GLP-1R and GCGR double-target-activated polypeptide or derivative thereof, pharmaceutically acceptable salt and application of polypeptide or derivative and pharmaceutically acceptable salt

Abstract

The invention provides a polypeptide or a derivative and a pharmaceutically acceptable salt thereof. The amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1. The polypeptide or the derivative and the pharmaceutically acceptable salt thereof disclosed by the invention can be combined with GLP-1R and GCGR, and is used for effectively activating the GLP-1R and the GCGR. Therefore, the polypeptide or the derivative and the pharmaceutically acceptable salt thereof can be used for detecting GLP-1R and / or GCGR, and can also be used for treating or preventing GLP-1R and / or GCGR related diseases (such as metabolic disorder related diseases, such as obesity, diabetes, fatty liver diseases, non-alcoholic fatty liver diseases and the like).

Description

Technical Field

[0001] This application belongs to the technical field of biopharmaceuticals, and specifically relates to GLP-1R and GCGR dual-target activating polypeptides or their derivatives, pharmaceutically acceptable salts, and their uses. Background Art

[0002] Glucagon-like peptide-1 (GLP-1) is a polypeptide expressed by the proglucagon gene in intestinal mucosal L cells and consists of 31 amino acids. GLP-1 mainly binds to the GLP-1 receptor (GLP-1R), stimulates insulin secretion, inhibits glucagon secretion, protects pancreatic islet β cells, and plays a role in regulating blood glucose homeostasis. In addition, GLP-1 can also inhibit appetite and gastric emptying through the central nervous system signaling pathway, increase satiety, and thus reduce body weight.

[0003] Glucagon (GCG) is a polypeptide containing 29 amino acids secreted by pancreatic islet α cells. It mainly acts on the glucagon receptor (GCGR) mainly distributed in the liver and kidneys. GCG can stimulate hepatic glycogenolysis, increase blood glucose levels, activate lipase, promote fat breakdown, and enhance fatty acid oxidation, thereby increasing ketone body production. Research results show that GCG has a certain effect on reducing food intake, increasing energy consumption in adipose tissue, and reducing body fat content.

[0004] In the prior art, GLP-1 receptor (GLP-1R) agonists and glucagon receptor (GCGR) agonists have been widely studied and applied. GLP-1 receptor agonists reduce blood glucose by mimicking the action of endogenous GLP-1, while GCGR agonists affect blood glucose levels by regulating glucagon secretion.

[0005] However, these single-target agonists have some limitations in the treatment of diabetes, such as short half-life and easy degradation. At present, in addition to the above-mentioned single agonists targeting GLP-1R or GCGR, experiments show that several GLP-1R / GCGR dual agonists may bring more significant weight loss effects, and also show potential advantages in the treatment of fatty liver disease and related complications, playing a positive role through synergistically regulating glucose and lipid metabolism. Therefore, the development of a novel GLP-1R and GCGR dual-target activating polypeptide has important uses in the treatment of diabetes, obesity, and other related diseases. Summary of the Invention

[0006] This application aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, this application provides a GLP-1R and GCGR dual-target activating polypeptide. In the first aspect of the present application, the present application provides a polypeptide or its derivative, and a pharmaceutically acceptable salt thereof. According to an embodiment of the present application, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1. The polypeptide or its derivative, and the pharmaceutically acceptable salt thereof according to the present application can bind to GLP-1R and GCGR, and are used for effectively activating GLP-1R and GCGR. Thus, the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt thereof can be used for detecting GLP-1R and / or GCGR, and can also be used for treating or preventing GLP-1R and / or GCGR-related diseases (such as diseases related to metabolic disorders, for example, diseases related to dyslipidemia, for example, metabolic syndrome, especially obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease, etc.).

[0007] According to an embodiment of the present application, the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt thereof may further include at least one of the following technical features: According to an embodiment of the present application, the derivative of the polypeptide includes a modifying group, and the polypeptide is linked to the modifying group.

[0008] According to an embodiment of the present application, the modifying group is linked to -NH2 of the amino acid side chain in the polypeptide.

[0009] According to an embodiment of the present application, the modifying group has at least one of the following structures: .

[0010] In the second aspect of the present application, the present application provides the use of the polypeptide or its derivative, and the pharmaceutically acceptable salt thereof according to the first aspect in the preparation of an activator for GLP-1R and GCGR dual targets. As can be seen from the above, the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt thereof can effectively activate GLP-1R and GCGR. Thus, the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt thereof can be made into an activator for GLP-1R and GCGR dual targets, and are used for treating or preventing GLP-1R and / or GCGR-related diseases, such as diseases related to metabolic disorders, for example, diseases related to dyslipidemia, for example, metabolic syndrome, especially obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease, etc.

[0011] In the third aspect of the present application, the present application provides a fusion protein. According to an embodiment of the present application, the fusion protein comprises the polypeptide or its derivative described in the first aspect, and a pharmaceutically acceptable salt. As can be seen from the foregoing, the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and can be used to effectively activate GLP-1R and GCGR. Thus, the fusion protein containing the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and can be used for detecting GLP-1R and / or GCGR or for activating GLP-1R and GCGR. It can also be used for treating or preventing GLP-1R and / or GCGR-related diseases, such as diseases related to metabolic disorders, such as diseases related to dyslipidemia, such as metabolic syndrome, especially obesity, diabetes, fatty liver disease and non-alcoholic fatty liver disease, etc.

[0012] In the fourth aspect of the present application, the present application provides a reagent or a kit. According to an embodiment of the present application, the reagent or the kit comprises the polypeptide or its derivative described in the first aspect, and a pharmaceutically acceptable salt, or the fusion protein described in the third aspect. As can be seen from the foregoing, the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and can be used to effectively activate GLP-1R and GCGR. Thus, the reagent or the kit containing the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and can be used for detecting GLP-1R and / or GCGR.

[0013] In the fifth aspect of the present application, the present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises the polypeptide or its derivative described in the first aspect, and a pharmaceutically acceptable salt, or the fusion protein described in the third aspect. As can be seen from the foregoing, the above-mentioned polypeptide or its derivative, and the pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and can be used to effectively activate GLP-1R and GCGR. Thus, the pharmaceutical composition containing the above-mentioned polypeptide can be used for treating or preventing GLP-1R and / or GCGR-related diseases, such as diseases related to metabolic disorders, such as diseases related to dyslipidemia, such as metabolic syndrome, especially obesity, diabetes, fatty liver disease and non-alcoholic fatty liver disease, etc.

[0014] According to an embodiment of the present application, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient.

[0015] In the sixth aspect of the present application, the present application provides the use of the polypeptide or its derivative, and the pharmaceutically acceptable salt described in the first aspect, the fusion protein described in the third aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug for treating or preventing GLP-1R and / or GCGR-related diseases, such as diseases related to metabolic disorders.

[0016] According to an embodiment of the present application, the GLP-1R and / or GCGR-related diseases include diseases related to metabolic disorders.

[0017] According to an embodiment of the present application, the diseases related to metabolic disorders include diseases related to dyslipidemia.

[0018] According to an embodiment of the present application, the diseases related to dyslipidemia include metabolic syndrome.

[0019] According to an embodiment of the present application, the metabolic syndrome includes at least one of obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease.

[0020] In a seventh aspect of the present application, the present application provides a method for detecting GLP-1R and / or GCGR. According to an embodiment of the present application, the method includes: contacting a sample to be detected with the polypeptide or its derivative, pharmaceutically acceptable salt, fusion protein described in the first aspect, or reagent or kit described in the fourth aspect; and determining whether GLP-1R and / or GCGR is contained in the sample to be detected based on the signal generated by the contact product. As can be seen from the foregoing, the above-mentioned polypeptide or its derivative, pharmaceutically acceptable salt can bind to GLP-1R and GCGR and can be used to effectively activate GLP-1R and GCGR. Thus, the above method can be used to bind to GLP-1R and GCGR for detecting GLP-1R and / or GCGR.

[0021] Additional aspects and advantages of the present application will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 is a schematic diagram of the polypeptide design and screening process of the present application; Figure 2 is the detection result of GA-Dual.R5.S3 activating the GLP-1R target to release cAMP in Example 2 of the present application; Figure 3 is the detection result of GA-Dual.R5.S3 activating the GCGR target to release luciferin in Example 2 of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, "a plurality of" means two or more than two.

[0024] In this text, the term "comprising" or "including" is an open-ended expression, that is, it includes the content specified in the present application, but does not exclude other aspects.

[0025] In this text, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where such events or conditions occur, as well as the cases where such events or conditions do not occur.

[0026] In this text, the term "target protein" refers to a protein that plays a key role in an organism, and is usually the target of action in drug research and development. By binding to the target protein, a drug can regulate its biological activity, thereby achieving the purpose of treating diseases.

[0027] In this text, the term "peptide" is a biological macromolecule composed of many amino acids, usually composed of 50 or fewer amino acids linked together by peptide bonds. Peptides have various functions in organisms, including participating in various physiological processes of organisms as enzymes, hormones, antibodies, etc. In addition, due to the good biocompatibility, selectivity and high biological activity of peptides, they have also been widely studied for drug discovery and treatment of various diseases, such as diseases related to metabolic disorders. In this text, the term "dual-target peptide" refers to a polypeptide molecule that can bind to two different targets simultaneously, which enables the dual-target peptide to have unique advantages in drug research and development and can enhance the therapeutic effect by regulating multiple biological pathways simultaneously.

[0028] In this text, the term "cyclic adenosine monophosphate (cAMP)" is a small molecule that is ubiquitously present in organisms and is converted from ATP (adenosine triphosphate) through the catalytic action of adenylate cyclase. As a key mediator of intracellular signal transduction, cAMP plays a crucial role in regulating various cell functions and physiological processes.

[0029] In this article, the term "peptide bio-activity" refers to the biological functions and activities of peptides in organisms. Peptides can regulate biological processes such as signal transduction, immune response and cell proliferation by binding to specific target proteins. The biological activity of a peptide depends on its amino acid sequence, structure and interaction with the target. Due to their good biocompatibility and selectivity, peptides have important potential in drug development, disease treatment and biotechnology applications.

[0030] In this article, the term “EC 50 " refers to the concentration of a polypeptide or its derivatives or pharmaceutically acceptable salts reaching 50% of the maximum reaction concentration under specific experimental conditions. This reaction can be the physiological reaction of an organism, cell growth, enzyme activity, etc. EC 50 EC values ​​are widely used in drug development, toxicology studies, and biological experiments. 50 Values, their relative potency can be assessed. 50 Lower values ​​indicate a more potent drug because a significant biological response can be elicited at lower concentrations. 50 It is usually determined by a dose-response curve. This curve depicts the effect of different concentrations of the drug on the biological response, usually showing an S-shaped curve. By analyzing this curve, the concentration that makes the response reach 50% can be found.

[0031] In this document, amino acids use the conventional single-letter and three-letter codes for natural amino acids, and the commonly accepted three-letter codes for other α-amino acids. Unless otherwise specified, in this application, capital letters represent amino acids in the L configuration and lowercase letters represent amino acids in the D configuration.

[0032] In this context, the structural formula of the term "αMeK" is .

[0033] In this context, the term "HoK" has the structural formula .

[0034] In this context, the structural formula of the term "N-Me-K" is .

[0035] In this document, the structural formula of the term "Orn" is .

[0036] In this context, the structural formula of the term "Dab" is .

[0037] As used herein, the structural formula of the term "Dap" is .

[0038] As used herein, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other ingredients contained in a polypeptide or its derivative and / or the mammal to be treated therewith. Preferably, "pharmaceutically acceptable" as used in this application refers to those approved by a federal regulatory agency or a national government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly in humans.

[0039] As used herein, the term "pharmaceutically acceptable salt" refers to organic and inorganic salts of the polypeptides or their derivatives of this application. Pharmaceutically acceptable salts are well known in the art. Salts formed from pharmaceutically acceptable non-toxic acids include, but are not limited to, inorganic acid salts formed by reacting with amino groups (such as hydrochloride, hydrobromide, phosphate, sulfate, perchlorate), and organic acid salts (such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate), or these salts can be obtained by other methods described in the literature, such as ion exchange.

[0040] As used herein, the term "pharmaceutical composition" can refer to the treatment of diseases and can also be used for in vitro cell culture experiments. When used for the treatment of diseases, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining the active ingredient with excipients that constitute one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active polypeptide or its derivative with a liquid excipient, a finely divided solid excipient, or both.

[0041] As used herein, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. Except for the scope where any conventional excipient is incompatible with the polypeptides or their derivatives, pharmaceutical compositions, or drugs containing them of this application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their uses are also within the scope contemplated by this application.

[0042] Except for any conventional excipient, the scope where it is incompatible with the polypeptides or their derivatives, pharmaceutical compositions, or drugs containing them of this application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their uses are also within the scope contemplated by this application.

[0043] The pharmaceutical compositions of the present disclosure include formulations suitable for parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the pharmaceutical art. The amount of the active ingredient combined with the excipient substances to prepare a single dosage form is generally the amount of the polypeptide or its derivative that produces a therapeutic effect.

[0044] As used herein, the term "agonist" refers to a substance (ligand) that activates the receptor type.

[0045] As used herein, the term "treatment" is used to refer to obtaining the desired pharmacological and / or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or an adverse effect caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting a disease, such as arresting the progression of the disease; or (c) alleviating a disease, such as relieving the symptoms associated with the disease. "Treatment" as used herein covers any administration of a polypeptide or its derivative, a pharmaceutically acceptable salt, or a medicament containing the same to an individual to treat, cure, relieve, improve, alleviate, or inhibit a disease of the individual, including but not limited to administering a medicament containing the polypeptide or its derivative, a pharmaceutically acceptable salt described herein to an individual in need thereof.

[0046] As used herein, the term "non-alcoholic fatty liver disease (NAFLD)" generally refers to a clinicopathological syndrome characterized by excessive intrahepatic fat deposition excluding that caused by alcohol and other definite liver-damaging factors, an acquired metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility, including but not limited to simple fatty liver (SFL), non-alcoholic steatohepatitis (NASH), and its related cirrhosis.

[0047] The present application provides a GLP-1R and GCGR dual-target activating polypeptide or its derivative, a pharmaceutically acceptable salt thereof, and uses thereof, which will be described in detail below.

[0048] Polypeptide or its derivative, pharmaceutically acceptable salt In one aspect of the present application, the present application provides a polypeptide or its derivative, a pharmaceutically acceptable salt thereof. According to an embodiment of the present application, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.

[0049] The inventors of the present application have optimized to obtain a brand-new GLP-1R and GCGR dual-target peptide through precise calculations, deep learning models, and iterative evolution models. Compared with traditional GLP-1 and GCGR agonists, it has a unique amino acid arrangement, and the above-mentioned polypeptide or its derivative, pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and can effectively activate GLP-1R and GCGR, showing high affinity and biological activity for GLP-1R and GCGR. Therefore, the above-mentioned polypeptide or its derivative, pharmaceutically acceptable salt can be used to detect GLP-1R and / or GCGR, and can also be used to treat or prevent GLP-1R and / or GCGR-related diseases (such as diseases related to metabolic disorders, such as diseases related to dyslipidemia, such as metabolic syndrome, especially obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease, etc.).

[0050] In particular, with the continuous increase in the prevalence of metabolic disorder-related diseases such as diabetes globally and the limitations of existing treatment regimens, the polypeptide or its derivative, pharmaceutically acceptable salt of the present application can not only provide new treatment options for diabetic patients, etc., but also is expected to reshape the market pattern of the treatment of diabetes and the like.

[0051] In this article, the term "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: A" includes the amino acid sequence of SEQ ID NO: A, the amino acid sequence of the conservative modification form of SEQ ID NO: A, or the sequence similarity with the amino acid sequence shown in SEQ ID NO: A is more than 90% (such as more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%), all within the protection scope of the present application. Without special explanation, "the amino acid sequence of the conservative modification form of SEQ ID NO: A, or the sequence similarity with the amino acid sequence shown in SEQ ID NO: A is more than 90% (such as more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%)" The conservative modified amino acids or similar amino acids with differences can be located at any position of the polypeptide of the present application, and can also be located at least one of the 2nd, 4th - 12th, 14th, 22nd, 25th - 26th positions, and can also be located at least one of the 1st, 3rd, 13th, 15th - 21st, 23rd - 24th, 27th - 30th positions. Such amino acid modifications or similar differences do not significantly affect or change the structural stability of the polypeptide containing the amino acid and / or its binding activity with the receptor, and are all within the protection scope of the present application.

[0052] Exemplarily, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1" means that the polypeptide is the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence of a conservative modification form of SEQ ID NO: 1, or the sequence similarity with the amino acid sequence shown in SEQ ID NO: 1 is more than 90% (such as more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%), and all are within the protection scope of this application.

[0053] In this article, "the amino acid sequence of a conservative modification form" refers to such amino acid modifications that do not significantly affect or change the characteristics of the polypeptide containing this amino acid sequence. Such modifications include amino acid substitution, addition, and deletion. The modifications can be introduced into the polypeptides of this application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitution is a substitution in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been determined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Exemplarily, the number of conservative modifications does not exceed 10% of the total number, preferably does not exceed 5% of the total number. In this article, "the amino acid sequence of a conservative modification form" also includes amino acid modifications caused by natural mutations. "Natural mutation" refers to mutations caused by changes such as alleles during the natural mutation process of the polypeptide.

[0054] In this article, the term "sequence similarity" is defined by the percentage similarity method, that is, it is calculated by comparing the number of identical or similar amino acids in two protein or polypeptide sequences to the total number of amino acids. 。

[0055] According to the embodiments of this application, the polypeptide has the amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence with at least 80% sequence similarity thereto; or, compared with the amino acid sequence shown in SEQ ID NO:1, the polypeptide has been substituted, deleted, or added with 1 to 3 amino acids and has GLP-1R and GCGR binding activities. Such as substituting, deleting, or adding 1, 2, or 3 amino acids.

[0056] It should be noted that in this article, "substituting, deleting or adding one or several amino acids" means that after substituting, deleting or adding such amino acids, it does not significantly affect or change the binding characteristics of the original amino acid sequence. Amino acid substitution is the substitution of an amino acid residue in the original peptide chain with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been determined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0057] According to an embodiment of the present application, the amino acid for substitution or addition is selected from amino acid X having -NH2, -SH, -OH or -COOH in its side chain.

[0058] According to an embodiment of the present application, the amino acid for substitution or addition is selected from amino acid X having -NH2 in its side chain. Furthermore, more modifiable sites are added to the original polypeptide, and the side chain -NH2 of the modifiable site can bind to a modifying group, thereby effectively extending the half-life of the polypeptide or its derivative, pharmaceutically acceptable salt in vivo.

[0059] In this article, the term "modifying group" should be understood in a broad sense, and can be a chemical group or an amino acid fragment, and the specific type is not limited, and all are within the protection scope of the present application.

[0060] According to an embodiment of the present application, the modifying group is connected to the -NH2 in the side chain of amino acid K in the polypeptide or amino acid X after substitution or addition; the modifying group has at least one of the following structures: .

[0061] In this article, the " " in the description of the chemical group is used to describe the position of group substitution. That is, the above chemical group is connected to the -NH2 of the amino acid through to form a -CO-NH- connection.

[0062] According to an embodiment of the present application, the amino acid X is selected from K, k, αMeK, HoK, Dap, Dab, Orn, or N-Me-K.

[0063] Use In the second aspect of the present application, the present application provides the use of the polypeptide or its derivative, or a pharmaceutically acceptable salt described in the first aspect in the preparation of an activator for dual targets of GLP-1R and GCGR. As can be seen from the foregoing, the above-mentioned polypeptide or its derivative, or a pharmaceutically acceptable salt can effectively activate GLP-1R and GCGR. Therefore, the above-mentioned polypeptide or its derivative, or a pharmaceutically acceptable salt can be formulated into an activator for dual targets of GLP-1R and GCGR, and is used for treating or preventing GLP-1R and / or GCGR-related diseases, such as diseases related to metabolic disorders, such as diseases related to dyslipidemia, such as metabolic syndrome, especially obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease, etc.

[0064] Fusion protein, reagent or kit, pharmaceutical composition In the third aspect of the present application, the present application provides a fusion protein. According to the embodiments of the present application, the fusion protein includes the polypeptide or its derivative, or a pharmaceutically acceptable salt described in the first aspect. As can be seen from the foregoing, the above-mentioned polypeptide or its derivative, or a pharmaceutically acceptable salt can bind to GLP-1R and GCGR and can be used to effectively activate GLP-1R and GCGR. Therefore, the fusion protein containing the above-mentioned polypeptide or its derivative, or a pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and is used for detecting GLP-1R and / or GCGR or for activating GLP-1R and GCGR, and can also be used for treating or preventing GLP-1R and / or GCGR-related diseases, such as diseases related to metabolic disorders, such as diseases related to dyslipidemia, such as metabolic syndrome, especially obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease, etc.

[0065] In an optional embodiment of the present application, the fusion protein further includes a functional fragment.

[0066] As used herein, the term "functional fragment" refers to an amino acid fragment, which can be a functionally active fragment or a protein tag, and the specific type is not limited, and all are within the protection scope of the present application.

[0067] It should be noted that the above-mentioned functionally active fragment can be used to exert its function in animals or in vitro. Exemplarily, when the functionally active fragment is used to exert its function in animals, it can be used for preventing and / or treating diseases; when the functionally active fragment is used to exert its function in vitro, it is used for specifically binding to a certain substance, can be used for detecting the substance, or can be used for in vitro diagnosis of diseases.

[0068] It should be noted that the above protein tag refers to a short peptide co-expressed with the target protein, which facilitates the expression, detection, tracing, or purification of the polypeptide of the present application. Exemplarily, the protein tag includes at least one of His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

[0069] In the fourth aspect of the present application, the present application proposes a reagent or a kit. According to an embodiment of the present application, the reagent or the kit includes the polypeptide or its derivative described in the first aspect, a pharmaceutically acceptable salt, or the fusion protein described in the third aspect. As can be seen from the foregoing, the above polypeptide or its derivative, pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and can be used to effectively activate GLP-1R and GCGR. Thus, a reagent or a kit containing the above polypeptide or its derivative, pharmaceutically acceptable salt can bind to GLP-1R and GCGR and be used to detect GLP-1R and / or GCGR.

[0070] In this article, the kit or reagent does not need to have a box structure, only requires being relatively independent and having a suitable loading or container, such as a tube, box, bottle, card; certain components are located in different containers, and if permitted, certain components can be combined in one container.

[0071] According to an embodiment of the present application, the kit includes reagents suitable for detection. In some embodiments, the kit may contain instructions for detection. In some embodiments, the kit may contain a calibrator or a control, such as a standard or a control sample. In some embodiments, the kit further contains containers in the kit, such as test tubes, microplates, or test strips.

[0072] In the fifth aspect of the present application, the present application proposes a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition includes the polypeptide or its derivative described in the first aspect, a pharmaceutically acceptable salt, or the fusion protein described in the third aspect. As can be seen from the foregoing, the above polypeptide or its derivative, pharmaceutically acceptable salt can bind to GLP-1R and GCGR, and can be used to effectively activate GLP-1R and GCGR. Thus, a pharmaceutical composition containing the above polypeptide can be used to treat or prevent GLP-1R and / or GCGR-related diseases, such as metabolic disorder-related diseases, such as dyslipidemia-related diseases, such as metabolic syndrome, especially obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease, etc.

[0073] According to an embodiment of the present application, the pharmaceutical composition may further include pharmaceutically acceptable excipients.

[0074] In an alternative embodiment of the present application, a pharmaceutically acceptable excipient refers to conventional pharmaceutical excipients in the pharmaceutical field, such as diluents, buffers, osmotic pressure regulators, pH regulators, etc.

[0075] In an alternative embodiment of the present application, a pharmaceutically acceptable carrier refers to conventional pharmaceutical carriers in the pharmaceutical field, such as protective agents, etc.

[0076] In an alternative embodiment of the present application, a pharmaceutically acceptable vehicle refers to conventional pharmaceutical vehicles in the pharmaceutical field, such as solutions (e.g., water) and liposomes.

[0077] In an alternative embodiment of the present application, examples of suitable pharmaceutically acceptable carriers, excipients, and vehicles are well-known in the art. Pharmaceutical compositions containing such carriers, excipients, and vehicles can be formulated by well-known conventional methods.

[0078] In some alternative embodiments of the present application, the pharmaceutical composition can be an oral preparation, such as a solid oral preparation or a liquid oral preparation, and the specific type is not limited and is within the protection scope of the present application.

[0079] In some alternative embodiments of the present application, the pharmaceutical composition of the present application can also contain other active ingredients for treatment.

[0080] The pharmaceutical composition of the present application can be administered in different ways, such as enterally, orally (e.g., liquid solution), via injection (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal). Preferably, the pharmaceutical composition of the present application is in the form of a freeze-dried preparation or an aqueous solution. The clinical dosing regimen will be determined by the attending physician and clinical factors. As is well-known in the medical field, the dose for any patient depends on many factors, including the patient's physique, body surface area, age, drug to be administered, gender, administration time and route, general health, and other drugs administered simultaneously. The pharmaceutical composition of the present application can be administered locally or systemically. Preferably, it can be administered intravenously or subcutaneously.

[0081] Use In the sixth aspect of the present application, the present application provides the use of the polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, the fusion protein described in the third aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a drug for treating or preventing GLP-1R and / or GCGR-related diseases, such as diseases related to metabolic disorders.

[0082] The present application provides the use of the polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, the fusion protein described in the third aspect, or the pharmaceutical composition described in the fifth aspect in treating or preventing GLP-1R and / or GCGR-related diseases.

[0083] The polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, the fusion protein described in the third aspect, or the pharmaceutical composition described in the fifth aspect of the present application is used for treating or preventing GLP-1R- and / or GCGR-related diseases.

[0084] According to an embodiment of the present application, the above use may further include at least one of the following technical features: According to an embodiment of the present application, the GLP-1R- and / or GCGR-related diseases include metabolic disorder-related diseases.

[0085] According to an embodiment of the present application, the metabolic disorder-related diseases include dyslipidemia-related diseases.

[0086] According to an embodiment of the present application, the dyslipidemia-related diseases include metabolic syndrome.

[0087] According to an embodiment of the present application, the metabolic disorder-related diseases include at least one of obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease.

[0088] Method In the seventh aspect of the present application, the present application provides a method for detecting GLP-1R and / or GCGR. According to an embodiment of the present application, the method includes: contacting a sample to be detected with the polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, the fusion protein described in the third aspect, or the reagent or kit described in the fourth aspect; and determining whether the sample to be detected contains GLP-1R and / or GCGR based on the signal generated by the contact product. As can be seen from the above, the above polypeptide or its derivative, pharmaceutically acceptable salt can bind to GLP-1R and GCGR and can be used to effectively activate GLP-1R and GCGR. Therefore, the above method can bind to GLP-1R and GCGR and is used for detecting GLP-1R and / or GCGR.

[0089] According to a specific embodiment of the present application, the signal includes a fluorescence signal.

[0090] According to a specific embodiment of the present application, it further includes determining the content value of GLP-1R and / or GCGR in the sample to be detected based on the signal generated by the contact product.

[0091] In the eighth aspect of the present application, the present application provides a method for treating or preventing metabolic disorder-related diseases. According to an embodiment of the present application, the method includes: administering to a subject a pharmaceutically acceptable dose of the polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, the fusion protein described in the third aspect, or the pharmaceutical composition described in the fifth aspect.

[0092] In an alternative embodiment of the present application, the pharmaceutically acceptable dose may be selected from an effective dose (or effective amount).

[0093] The effective amount of the polypeptide or its derivative, pharmaceutically acceptable salt of the present application may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art based on various factors (such as through clinical trials). Such factors include but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0094] The polypeptide or its derivative, pharmaceutically acceptable salt, polypeptide derivative or its pharmaceutically acceptable salt, or pharmaceutical composition of the present application can be incorporated into drugs suitable for use in medicine, and these drugs can be prepared in various forms, such as liquid, semi-solid and solid dosage forms, etc., including but not limited to solid dosage forms, semi-solid dosage forms, liquid dosage forms and gas dosage forms, etc. Various ways of the polypeptide or its derivative, pharmaceutically acceptable salt, polypeptide derivative or its pharmaceutically acceptable salt, pharmaceutical composition or drug administration of the present application are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, topical, nasal, pulmonary, rectal and topical applications, but the present application is not limited to these exemplified administration methods.

[0095] According to an embodiment of the present application, the metabolic disorder-related diseases include dyslipidemia-related diseases.

[0096] According to an embodiment of the present application, the dyslipidemia-related diseases include metabolic syndrome.

[0097] According to an embodiment of the present application, the metabolic disorder-related diseases include at least one of obesity, diabetes, fatty liver disease and non-alcoholic fatty liver disease.

[0098] The solution of the present application will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those not specified in the embodiments regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained commercially.

[0099] Example 1: The schematic diagram of the polypeptide design and screening process of the present application is as Figure 1, which covers a series of steps from the generation to screening and optimization of GLP-1R and GCGR dual-target peptide sequences. The whole method consists of multiple iterative evolution processes. After each iterative evolution, the polypeptides designed by the model can obtain effective improvement in biological activity. Each iterative evolution includes three parts, namely, the generation and optimization of new polypeptides based on artificial intelligence, the determination of polypeptide biological activity through wet experiments, and feedback to strengthen the model's perception of biological activity.

[0100] First, through an innovative deep learning method TPDiffusion, it is possible to generate polypeptide sequences that can bind to the target protein according to the amino acid sequence of the target protein. The process of transforming the target protein sequence into a polypeptide sequence can be regarded as a process of giving specific answers to specific questions. By training the conditional diffusion model TPDiffusion to learn the relationship rules between the target protein and the polypeptide sequence, specific polypeptides (i.e., GLP-1R and GCGR dual-target peptides) can be generated for specific target proteins (i.e., GLP-1R and GCGR). The training of the polypeptide sequence generation model TPDiffusion mainly includes the forward diffusion process and the reverse diffusion process. The forward diffusion process includes the following steps: 1. Encoding the amino acid sequence: To model the joint feature space of protein-polypeptide, the target protein and polypeptide sequences are spliced together as a whole, and an embedding transformation function is introduced to map each discrete amino acid word or character to a continuous vector encoding space.

[0101] 2. Gradually adding noise to the polypeptide sequence: Gaussian noise is gradually added to the polypeptide part in the vector encoding based on the Markov chain until the polypeptide sequence is completely destroyed.

[0102] After the forward diffusion process is completed, a set of clean target protein and noisy polypeptide sequences will be obtained. Then, the polypeptide sequence is restored through the reverse diffusion process, which mainly includes the following steps: 1. Gradually denoising the polypeptide sequence: By constructing a denoising network to estimate the noise distribution added in the forward diffusion process and gradually denoising the polypeptide part until the polypeptide sequence is completely restored.

[0103] 2. Calculating the loss function: The model outputs the probability distribution of the predicted polypeptide sequence, and the mean square error loss function is used to calculate the difference between the model prediction result and the real result, and the model parameters are updated through backpropagation.

[0104] The denoising network in TPDiffusion adopts the BERT (Devlin et al. 2018) model. The model architecture of BERT is mainly composed of multiple layers of Transformer (Vaswani et al. 2017) encoders. Each Transformer block contains two parts: self-attention and feed-forward neural network. Through the self-attention mechanism, TPDiffusion will introduce target sequence information during the reverse diffusion process of restoring the polypeptide sequence, thereby implicitly modeling the relationship characteristics between the target protein and the polypeptide sequence, and realizing the relationship mapping from the target protein to the polypeptide sequence. During the generation process, given any target protein sequence, the model will first randomly sample from Gaussian noise and then perform the reverse diffusion process. Guided by the target protein sequence, the noise is gradually eliminated through a fixed number of time steps, and finally, a binding polypeptide sequence for the given target can be generated. By using the trained TPDiffusion, the sequences of two targets, GLP-1R and GCGR, are used as inputs to generate a batch of candidate polypeptide sequences that may have high affinity for the two targets, GLP-1R and GCGR.

[0105] Second, affinity maturation is performed on these high-affinity candidate polypeptide sequences. This method is an innovative deep reinforcement learning scheme dedicated to optimizing polypeptide sequences, guiding the evolution of candidate polypeptide sequences by simulating the environment and defining actions. This deep reinforcement learning framework can combine some reward models as prior knowledge to guide the evolution of polypeptides. The reward model can be an affinity prediction model, a solubility prediction model, a toxicity prediction model, etc.

[0106] The reward model is an innovative technical solution, PepAF, which effectively predicts the binding affinity between the target protein and the polypeptide by comprehensively using structural information, flexibility characteristics, and advanced pre-training strategies. PepAF first learns on two pre-training tasks: 1) estimating the binding free energy of the protein (GLP-1R and GCGR)-polypeptide complex; 2) predicting the affinity of the protein (GLP-1R and GCGR)-polypeptide complex. The first task enables the model to learn the complex interactions between the protein (GLP-1R and GCGR) and the polypeptide, as well as their structures and physicochemical properties, which provides a basis for understanding the binding mode and key features of the protein (GLP-1R and GCGR)-polypeptide interaction. The second task enables the model to capture a wide range of molecular interactions at the atomic scale. PepAF also improves the prediction accuracy by modeling the target protein structure and the polypeptide flexibility. The core technology PepAF in this patent is used as the reward model of the deep reinforcement learning method to guide the mutation of candidate polypeptide sequences.

[0107] After obtaining the affinity-matured polypeptide, its biological activity is evaluated, and the results are then fed back to the model to continue the next round of polypeptide generation and optimization. After one round of iteration, a polypeptide that effectively activates the two target proteins GLP-1R and GCGR is designed, and the sequence is shown in SEQ ID NO:1. Among them, the amino acid sequence shown in SEQ ID NO:1 is as follows:

[0108] Example 2: Evaluation of the activation ability of candidate polypeptides on GLP-1R and GCGR target proteins simultaneously 1. It is aimed to evaluate the ability of the candidate polypeptide (GA-Dual.R5.S3) obtained in Example 1 to activate cAMP activity on huGLP-1R-FL-CRE-HEK293-A5 cells overexpressing. By quantitatively measuring the production level of cAMP, this experiment can accurately evaluate the activation effect of the candidate sample on the GLP-1R receptor, providing important biological activity data for the drug development of metabolic diseases such as diabetes. The experimental procedure is as follows: Polypeptide synthesis and purification: According to the designed specific amino acid sequence, the candidate polypeptides were synthesized respectively using solid-phase peptide synthesis (Fmoc-SPPS) technology. The purification process used high-performance liquid chromatography (HPLC) to ensure that the synthesized polypeptides had high purity and no obvious impurities. In addition, the exact molecular weight of the polypeptides was further verified by mass spectrometry (MS).

[0109] Polypeptide dissolution conditions: The purified candidate polypeptides were dissolved in DMSO (dimethyl sulfoxide) solvent respectively to prepare a stock solution of 1 mg / mL for subsequent experiments.

[0110] Cell starvation treatment: A huGLP-1R-FL-CRE-HEK293-A5 cell line was constructed, and a huGLP-1R-FL-CRE-HEK293-A5 cell line with high viability was selected for starvation treatment with DMEM medium containing 0% fetal bovine serum (FBS) to standardize the experimental conditions and exclude the interference of other factors in the serum.

[0111] Cell digestion, washing and plating: The adherent cells were gently digested with Accutase enzyme and washed with PBS buffer to remove the residual culture medium and enzyme. The cell suspension was accurately plated in a HTRF 96-well low-volume detection plate to ensure the consistency of the number of cells per well, providing a uniform starting condition for the subsequent cAMP activity detection.

[0112] cAMP activity detection: After cell plating, a precisely diluted candidate polypeptide was added to each well (for specific concentrations, see Figure 2(abscissa), incubate at 37 °C for 20 minutes to simulate the in-vivo environment and activate the GLP-1R and GCGR receptors. Add the cAMP d2 reagent and the working solution of anti-cAMP Eu Cryptate antibody (PerkinElmer, catalog number 62AM4PEB) to each well and incubate at room temperature for 1 hour. This step binds cAMP to the fluorescently labeled antibody to form an energy transfer complex.

[0113] Signal detection: Using the TR-FRET technique, set the excitation wavelength to 340 nm and detect the emission signals at 665 nm and 620 nm respectively. This technique has high sensitivity and anti-interference ability. The FRET signal value is calculated by the ratio of the light intensities at 665 nm and 620 nm, which reflects the concentration change of cAMP. The change in the ratio is inversely proportional to the cAMP concentration.

[0114] It was found that the synthesized candidate polypeptides all had good cAMP activation activity functions, indicating that the candidate polypeptides all had good cAMP activation activity functions and had high GLP-1R activation biological activity. The detection results are as Figure 2 shown, and the EC of some candidate polypeptides 50 is shown in Table 1.

[0115] Table 1

[0116] 2. Aim to evaluate the activation ability of the candidate polypeptide (GA-Dual.R5.S3) obtained in Example 1 on the GCGR target. The experimental procedure is as follows: First, dilute the candidate polypeptides separately using DMEM complete medium to prepare a series of dilutions, and add 50 μL of the dilution to the culture plate. Add human GCGR-CRE-luci-HEK293-A5 cells at a concentration of 1×10 5 cells / well, add 50 μL to the culture plate, and mix with the diluted agonist. Place the culture plate in an incubator at 37 °C and incubate for 6 hours to ensure that the cells fully absorb the agonist and activate GCGR. Add 50 μL Bright-Lite (Vazyme, DD1204-04-AA) to each well, and then use a luminometer to read the luciferase signal to evaluate the activation effect of GCGR.

[0117] It was found that the synthesized candidate polypeptides had good luciferase activation activity functions, indicating that the candidate polypeptides had high GCGR activation biological activity. The detection results are as Figure 3 shown, and the EC of some candidate polypeptides 50 is shown in Table 2.

[0118] Table 2

[0119] Example 3: This example aims to verify the therapeutic effects of the candidate polypeptide (GA-Dual.R5.S3) in Example 1 on obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease in vitro. The specific steps are as follows: 1) For obesity: Using 3T3-L1 adipocytes, first induce cell differentiation into adipocytes with high-glucose DMEM medium (containing 4.5 g / L glucose, supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, Thermo Fisher Scientific, catalog number 11995065) to establish an obesity model. When the confluence of 3T3-L1 preadipocytes reaches 80 - 90%, digest with 0.25% trypsin and inoculate at a density of 2 - 3×10 4 cells / cm 2 . When the cell confluence reaches 80 - 95%, add adipogenic induction differentiation medium for induction. After 2 - 3 days of induction, change to adipogenic induction differentiation maintenance medium and maintain for 1 day, and alternate induction 3 - 5 times until obvious lipid droplets appear in the cells, indicating that the obesity model is successfully constructed.

[0120] Then add the candidate polypeptide to adipocyte culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), and control the cell density at 2 - 3×10 4 cells / cm 2 to ensure the normal growth and differentiation of cells, and culture for 24 - 48 hours. Detect the content of lipid droplets in the cells by Oil Red O staining: aspirate the culture medium, rinse with 1×PBS, add 4% neutral formaldehyde solution to fix for 30 min, prepare Oil Red O working solution (saturated Oil Red O solution: distilled water = 3:2), filter and use for staining, add 1 mL of Oil Red O working solution to each well, stain at room temperature for 30 min, aspirate the staining solution, rinse with 1×PBS, observe and take pictures under a microscope. At the same time, use a qPCR kit (Takara RR420A) to detect the expression levels of adipogenesis-related genes (PPARγ, C / EBPα). If the candidate polypeptide can significantly reduce the content of lipid droplets in the cells and decrease the expression of adipogenesis-related genes, it indicates that it has potential therapeutic effects on obesity and may play a role by inhibiting adipocyte differentiation and lipid accumulation.

[0121] 2) For diabetes: Using pancreatic islet β cell line (MIN6 cells), the cells were cultured in high-glucose DMEM medium (containing 4.5 g / L glucose, supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, Thermo Fisher Scientific, catalog number 11995065) to simulate the high-glucose environment of diabetes. The candidate polypeptides were added to cell culture media at different concentrations (10 nM, 100 nM, 1 μM and 10 μM) respectively, and the cell density was controlled at 5×10 3 cells / cm 2 , and cultured for 24 - 48 hours. An insulin ELISA kit was used to detect the insulin secretion in the cell culture supernatant. At the same time, a GLU2 kit (YUANJU BIO, catalog number YJ24519) and an IRS1 kit (Thermo Fisher Scientific, catalog number KHO0521) were used to detect the expression levels of proteins related to the insulin signaling pathway such as glucose transporter (GLUT2) and insulin receptor substrate (IRS). If the candidate polypeptide can increase the insulin secretion and up-regulate the expression levels of GLUT2 and IRS, it indicates that it may have the effects of improving insulin resistance and promoting insulin secretion, and has potential value for the treatment of diabetes.

[0122] 3) For dyslipidemia-related diseases: The hepatocyte cell line (HepG2 cells) was selected and cultured with a medium containing high concentrations of fatty acids (Wuhan Shang'en Biotechnology Co., Ltd., special medium for HepG2 cells, the components of which include EMEM (MEM + NEAA) treated cells + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) to establish a dyslipidemia model. The candidate polypeptides were added to cell culture media at different concentrations (10 nM, 100 nM, 1 μM and 10 μM) respectively, and the cell density was controlled at 5×10 3 cells / cm 2, incubate for 24 - 48 hours. Use an enzymatic assay kit (Total Cholesterol Assay Kit from Sigma - Aldrich, catalog number MAK043) to detect the intracellular total cholesterol content, and use a triglyceride assay kit (Triglyceride Assay Kit from Sigma - Aldrich, catalog number MAK041) to detect the triglyceride content. Meanwhile, use an LDLR kit (from Thermo Fisher Scientific, catalog number EHLDLR) and a CYP7A1 kit (from proteintech, catalog number 18054 - 1 - AP) to detect the expression levels of genes related to cholesterol metabolism such as low - density lipoprotein receptor (LDLR) and cholesterol 7α - hydroxylase (CYP7A1). If the candidate polypeptide can reduce the intracellular total cholesterol and triglyceride contents and up - regulate the expression of LDLR and CYP7A1 simultaneously, it indicates that it may have a certain therapeutic effect on dyslipidemia - related diseases by regulating cholesterol synthesis and metabolism.

[0123] 4) For fatty liver disease: Also use HepG2 cells, and induce cell steatosis with a high - fat medium (a special medium for HepG2 cells from Wuhan Shangen Biotechnology Co., Ltd., whose components include EMEM (MEM + NEAA) + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) to establish a fatty liver disease model. Add the candidate polypeptide to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM) respectively, and control the cell density at 5×10 3 cells / cm 2 , incubate for 24 - 48 hours. Use Oil Red O staining to detect the intracellular lipid droplet content: Aspirate the medium, wash with 1×PBS, add 4% neutral formaldehyde solution to fix for 30 min, prepare an Oil Red O working solution (saturated Oil Red O solution: distilled water = 3:2), filter it and use it for staining. Add 1 mL of the Oil Red O working solution to each well, stain at room temperature for 30 min, aspirate the staining solution, wash with 1×PBS, and observe and take pictures under a microscope. Meanwhile, use ELISA kits (TNF - α and IL - 6 ELISA kits from Thermo Fisher Scientific, catalog numbers K1480 and K1501 respectively) to detect the secretion levels of inflammatory factors, and use colorimetric assay kits (MDA Assay Kit from Cayman Chemical, catalog number 700455 and SOD Assay Kit, catalog number 705125) to detect oxidative stress indicators. If the candidate polypeptide can reduce the accumulation of intracellular lipid droplets, decrease the secretion of inflammatory factors, and improve the oxidative stress state, it indicates that it may have a therapeutic effect on fatty liver disease and can reduce hepatocyte steatosis and inflammatory damage.

[0124] 5) For metabolic syndrome: Construct a cell model of metabolic syndrome, and multiple cells (such as adipocytes, hepatocytes, muscle cells, etc.) can be used in combination to simulate the complex pathological environment of metabolic syndrome.

[0125] Adipocytes: Use 3T3-L1 adipocytes and induce their differentiation into adipocytes with high-glucose DMEM medium (containing 4.5 g / L glucose, supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) to establish an obesity model.

[0126] Hepatocytes: Use HepG2 cells and treat them with a high-fat medium (EMEM + 10% fetal bovine serum + 1% penicillin / streptomycin, supplemented with a high concentration of fatty acids) to simulate the environment of fatty liver disease.

[0127] Muscle cells: Use C2C12 muscle cells and culture them with high-glucose DMEM medium to simulate the environment of insulin resistance.

[0128] Add the candidate polypeptides to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM) respectively, and control the cell density at 5×10 3 cells / cm 2 , and culture for 24 - 48 hours. After culture, detect the indexes related to metabolic syndrome in each cell, such as insulin resistance index (HOMA-IR, calculated by detecting insulin and glucose levels), blood lipid levels (detect triglyceride and total cholesterol levels using a triglyceride assay kit (Triglyceride Assay Kit from Sigma-Aldrich, catalog number MAK041) and a total cholesterol assay kit (Total Cholesterol Assay Kit from Sigma-Aldrich, catalog number MAK043)), inflammatory factors (detect the secretion levels of TNF-α and IL-6 using ELISA kits (TNF-α and IL-6 ELISA kits from Thermo Fisher Scientific, catalog numbers K1480 and K1501) respectively). If the candidate polypeptide can improve insulin resistance, reduce blood lipid levels, and decrease the secretion of inflammatory factors, it indicates that it has potential therapeutic effects on metabolic syndrome and may comprehensively regulate metabolic disorders through a multi-target mechanism of action.

[0129] 6) For non-alcoholic fatty liver disease: HepG2 cells were used and high-fat and high-glucose culture medium (Wuhan Shanen Biotechnology Co., Ltd., HepG2 cell-specific culture medium, whose components include EMEM (MEM+NEAA) treated cells + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) was used to induce non-alcoholic fatty liver disease-like changes in cells. The candidate peptides were added to the cell culture medium at different concentrations (10 nM, 100 nM, 1 μM and 10 μM), and the cell density was controlled at 5×10 3 cells / cm 2 , cultured for 24-48 hours. The content of intracellular lipid droplets was detected by Oil Red O staining, and the secretion level of inflammatory factors was detected by ELISA kits (IL-1β and IL-8 ELISA kits from Thermo Fisher Scientific, catalog numbers K1480 and K1501, respectively). The level of hepatocyte injury markers was detected by colorimetric kits (ALT and AST AssayKit from Cayman Chemical, catalog numbers 700455 and 705125, respectively). The expression level of indicators related to liver fibrosis (collagen I and α-smooth muscle actin) was detected by Western blot and qPCR methods. If the candidate polypeptide can reduce the intracellular fat content, reduce the secretion of inflammatory factors, reduce the level of hepatocyte injury markers, and inhibit the expression of liver fibrosis-related indicators, it means that it has a certain therapeutic effect on non-alcoholic fatty liver disease, and may play a role by reducing fat accumulation, inhibiting inflammation and fibrosis.

[0130] It was found that the candidate polypeptide in Example 1 had the following effects: 1) Both can significantly reduce the content of lipid droplets in 3T3-L1 adipocytes and reduce the expression of genes related to adipogenesis; 2) Both can increase the secretion of insulin in pancreatic β-cells (such as MIN6 cells) and upregulate the expression levels of proteins related to the insulin signaling pathway, such as glucose transporter (GLUT2) and insulin receptor substrate (IRS); 3) Reduce the levels of total cholesterol and triglycerides in the hepatocyte lineage, and upregulate the expression of genes related to cholesterol metabolism, such as low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1); 4) Reduce the accumulation of lipid droplets in HepG2 cells, reduce the secretion of inflammatory factors, and improve oxidative stress; 5) Improve indicators related to metabolic syndrome in various cells (such as fat cells, liver cells, muscle cells, etc.); 6) Reduce the fat content in HepG2 cells, decrease the secretion of inflammatory factors, lower the levels of hepatocyte injury markers, and inhibit the expression of liver fibrosis-related indicators.

[0131] In summary, it can be shown that the above-mentioned candidate polypeptide GA-Dual.R5.S3 has therapeutic effects on obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0132] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0133] Although the embodiments of this application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting this application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of this application.

Claims

1. A polypeptide or its derivative, a pharmaceutically acceptable salt, characterized in that, The amino acid sequence of the polypeptide is as shown in SEQ ID NO:

1.

2. The polypeptide or its derivative, pharmaceutically acceptable salt according to claim 1, characterized in that, The derivative of the polypeptide includes a modifying group, and the polypeptide is linked to the modifying group.

3. The polypeptide or its derivative, pharmaceutically acceptable salt according to claim 2, characterized in that, The modifying group is linked to -NH2 of the amino acid side chain in the polypeptide; The modifying group has at least one of the following structures: 。 4. Use of the polypeptide or its derivative, pharmaceutically acceptable salt according to any one of claims 1 to 3 in the preparation of an activator for dual targets of GLP-1R and GCGR.

5. A fusion protein, characterized in that, Comprising the polypeptide or its derivative, pharmaceutically acceptable salt according to any one of claims 1 to 3.

6. A reagent or kit, characterized in that, Comprising the polypeptide or its derivative, pharmaceutically acceptable salt according to any one of claims 1 to 3.

7. A pharmaceutical composition, characterized in that, Comprising the polypeptide or its derivative, pharmaceutically acceptable salt according to any one of claims 1 to 3; and Optionally, pharmaceutically acceptable excipients.

8. Use of the polypeptide or its derivative, pharmaceutically acceptable salt according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating or preventing GLP-1R and / or GCGR-related diseases.

9. The use according to claim 8, wherein The GLP-1R and / or GCGR-related diseases include diseases related to metabolic disorders.

10. The use according to claim 9, characterized in that, The diseases related to metabolic disorders include diseases related to dyslipidemia.

11. The use according to claim 10, characterized in that, The diseases related to dyslipidemia include metabolic syndrome.

12. The use according to claim 11, wherein, The metabolic syndrome includes at least one of obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease.

13. A method for detecting GLP-1R and / or GCGR, characterized in that, Comprising: Contacting a sample to be detected with the polypeptide or its derivative, pharmaceutically acceptable salt according to any one of claims 1 to 3, the fusion protein according to claim 5, or the reagent or kit according to claim 6; Based on the signal generated by the contact product, determining whether GLP-1R and / or GCGR is contained in the sample to be detected.

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