non-alcoholic fatty liver disease, and atherosclerosis.

[0138] In tumors, existing research has shown that under the stimulation of insulin-like growth factor (IGF), overactivated AKT can phosphorylate PCK1 protein. Phosphorylated PCK1 can phosphorylate Insig1 / 2 protein (Insig1 / 2S207), thereby promoting the translocation of the SCAP-SREBP complex from the endoplasmic reticulum to the Golgi apparatus, activating the transcription of SREBP protein and downstream lipid-related genes, thereby promoting cellular lipid synthesis and providing conditions for tumor proliferation.

[0139] Sterol-regulated element-binding protein (SREBP) is an 8-base motif that binds to transcription factors to regulate various lipid metabolism-related genes. Humans express two different SREBPs, called sterol-regulated element-binding protein 1 (SREBP1) and SREBP2. Mammalian SREBP1 has two transcripts, SREBP-1a and SREBP-1c, with SREBP-1a being the gene that regulates all SREBP responses in cholesterol and fatty acid biosynthesis pathways.

[0140] The pharmaceutical compositions of the present invention and their applications

[0141] In one aspect of the invention, a pharmaceutical composition is also provided. In one embodiment of the invention, the pharmaceutical composition is a protein and / or nucleic acid pharmaceutical composition.

[0142] There are many methods for delivering protein or nucleic acid drugs. Scientists have developed methods such as liposome delivery, polymer delivery, peptide chain delivery, virus-like replicon particle delivery, and cationic nanoemulsion delivery. In addition, naked proteins or nucleic acids can also be directly injected into cells. The most commonly used delivery method is lipid nanoparticle delivery, which has advantages such as low toxicity and high delivery efficiency.

[0143] The "active ingredient" in the pharmaceutical composition of this invention refers to the polypeptide described in the first aspect of this invention, the nucleic acid molecule described in the second aspect of this invention, or the expression vector described in the third aspect of this invention. In a preferred embodiment, the "active ingredient" further comprises other drugs for inhibiting Insig1 / 2 phosphorylation or drugs for treating tumors.

[0144] The "active ingredients," formulations, and / or compositions described in this invention can be used to prevent and / or treat diseases or conditions such as tumors, obesity, diabetes, pancreatitis, non-alcoholic fatty liver disease, lipid metabolism disorders, atherosclerosis, and cardiovascular diseases. "Safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances suitable for human use, and which must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredients of this invention without significantly reducing the efficacy of the active ingredients.

[0145] The pharmaceutical composition can be a liquid or a solid, such as a powder, gel, or paste. Preferably, the composition is a liquid, and more preferably an injectable liquid.

[0146] Pharmaceutically acceptable examples of carrier components include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as Tween). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0147] Pharmaceutical compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0148] The pharmaceutical compositions of the present invention can be formulated into dosage forms such as injections, lyophilized formulations, nebulized inhalers, oral formulations, and topical formulations. The pharmaceutical compositions of the present invention can be delivered (administered) by any suitable method, including oral, parenteral, and topical methods. The pharmaceutical compositions of the present invention can also be administered by injection, i.e., intravenous, intratumoral, intramuscular, intradermal, subcutaneous, or intraperitoneal injection. Furthermore, the pharmaceutical compositions of the present invention can be administered transdermally. Transdermal administration via a local route can be formulated into medicated sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, gels, paints, powders, and aerosols. In addition, the pharmaceutical compositions of the present invention can be actively administered to intradermal, subcutaneous, intramuscular, tumor, tissue, organ, and central nervous system sites via electrodes / electric fields / potential differences.

[0149] The pharmaceutical compositions of the present invention can be co-administered with another active agent. Co-administration includes administering the compound and active agent of the present invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of each other. Co-administration also includes administering the compound and active agent of the present invention simultaneously, substantially simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition comprising both the active ingredient of the present invention (the mRNA transcription template construct or optimized mRNA described in the present invention) and the active agent. In other embodiments, the active ingredient and active agent of the present invention can be formulated separately.

[0150] The present invention also provides the use of the pharmaceutical composition for preparing a medicament for the prevention and / or treatment of diseases, including (but not limited to) diseases of PCK1 S90 phosphorylation, such as PCK1 S90 phosphorylation cancers or tumors and diseases of lipid metabolism disorders. In a preferred embodiment, the cancers or tumors include: gastrointestinal cancer, central or peripheral nervous system tissue cancers, endocrine or neuroendocrine system cancers or hematopoietic system cancers, gliomas, sarcomas, epithelial cancers, lymphomas, melanomas, fibromas, meningiomas, brain cancers, kidney cancers, thyroid cancers, parathyroid cancers, pituitary adenomas, adrenal adenomas, bone-derived sarcomas, neuroendocrine system tumors, breast cancers, lung cancers, head and neck cancers, prostate cancers, esophageal cancers, tracheal cancers, liver cancers, bladder cancers, stomach cancers, pancreatic cancers, ovarian cancers, uterine cancers, cervical cancers, testicular cancers, colon cancers, rectal cancers, skin cancers, leukemias, or combinations thereof.

[0151] Detection methods

[0152] The present invention also relates to a method for detecting PCK1 pS90 protein. The method comprises the following steps: obtaining cell and / or blood samples; dissolving the samples in a medium; and detecting the level of PCK1 pS90 protein in the dissolved samples.

[0153] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0154] Reagent test kit

[0155] The present invention also provides a kit containing the peptide or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0156] This invention also provides a detection kit for detecting PCK1 pS90 levels. The kit may further include an antibody that recognizes the PCK1 pS90 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0157] application

[0158] As described above, the peptides of the present invention have broad biological and clinical application value, and their applications involve multiple fields such as the diagnosis and treatment of lipid synthesis-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of PCK1 S90.

[0159] In a preferred embodiment, the polypeptide of the present invention is used to inhibit lipid synthesis in tumor cells, thereby inhibiting the growth of tumor cells and thus treating and / or alleviating tumors.

[0160] In another preferred embodiment, the tumor is selected from the group consisting of: gastrointestinal cancer, central or peripheral nervous system cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial cancer, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, osteosarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, leukemia, or combinations thereof.

[0161] In a preferred embodiment, the polypeptide of the present invention can be encapsulated in lipid nanoparticles for delivery to tumor cells. In a preferred embodiment, the lipid nanoparticles further include a targeting element for targeting tumor cells.

[0162] The main advantages of this invention include:

[0163] (1) The polypeptide of the present invention can inhibit lipid synthesis by inhibiting Insig1 / 2 phosphorylation, thereby inhibiting tumor cell growth, and can be used to prepare targeted drug delivery for efficient treatment of tumors.

[0164] (2) The polypeptides of the present invention can also be used to treat lipid metabolism disorders (such as obesity) by inhibiting lipid synthesis, thereby effectively treating the disease.

[0165] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0166] Materials and methods

[0167] The various tumor cell lines of this invention specifically include: human liver cancer cells (Huh7 cells), human liver cancer cells (Hep3b cells), human highly metastatic liver cancer cells (LM3 cells), human lung cancer cells (H292 cells), human pancreatic cancer cells (CFPAC-1 cells), human prostate cancer cells (DU145 cells), human breast cancer cells (MDB-231 cells), human acute myeloid leukemia cells (Molm13 cells), human acute myeloid leukemia cells (KG-1 cells), human colon cancer cells (HCT116 cells), mouse colon cancer cells (CT26 cells), human gastric cancer cells (HGC-27 cells), human cervical cancer cells (HeLa cells), human glioma cells (U251 cells), human ovarian cancer cells (SKOV-3 cells), and human esophageal cancer cells (TE-1 cells). All are derived from laboratory cell banks.

[0168] In cell experiments, to increase the uptake of peptides by tumor cells, a transmembrane peptide TAT sequence (RKKRRQRRR (SEQ ID NO:3)) was introduced into the N-terminus of the naked peptide for cell treatment, thereby promoting peptide absorption.

[0169] In animal experiments, since the inhibitory peptide naked peptide has an extremely short half-life in vivo, LNP is used to encapsulate the inhibitory peptide of the present invention (LNPcRGD, TAT@Peptide) for in vivo delivery.

[0170] The preparation method of LNPcRGD,TAT@Peptide is as follows:

[0171] 1 mL of DSPE-PEG-cRGD solution was added to a 50 mL round-bottom flask, and then the flask was placed in a rotary evaporator to allow the solution to slowly evaporate and form a film. 6 mL of cholesterol solution, 2 mL of lecithin solution, and 2 mL of DSPE-PEG solution were added to the same round-bottom flask, and a film was formed using the same method. 5 mL of DSPE-PEG-TAT solution and 5 mL of peptide solution were added to the round-bottom flask. The resulting mixture was then sonicated in an ice-water bath for 20 minutes and dialyzed in a dialysis bag for 48 hours to prepare the targeted nanomaterial LNPcRGD,TAT@Peptide loaded with peptide (the peptide of this invention).

[0172] Example 1

[0173] The transcriptional activity of SREBP1 was detected using a luciferase reporter gene assay. The detection method included the following steps:

[0174] (1) Huh7 cells were seeded in 24-well plates and transfected with 0.1 μg SRE luciferase reporter gene and 0.075 μg β-galactosidase.

[0175] (2) 24 hours after transfection, the cells were divided into three groups: control group, IGF group, and PCK1 peptide (blocking peptide) + IGF group. Each group had three replicates. The PCK1 peptide + IGF group was pretreated with inhibitory peptide (Insig1 / 2 blocking peptide) for 2 hours.

[0176] (3) After the pretreatment, IGF was added to the IGF group and the PCK1 peptide + IGF group for 6 hours to stimulate them.

[0177] (4) Collect cell lysates and use a kit to detect the luciferase and β-galactosidase activities in the cell lysates.

[0178] The test results are shown in Figure 1. The results show that the Insig1 / 2 blocking peptide can block the IGF-mediated transcriptional activation of SREBP1 in liver cancer cells, thereby inhibiting lipid synthesis in tumor cells.

[0179] Example 2

[0180] Huh7 cells were treated with different concentrations of Insig1 / 2 blocking peptide, and the phosphorylation level of the downstream protein Insig was detected by Western blotting.

[0181] The test results are shown in Figure 2. The results indicate that the Insig1 / 2 blocking peptide can inhibit IGF (insulin-like growth factor)-mediated phosphorylation of Insig1 / 2 protein in Huh7 liver cancer cells.

[0182] Example 3

[0183] Octet was used to detect the affinity of the Insig1 / 2 blocking peptide for PCK1 pS90 protein.

[0184] The specific experimental steps are as follows:

[0185] (1) PCK1 pS90 protein was purified from Escherichia coli and biotinylated using a biotinylation kit.

[0186] (2) The biotinylated PCK1 pS90 protein was linked to the SSA probe.

[0187] (3) Prepare a 2mM stock solution of Insig1 / 2 blocking peptide, and add it to 96 plates after serial dilution.

[0188] (4) The probe is tested with different concentrations of peptides to obtain the affinity KD value.

[0189] The test results are shown in Figure 3. The results indicate that the blocking peptide can bind to the PCK1 pS90 protein with an affinity KD of 1.6E-04M.

[0190] Example 4

[0191] Octet was used to detect the affinity of the Insig1 / 2 blocking peptide mutant for PCK1 pS90 protein.

[0192] The specific experimental steps are as follows:

[0193] (1) PCK1 pS90 protein was purified from Escherichia coli and biotinylated using a biotinylation kit.

[0194] (2) The biotinylated PCK1 pS90 protein was linked to the SSA probe.

[0195] (3) Prepare a 2mM stock solution of the Insig1 / 2 blocking peptide mutant, and add it to 96 plates after serial dilution.

[0196] (4) The probe is tested with different concentrations of peptides to obtain the affinity KD value.

[0197] The test results are shown in Figure 4. The results indicate that the mutant can still bind to the PCK1 pS90 protein with an affinity KD of 2.1E-05M.

[0198] Example 5

[0199] In this embodiment, crystal violet staining was used to detect the inhibitory effect of different concentrations of Insig1 / 2 blocking peptide on the proliferation of various tumors. The detection method includes the following steps:

[0200] Various tumor cells were collected, including: Huh7 cells, Hep3b cells, LM3 cells, H292 cells, CFPAC-1 cells, DU145 cells, MDB-231 cells, Molm13 cells, KG-1 cells, HCT116 cells, CT26 cells, HGC-27 cells, HeLa cells, U251 cells, SKOV-3 cells, and TE-1 cells.

[0201] Non-suspension tumor cells from various tumor cell types were digested with digestive enzymes and seeded at a density of 2000 cells / well in 96-well plates. After 24 hours of cell adhesion, various tumor cell types were treated with different concentrations of Insig1 / 2 blocking peptide. The experimental groups included 0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM. After 72 hours of drug treatment, the original culture medium was discarded, and the cells were washed three times with PBS. After staining with crystal violet solution prepared with methanol for 30 minutes, the cells were rinsed with water to remove excess staining solution. Cells that could be stained were considered viable cells.

[0202] After drying the 96-well plate in an oven, the dye was dissolved in 33% acetic acid, and the OD value was measured at 582 nm. The inhibition rate of different concentrations of the drug on tumor cells was calculated according to the inhibition rate formula.

[0203] Inhibition rate (%) = (OD of drug experimental group - average OD of drug control well) / (average OD of cell control well - average OD of drug control well) x 100%. The test results are shown in Table 1.

[0204] Tumor cells growing in suspension (KG-1 cells and MOLM13 cells) were detected using the MTT assay. The detection method included the following steps:

[0205] Human leukemia cells KG-1 / MOLM13 were used as suspension cells. After mixing the KG-1 / MOLM13 cells by pipetting, they were seeded into 96-well plates at a density of 2000 cells / well. After culturing for 24 h, the tumor cells were treated with different concentrations of Insig1 / 2 blocking peptide. The experimental groups included 0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM. After 72 h of drug treatment, 20 μl of MTT solution (5 mg / ml) was added to each well, and the cells were cultured for another 4 h. After 4 h, the 96-well plates were centrifuged at 2000 rpm for 30 min. After centrifugation, the medium was discarded, and 150 μl of DMSO was added to each well. The plates were shaken at low speed for 10 min to fully dissolve the crystals. The absorbance was measured at OD 490 nm.

[0206] The inhibition rate of different drug concentrations on tumor cells was calculated using the inhibition rate formula.

[0207] Inhibition rate (%) = (OD of drug experimental group - average OD of drug control well) / (average OD of cell control well - average OD of drug control well) x 100%. The test results are shown in Table 1.

[0208] Table 1. Insig1 / 2 blocking peptides inhibit the proliferation of various tumor cells.

[0209] The results showed that the Insig1 / 2 blocking peptide of the present invention has an inhibitory effect on the proliferation of various tumor cells, and the inhibition rate increases with the increase of the concentration of the Insig1 / 2 blocking peptide.

[0210] Example 6

[0211] The inhibitory effects of different concentrations (0 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM) of the Insig1 / 2 blocking peptide mutant on the proliferation of various tumor cells were detected using crystal violet staining and the MTT assay. The detection methods were the same as in Example 5. These mutants were all obtained through affinity screening and optimization based on the wild-type Insig1 / 2 blocking peptide (SEQ ID NO:1) to improve the efficacy of the peptide.

[0212] This example lists seven Insig1 / 2 blocking peptide mutants and their inhibitory effects on the proliferation of various tumor cells.

[0213] The seven Insig1 / 2 blocking peptide mutant sequences are as follows:

[0214] Insig1 / 2 blocking peptide mutant 1: LYPCIDSHLAAAHKFKRE (SEQ ID NO:4), where the wavy underlined part indicates the mutation site (the amino acid GEP at the mutation site is mutated to three alanine AAAs);

[0215] Insig1 / 2 blocking peptide mutant 2: LYPCIWTHLGEPHKFKRE (SEQ ID NO:5), where the wavy underlined part indicates the mutation site (the amino acids at the mutation site have the following mutations: D->W, S->T);

[0216] Insig1 / 2 blocking peptide mutant 3: LYPCIWTKGPEPHKFKRE (SEQ ID NO:6), where the underlined part is the mutation site (the amino acids at the mutation site have the following mutations: D->W, S->T, H->K, L->G, G->P);

[0217] Insig1 / 2 blocking peptide mutant 4: LYPCWMSHLGEVHKFKRE (SEQ ID NO:7), where the wavy underlined part is the mutation site (the amino acids at the mutation site have the following mutations: I->W, D->M, and P->V);

[0218] Insig1 / 2 blocking peptide mutant 5: LYPMSHSHLCQGWKFKRE (SEQ ID NO:8), where the wavy underlined part is the mutation position (the amino acids at the mutation positions are respectively mutated as follows: C->M, I->S, D->H, and G->C, E->Q, P->G, H->W);

[0219] Insig1 / 2 blocking peptide mutant 6: LYPVCHSHLGNTLKFKRE (SEQ ID NO:9), where the wavy underlined part is the mutation position (the amino acids at the mutation positions are respectively mutated as follows: C->V, I->C, D->H, and E->N, P->T, H->L);

[0220] Insig1 / 2 blocking peptide mutant 7: LYPCMMSHLIGASKFKRE (SEQ ID NO:10), where the wavy underlined part is the mutation position (the amino acids at the mutation positions are respectively mutated as follows: I->M, D->M, and G->I, E->G, P->A, H->S);

[0221] The test results of Insig1 / 2 blocking peptide mutant 1 on various tumor cells are shown in Table 2.

[0222] Table 2

[0223] The results showed that the Insig1 / 2 blocking peptide mutant 1 of the present invention has a more effective efficacy than the wild-type Insig1 / 2 blocking peptide (SEQ ID NO:1), and its inhibition rate on tumor cell proliferation is superior to that of the wild-type Insig1 / 2 blocking peptide. Furthermore, the Insig1 / 2 blocking peptide mutant 1 also inhibits the proliferation of various tumor cells, and the inhibition rate increases with increasing Insig1 / 2 blocking peptide concentration.

[0224] The test results of Insig1 / 2 blocking peptide mutants 2–7 are shown in Tables 3 and 4.

[0225] Table 3. Inhibition of Huh7 cell proliferation by Insig1 / 2 blocking peptides and their mutants 2–7.

[0226] Table 4. Inhibition of A549 cell proliferation by Insig1 / 2 blocking peptides and their mutants 2–7.

[0227] As shown in Tables 3 and 4, the Insig1 / 2 blocking peptide mutants 2–7 exhibit more potent efficacy than the wild-type Insig1 / 2 blocking peptide (SEQ ID NO:1), demonstrating superior inhibition rates against tumor cell proliferation. Furthermore, the Insig1 / 2 blocking peptide mutants 2–7 also inhibited the proliferation of various tumor cell types, with the inhibition rate increasing with increasing Insig1 / 2 blocking peptide concentration.

[0228] Example 7 Animal Experiment

[0229] Insig1 / 2 blocking peptides can inhibit the growth of liver cancer in nude mice.

[0230] 7.1 Experimental Methods

[0231] This experiment used Huh7 liver cancer cells and nude mice (BALB / C-nude, 4 weeks old, male, Jiangsu Jicui Pharmaceutical Co., Ltd.) as experimental subjects (6 mice in each of the control group, liposome group, and experimental group). Tumor cells were injected subcutaneously at a dose of 1.5*102. 6 Subcutaneous tumor formation is performed using this method.

[0232] Five days after the initial injection of tumor cells, small tumors were observed to grow subcutaneously in the nude mice. The mice in each group then underwent the following treatments:

[0233] Control group: 100 μL of 0.9% normal saline was injected via the tail vein;

[0234] Liposome group: 100 μL of empty liposomes were injected via tail vein;

[0235] Liposome-peptide group: The blocking peptide was delivered by encapsulation in liposomes via tail vein injection at a dose of 10 mg / kg, every other day, until the tumor in the control group mice grew to about 1.5 cm * 1.5 cm and then the administration was stopped.

[0236] After the drug was discontinued, the mice were euthanized by cervical dislocation, the tumors were removed, photographed, weighed, and a tumor growth curve was plotted.

[0237] The extracted tumor tissue was then added to RIPA lysis buffer for tissue disruption. After centrifugation, the supernatant was collected for Western blotting to detect p-Insig1 S207 levels.

[0238] 7.2 Experimental Results

[0239] The experimental results are shown in Figures 5 and 6. Figure 5 shows that, compared to the control group, the tumors in the peptide-treated group grew more slowly and had lower tumor weight. Figure 6 shows that the expression level of p-Insig1 S207 in the peptide-treated group was significantly lower than that in the control group.

[0240] Therefore, it can be concluded that the inhibitory peptide (or Insig1 / 2 blocking peptide) of the present invention can inhibit the phosphorylation of Insig protein in tumor tissue, thereby inhibiting lipid synthesis in tumor cells and thus inhibiting the formation of liver cancer.

[0241] The sequence information of this invention is shown in Table 5:

[0242] Table 5

[0243] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A polypeptide, characterized in that, The polypeptide is selected from the following group: (P1) comprises polypeptide A, which has the amino acid sequence shown in SEQ ID NO:1; (P2) A polypeptide B that has the same function as polypeptide A, which is formed by adding, deleting, modifying and / or substituting at least one amino acid, or cyclizing it. (P3) The combination of (P1) and (P2) above.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1.

3. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 2.

4. A host cell, characterized in that, The host cell contains the expression vector of claim 3, or the nucleic acid molecule of claim 2 is integrated into its genome.

5. The use of the polypeptide of claim 1, or the nucleic acid molecule of claim 2, or the expression vector of claim 3, characterized in that, For use in the preparation of formulations or compositions, said formulations or compositions being used for: (a) Competitively binds to phosphorylated PCK1 S90 (pS90) protein; (b) Prevention and / or treatment of tumors; (c) Prevention and / or treatment of diseases caused by lipid metabolism disorders.

6. The use as described in claim 5, characterized in that, The formulation or composition is used for: (i) Block phosphorylation of Insig1 / 2 protein; (ii) Inhibit the activation of SREBP protein; (iii) Inhibits lipid synthesis in cells; and / or (iv) Inhibit the growth of tumor cells.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (a1) the polypeptide of claim 1, or the nucleic acid molecule of claim 2, or the expression vector of claim 3; and (b) Pharmaceutically acceptable carriers.

8. A reagent combination, characterized in that, The reagent combination comprises: (a) the polypeptide of claim 1, the nucleic acid molecule of claim 2, or the expression vector of claim 3; and (b) Detection reagent for detecting PCK1 pS90 phosphorylation.

9. A reagent kit, characterized in that, The kit comprises the reagent combination as described in claim 8.

10. A method for inhibiting Insig1 / 2 phosphorylation in vitro in the presence of PCK1 pS90, characterized in that, Including the following steps: (s1) Contacting PCK1 pS90 with the polypeptide of claim 1 thereby inhibiting Insig1 / 2 phosphorylation.

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