GLP-1R and GCGR dual-target activating polypeptide or its derivatives, pharmaceutically acceptable salts and uses thereof

Abstract

The present application proposes a polypeptide or its derivative, or a pharmaceutically acceptable salt, the amino acid sequence of which is shown in SEQ ID NO: 1. The polypeptide or its derivative, or pharmaceutically acceptable salt of the present application can bind to GLP-1R and GCGR to effectively activate GLP-1R and GCGR. Thus, the above-mentioned polypeptide or its derivative, or 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 metabolic disorders, such as obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease).

Description

Technical Field

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

[0002] Glucagon-like peptide-1 (GLP-1) is a 31-amino acid polypeptide expressed by the proglucagon gene in intestinal mucosal L cells. GLP-1 primarily stimulates insulin secretion and inhibits glucagon secretion by binding to the GLP-1 receptor (GLP-1R), protecting pancreatic beta cells and regulating blood glucose homeostasis. Furthermore, GLP-1 can suppress appetite and gastric emptying through central nervous system signaling pathways, increasing satiety and thereby reducing weight.

[0003] Glucagon (GCG) is a 29-amino acid polypeptide secreted by pancreatic α-cells. It primarily targets the glucagon receptor (GCGR), which is primarily located in the liver and kidneys. GCG stimulates hepatic glycogenolysis, elevates blood glucose levels, activates lipase, promotes fat breakdown, and enhances fatty acid oxidation, thereby increasing ketone body production. Research results have shown that GCG is effective in reducing food intake, increasing adipose tissue energy expenditure, and reducing body fat.

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

[0005] However, these single-target agonists have limitations in the treatment of diabetes, such as short half-lives and susceptibility to degradation. Currently, in addition to the aforementioned single agonists targeting GLP-1R or GCGR, trials have shown that several GLP-1R / GCGR dual agonists may lead to more significant weight loss effects and also show potential advantages in the treatment of fatty liver disease and related complications, exerting positive effects by synergistically regulating glucose and lipid metabolism. Therefore, the development of a novel GLP-1R and GCGR dual-target agonist peptide has important applications in the treatment of diabetes, obesity, and other related diseases. Summary of the Invention

[0006] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a GLP-1R and GCGR dual-target activating polypeptide.

[0007] In the first aspect of the present application, a polypeptide or a derivative thereof, or a pharmaceutically acceptable salt thereof is provided. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. The polypeptide or its derivative or pharmaceutically acceptable salt of the present application can bind to GLP-1R and GCGR to effectively activate GLP-1R and GCGR. Thus, the above-mentioned polypeptide or its derivative or 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 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.).

[0008] According to the embodiments of the present application, the above-mentioned polypeptide or its derivatives or pharmaceutically acceptable salts may further include at least one of the following technical features:

[0009] According to an embodiment of the present application, the polypeptide derivative includes a modification group, and the polypeptide is connected to the modification group.

[0010] According to an embodiment of the present application, the modifying group is connected to the -NH2 of the amino acid side chain in the polypeptide.

[0011] According to an embodiment of the present application, the modifying group has at least one of the following structures:

[0012] .

[0013] In the second aspect of the present application, the present application proposes the use of the polypeptide or its derivatives or pharmaceutically acceptable salts described in the first aspect in the preparation of a dual-target activator of GLP-1R and GCGR. As previously mentioned, the polypeptide or its derivatives or pharmaceutically acceptable salts can effectively activate GLP-1R and GCGR. Thus, the polypeptide or its derivatives or pharmaceutically acceptable salts can be prepared as a dual-target activator of GLP-1R and GCGR for the treatment or prevention of GLP-1R and / or GCGR-related diseases, such as metabolic disorders, such as dyslipidemia-related diseases, such as metabolic syndrome, especially obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease.

[0014] In the third aspect of the present application, the present application proposes a fusion protein. According to an embodiment of the present application, the fusion protein includes the polypeptide or its derivatives and pharmaceutically acceptable salts described in the first aspect. As mentioned above, the above-mentioned polypeptide or its derivatives and pharmaceutically acceptable salts 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 derivatives and pharmaceutically acceptable salts can bind to GLP-1R and GCGR, and can be used to detect GLP-1R and / or GCGR or to activate GLP-1R and GCGR, and can also 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.

[0015] In a fourth aspect of the present application, a reagent or kit is provided. According to an embodiment of the present application, the reagent or kit includes the polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, or the fusion protein described in the third aspect. As previously mentioned, the 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 kit containing the polypeptide or its derivative, pharmaceutically acceptable salt can bind to GLP-1R and GCGR and be used to detect GLP-1R and / or GCGR.

[0016] In the fifth aspect of the present application, the present application proposes a pharmaceutical composition. According to the embodiments of the present application, the pharmaceutical composition comprises the polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, or the fusion protein described in the third aspect. As mentioned above, 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. Therefore, the pharmaceutical composition containing the above-mentioned 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.

[0017] According to an embodiment of the present application, the pharmaceutical composition may further include pharmaceutically acceptable excipients.

[0018] In the sixth aspect of the present application, the present application proposes the use of the polypeptide or its derivatives, pharmaceutically acceptable salts 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, which is used to treat or prevent GLP-1R and / or GCGR related diseases, such as metabolic disorder-related diseases.

[0019] According to an embodiment of the present application, the GLP-1R and / or GCGR related diseases include metabolic disorder related diseases.

[0020] According to an embodiment of the present application, the metabolic disorder-related diseases include dyslipidemia-related diseases.

[0021] According to an embodiment of the present application, the dyslipidemia-related disease includes metabolic syndrome.

[0022] 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.

[0023] In the seventh aspect of the present application, the present application proposes a method for detecting GLP-1R and / or GCGR. According to an embodiment of the present application, the method comprises: contacting the 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; based on the signal generated by the contact product, determining whether the sample to be detected contains GLP-1R and / or GCGR. 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. Therefore, the above-mentioned method can be used to bind to GLP-1R and GCGR for detecting GLP-1R and / or GCGR.

[0024] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0026] Figure 1 Schematic diagram of the peptide design and screening process of this application;

[0027] Figure 2 This is the detection result of GA-Dual.R5.S3 activating the GLP-1R target to release cAMP in Example 2 of the present application;

[0028] Figure 3 This is the detection result of GA-Dual.R5.S3 activating the GCGR target to release fluorescein in Example 2 of this application. DETAILED DESCRIPTION

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0030] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in this application, but not excluding other contents.

[0031] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0032] In this article, the term "target protein" refers to a protein that plays a key role in an organism and is typically the target of drug development. By binding to the target protein, drugs can modulate its biological activity, thereby achieving the goal of treating the disease.

[0033] In this article, the term "peptide" refers to a biological macromolecule composed of many amino acids, typically 50 or fewer linked together by peptide bonds. Peptides have diverse functions within organisms, including participating in various physiological processes as enzymes, hormones, and antibodies. Furthermore, due to their excellent biocompatibility, selectivity, and high bioactivity, peptides are widely studied for drug discovery and treatment of various diseases, such as those associated with metabolic disorders.

[0034] In this article, the term "dual-target peptide" refers to a polypeptide molecule that can simultaneously bind to two different targets. This gives dual-target peptides unique advantages in drug development and can enhance therapeutic effects by simultaneously regulating multiple biological pathways.

[0035] In this article, the term "cyclic adenosine monophosphate (cAMP)" refers to a small molecule ubiquitous in living organisms, converted from ATP (adenosine triphosphate) by the enzyme adenylate cyclase. As a key mediator of intracellular signaling, cAMP plays a crucial role in regulating a variety of cellular functions and physiological processes.

[0036] In this article, the term "peptide bioactivity" refers to the biological functions and activities exerted by peptides in organisms. Peptides can regulate biological processes such as signal transduction, immune responses, 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 its target. Due to their excellent biocompatibility and selectivity, peptides have significant potential in drug development, disease treatment, and biotechnology applications.

[0037] In this article, the term “EC 50 " refers to the concentration of a polypeptide or its derivatives or pharmaceutically acceptable salts that reaches 50% of the maximum reaction under specific experimental conditions. This reaction can be a 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 This is usually determined using a dose-response curve. This curve depicts the effect of varying drug concentrations on a biological response, often exhibiting an S-shaped curve. By analyzing this curve, the concentration that achieves a 50% response can be found.

[0038] 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.

[0039] In this context, the structural formula of the term "αMeK" is .

[0040] In this context, the structural formula of the term "HoK" is .

[0041] In this context, the structural formula of the term "N-Me-K" is .

[0042] In this context, the structural formula of the term "Orn" is .

[0043] In this context, the structural formula of the term "Dab" is .

[0044] In this context, the structural formula of the term "Dap" is .

[0045] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the polypeptide or its derivative and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein refers to a substance approved by a federal regulatory agency or a national government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0046] As used herein, the term "pharmaceutically acceptable salt" refers to organic and inorganic salts of the polypeptides or derivatives thereof of the present application. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts formed with non-toxic acids include, but are not limited to, inorganic acid salts (e.g., hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates) and organic acid salts (e.g., acetates, oxalates, maleates, tartrates, citrates, succinates, malonates) formed by reaction with amino groups, or other methods described in the literature, such as ion exchange methods, to obtain these salts.

[0047] As used herein, the term "pharmaceutical composition" may refer to a composition used for the treatment of a disease or for in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more auxiliary ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active polypeptide or its derivative with a liquid excipient, a finely divided solid excipient, or both.

[0048] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. Except to the extent that any conventional excipient is incompatible with the polypeptide or derivative thereof, pharmaceutical composition, or medicament containing the same, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present application.

[0049] In addition to any conventional excipients, the use of excipients that are incompatible with the polypeptides or derivatives thereof, pharmaceutical compositions or drugs containing them of the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, are also within the scope of consideration of the present application.

[0050] The pharmaceutical compositions of the present disclosure include formulations suitable for parenteral administration. The formulations can be conveniently presented in unit dosage form and can be prepared by any method known in the pharmaceutical art. The amount of active ingredient that can be combined with excipients to prepare a single dose form is generally the amount of polypeptide or its derivative that produces a therapeutic effect.

[0051] As used herein, the term "agonist" refers to a substance (ligand) that activates the receptor type in question.

[0052] As used herein, the term "treatment" refers to the process used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a polypeptide or its derivatives, pharmaceutically acceptable salts, or a drug containing the same to an individual to treat, cure, alleviate, improve, mitigate, or inhibit the individual's disease, including but not limited to administering a drug containing a polypeptide or its derivatives, pharmaceutically acceptable salts, as described herein to an individual in need.

[0053] In this article, the term "non-alcoholic fatty liver disease (NAFLD)" generally refers to a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes excluding those caused by alcohol and other clear liver-damaging factors. It is 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.

[0054] The present application proposes a GLP-1R and GCGR dual-target activating polypeptide or its derivatives, pharmaceutically acceptable salts and uses thereof, which will be described in detail below.

[0055] Polypeptide or its derivative, pharmaceutically acceptable salt

[0056] In one aspect of the present application, the present application provides a polypeptide or a derivative thereof, or a pharmaceutically acceptable salt thereof. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0057] The inventors of this application have obtained novel GLP-1R and GCGR dual-targeting peptides through precise calculations, deep learning models, and iterative evolutionary model optimization. Compared to traditional GLP-1 and GCGR agonists, these peptides have a unique amino acid arrangement. Furthermore, these peptides, or their derivatives, or pharmaceutically acceptable salts, can bind to and effectively activate GLP-1R and GCGR, exhibiting high affinity and biological activity for these two groups. Consequently, these peptides, or their derivatives, or pharmaceutically acceptable salts, 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 (e.g., metabolic disorders, such as dyslipidemia, metabolic syndrome, and particularly obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease).

[0058] In particular, with the increasing prevalence of metabolic disorders such as diabetes worldwide and the limitations of existing treatment options, the polypeptides or their derivatives and pharmaceutically acceptable salts of the present application can not only provide new treatment options for diabetic patients, but also have the potential to reshape the market landscape for the treatment of diabetes and other diseases.

[0059] As used herein, the term "the amino acid sequence of a polypeptide is as shown in SEQ ID NO: A" includes the amino acid sequence of SEQ ID NO: A, the amino acid sequence of conservatively modified forms of SEQ ID NO: A, or a sequence having a similarity of more than 90% (e.g., 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%) to the amino acid sequence shown in SEQ ID NO: A, all of which are within the scope of protection of this application. Without special explanation, the conservatively modified amino acids or amino acids with similarity differences in the phrase "an amino acid sequence of a conservatively modified form of SEQ ID NO: A, or an amino acid sequence shown in SEQ ID NO: A having a sequence similarity of 90% or more (e.g., 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more)" may be located at any position of the polypeptide of the present application, and may also be located at at least one of positions 2, 4-12, 14, 22, and 25-26, or at least one of positions 1, 3, 13, 15-21, 23-24, and 27-30. Such amino acid modifications or similarity differences do not significantly affect or alter the structural stability and / or receptor binding activity of the polypeptide comprising the amino acids, and are within the scope of protection of the present application.

[0060] Illustratively, "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 conservatively modified form of SEQ ID NO: 1, or the sequence similarity to the amino acid sequence shown in SEQ ID NO: 1 is greater than 90% (for example, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%), all of which are within the scope of protection of this application.

[0061] As used herein, "conservatively modified forms of an amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the properties of the amino acid sequence comprising the amino acid sequence, including amino acid substitutions, additions, and deletions. Modifications can be introduced into the polypeptides of the present application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been identified 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). Exemplarily, the number of conservative modifications does not exceed 10% of the total number, and preferably does not exceed 5% of the total number. In this article, "conservatively modified amino acid sequences" also include amino acid modifications of natural mutations, and "natural mutations" refer to mutations caused by changes in alleles, etc., caused by natural mutations of polypeptides.

[0062] In this article, the term "sequence similarity" is defined by the percentage similarity method, which 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.

[0063] .

[0064] According to an embodiment of the present application, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence similarity thereto; or, compared to the amino acid sequence as shown in SEQ ID NO: 1, the polypeptide has 1 to 3 amino acid substitutions, deletions, or additions, such as substitutions, deletions, or additions of 1, 2, or 3 amino acids, and has GLP-1R and GCGR binding activity.

[0065] It should be noted that, in this article, "after substitution, deletion or addition of one or more amino acids" means that after substitution, deletion or addition of such amino acids, it does not significantly affect or change the binding properties of the original amino acid sequence. Amino acid substitution is a substitution in which an amino acid residue in the original peptide chain is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been identified 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).

[0066] According to an embodiment of the present application, the amino acid used for substitution or addition is selected from amino acid X whose side chain contains -NH2, -SH, -OH or -COOH.

[0067] According to the embodiments of the present application, the amino acid used for substitution or addition is selected from amino acid X having a side chain containing -NH2. This increases the number of modifiable sites in the original polypeptide, and the side chain -NH2 of the modification site can bind to the modifying group, thereby effectively extending the half-life of the polypeptide or its derivatives or pharmaceutically acceptable salts in vivo.

[0068] 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. The specific type is not limited and all are within the scope of protection of this application.

[0069] According to an embodiment of the present application, the modifying group is connected to the -NH2 of the side chain of the amino acid K or the substituted or added amino acid X in the polypeptide; the modifying group has at least one of the following structures:

[0070] .

[0071] In this article, the chemical group description " " is used to describe the position of the group substitution. That is, the above chemical groups are replaced by It is connected to the -NH2 of amino acids to form a -CO-NH- linkage.

[0072] 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.

[0073] use

[0074] In the second aspect of the present application, the present application proposes the use of the polypeptide or its derivatives or pharmaceutically acceptable salts described in the first aspect in the preparation of a dual-target activator of GLP-1R and GCGR. As previously mentioned, the polypeptide or its derivatives or pharmaceutically acceptable salts can effectively activate GLP-1R and GCGR. Thus, the polypeptide or its derivatives or pharmaceutically acceptable salts can be prepared as a dual-target activator of GLP-1R and GCGR for the treatment or prevention of GLP-1R and / or GCGR-related diseases, such as metabolic disorders, such as dyslipidemia-related diseases, such as metabolic syndrome, especially obesity, diabetes, fatty liver disease, and non-alcoholic fatty liver disease.

[0075] Fusion protein, reagent or kit, pharmaceutical composition

[0076] In the third aspect of the present application, the present application proposes a fusion protein. According to an embodiment of the present application, the fusion protein includes the polypeptide or its derivatives and pharmaceutically acceptable salts described in the first aspect. As mentioned above, the above-mentioned polypeptide or its derivatives and pharmaceutically acceptable salts 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 derivatives and pharmaceutically acceptable salts can bind to GLP-1R and GCGR, and can be used to detect GLP-1R and / or GCGR or to activate GLP-1R and GCGR, and can also 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.

[0077] In an optional embodiment of the present application, the fusion protein further includes a functional fragment.

[0078] In this article, the term "functional fragment" refers to an amino acid fragment, which can be a functionally active fragment or a protein tag. The specific type is not limited and all are within the scope of protection of this application.

[0079] It should be noted that the functionally active fragments described above can be used to exert effects in animals or in vitro. For example, when the functionally active fragments are used to exert effects in animals, they can be used to prevent and / or treat diseases; when the functionally active fragments are used to exert effects in vitro, they can be used to specifically bind to a substance, detect the substance, or diagnose diseases in vitro.

[0080] It should be noted that the above-mentioned protein tag refers to a short peptide expressed together 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 a His tag, a Flag tag, a GST tag, an MBP tag, a SUMO tag and a C-Myc tag.

[0081] In a fourth aspect of the present application, a reagent or kit is provided. According to an embodiment of the present application, the reagent or kit includes the polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, or the fusion protein described in the third aspect. As previously mentioned, the 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 kit containing the polypeptide or its derivative, pharmaceutically acceptable salt can bind to GLP-1R and GCGR and be used to detect GLP-1R and / or GCGR.

[0082] In this article, the kit or reagent does not need to have a box structure, but only requires to be relatively independent and have a suitable loading or container, such as a tube, box, bottle, or card; some components are separated into different containers, and if permitted, some components can be combined in one container.

[0083] According to the embodiments of the present application, the kit includes reagents suitable for detection. In some embodiments, the kit may include instructions for detection. In some embodiments, the kit may include calibrators or controls, such as standards or control samples. In some embodiments, the kit also includes containers such as test tubes, microplates, or test strips in the kit.

[0084] In the fifth aspect of the present application, the present application proposes a pharmaceutical composition. According to the embodiments of the present application, the pharmaceutical composition comprises the polypeptide or its derivative, pharmaceutically acceptable salt described in the first aspect, or the fusion protein described in the third aspect. As mentioned above, 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. Therefore, the pharmaceutical composition containing the above-mentioned 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.

[0085] According to an embodiment of the present application, the pharmaceutical composition may further include pharmaceutically acceptable excipients.

[0086] In an optional embodiment of the present application, pharmaceutically acceptable excipients refer to conventional pharmaceutical excipients in the pharmaceutical field, such as diluents, buffers, osmotic pressure regulators, pH regulators, and the like.

[0087] In an optional embodiment of the present application, the pharmaceutically acceptable carrier refers to a conventional drug carrier in the pharmaceutical field, such as a protective agent.

[0088] In an optional embodiment of the present application, the pharmaceutically acceptable vehicle refers to a conventional drug vehicle in the pharmaceutical field, for example, a solution (such as water) and a liposome.

[0089] In an optional 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.

[0090] In some optional embodiments of the present application, the pharmaceutical composition may be an oral dosage form, such as a solid oral dosage form or a liquid oral dosage form. The specific type is not limited and all fall within the scope of protection of the present application.

[0091] In some optional embodiments of the present application, the pharmaceutical composition of the present application may also contain other active ingredients for treatment.

[0092] The pharmaceutical composition of the present application can be administered in different ways, for example, enterally, orally (for example, liquid solution), via injection (for example, intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally). Preferably, the pharmaceutical composition of the present application is in the form of a lyophilized preparation or an aqueous solution. The clinical dosage regimen can be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including patient size, body surface area, age, medicine to be administered, sex, application time and path, 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.

[0093] use

[0094] In the sixth aspect of the present application, the present application proposes the use of the polypeptide or its derivatives, pharmaceutically acceptable salts 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, which is used to treat or prevent GLP-1R and / or GCGR related diseases, such as metabolic disorder-related diseases.

[0095] The present application proposes the use of the polypeptide or its derivatives, pharmaceutically acceptable salts described in the first aspect, the fusion protein described in the third aspect, or the pharmaceutical composition described in the fifth aspect in the treatment or prevention of GLP-1R and / or GCGR related diseases.

[0096] The present application proposes 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, for treating or preventing GLP-1R and / or GCGR related diseases.

[0097] According to an embodiment of the present application, the above-mentioned use may further include at least one of the following technical features:

[0098] According to an embodiment of the present application, the GLP-1R and / or GCGR related diseases include metabolic disorder related diseases.

[0099] According to an embodiment of the present application, the metabolic disorder-related diseases include dyslipidemia-related diseases.

[0100] According to an embodiment of the present application, the dyslipidemia-related disease includes metabolic syndrome.

[0101] According to an embodiment of the present application, the metabolic disorder-related disease includes at least one of obesity, diabetes, fatty liver disease and non-alcoholic fatty liver disease.

[0102] method

[0103] In the seventh aspect of the present application, the present application proposes a method for detecting GLP-1R and / or GCGR. According to an embodiment of the present application, the method comprises: contacting the 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; based on the signal generated by the contact product, determining whether the sample to be detected contains GLP-1R and / or GCGR. 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. Therefore, the above-mentioned method can be used to bind to GLP-1R and GCGR for detecting GLP-1R and / or GCGR.

[0104] According to a specific embodiment of the present application, the signal includes a fluorescent signal.

[0105] According to a specific embodiment of the present application, the method further comprises determining the content value of GLP-1R and / or GCGR in the sample to be tested based on the signal generated by the contact product.

[0106] In an eighth aspect of the present application, a method for treating or preventing a disease associated with a metabolic disorder is provided. According to an embodiment of the present application, the method comprises administering to a subject a pharmaceutically acceptable dose of the polypeptide or derivative thereof, or a pharmaceutically acceptable salt thereof, of the first aspect, the fusion protein of the third aspect, or the pharmaceutical composition of the fifth aspect.

[0107] In an optional embodiment of the present application, the pharmaceutically acceptable dose can be selected from an effective dose (or effective amount).

[0108] The effective amount of the polypeptides, derivatives thereof, or pharmaceutically acceptable salts of the present invention may vary depending on the mode of administration and the severity of the disease being treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease being treated, the patient's weight, the patient's immune status, and the route of administration. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be proportionally reduced.

[0109] The polypeptides or derivatives thereof, pharmaceutically acceptable salts, polypeptide derivatives or pharmaceutically acceptable salts thereof, or pharmaceutical compositions of the present application can be incorporated into suitable drugs, which can be prepared into various forms, such as liquid, semi-solid and solid dosage forms, including but not limited to solid dosage forms, semi-solid dosage forms, liquid dosage forms and gaseous dosage forms. Various modes of administration of the polypeptides or derivatives thereof, pharmaceutically acceptable salts, polypeptide derivatives or pharmaceutically acceptable salts thereof, pharmaceutical compositions or drugs of the present application are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, nasal, pulmonary, rectal and topical administration, but the present application is not limited to these exemplified modes of administration.

[0110] According to an embodiment of the present application, the metabolic disorder-related diseases include dyslipidemia-related diseases.

[0111] According to an embodiment of the present application, the dyslipidemia-related disease includes metabolic syndrome.

[0112] According to an embodiment of the present application, the metabolic disorder-related disease includes at least one of obesity, diabetes, fatty liver disease and non-alcoholic fatty liver disease.

[0113] Below in conjunction with embodiment, the scheme of the application will be explained. Those skilled in the art will appreciate that the following examples are merely for illustration of the application and should not be considered as limiting the scope of the application. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents used or instruments not indicated by the manufacturer are conventional products that can be obtained commercially.

[0114] Example 1:

[0115] The schematic diagram of the polypeptide design and screening process of this application is as follows Figure 1 , which covers a series of steps from GLP-1R and GCGR dual-target peptide sequence generation to screening and optimization. The entire method consists of multiple iterative evolutionary processes, and after each iteration, the model-designed peptides can achieve effective bioactivity enhancement. Each iteration of evolution consists of three parts: AI-based generation and optimization of new peptides, wet experiments to determine the peptide's bioactivity, and feedback to strengthen the model's perception of bioactivity.

[0116] First, through an innovative deep learning method TPDiffusion, it is possible to generate a peptide sequence that can bind to the target protein based on the amino acid sequence of the target protein. This method converts the target protein sequence into a peptide sequence, which can be seen as a process of giving a specific answer to a specific question. By training the conditional diffusion model TPDiffusion, the relationship rules between target proteins and peptide sequences are learned, and specific peptides (i.e., GLP-1R and GCGR dual-target peptides) are generated for specific target proteins (i.e., GLP-1R and GCGR). The training of the peptide sequence generation model TPDiffusion mainly includes the forward diffusion process and the reverse diffusion process, where the forward diffusion process includes the following steps:

[0117] 1. Encoding amino acid sequences: To model the joint feature space of proteins and peptides, the target protein and peptide sequences are concatenated into a whole, and an embedding transformation function is introduced to map each discrete amino acid word or character into a continuous vector encoding space.

[0118] 2. Gradually add noise to the polypeptide sequence: Gaussian noise is gradually added to the polypeptide part of the vector encoding based on the Markov chain until the polypeptide sequence is completely destroyed.

[0119] After the forward diffusion process is completed, a set of clean target protein and noise peptide sequences will be obtained. Then, the peptide sequences will be restored through the reverse diffusion process, which mainly includes the following steps:

[0120] 1. Gradually denoise the peptide sequence: By constructing a denoising network, the noise distribution added by the forward diffusion process is estimated, and the peptide part is gradually denoised until the peptide sequence is completely restored.

[0121] 2. Calculate the loss function: The model outputs the predicted probability distribution of the peptide sequence, uses the mean square error loss function to calculate the difference between the model prediction result and the actual result, and performs backpropagation to update the model parameters.

[0122] The denoising network in TPDiffusion uses the BERT (Devlin et al. 2018) model. The BERT model architecture primarily consists of a multi-layer Transformer (Vaswani et al. 2017) encoder, with each Transformer block comprising a self-attention mechanism and a feed-forward neural network. Through the self-attention mechanism, TPDiffusion incorporates target sequence information during the reverse diffusion process to recover peptide sequences, implicitly modeling the relationship between the target protein and peptide sequence and achieving a mapping from target protein to peptide sequence. During the generation process, given an arbitrary target protein sequence, the model first randomly samples from Gaussian noise and then performs a reverse diffusion process. Guided by the target protein sequence, the noise is gradually eliminated over a fixed time step, ultimately generating peptide sequences that bind to the given target. Using the trained TPDiffusion, the GLP-1R and GCGR target sequences were used as input to generate a batch of candidate peptide sequences with high affinity for both targets.

[0123] Second, affinity maturation is performed on these high-affinity candidate peptide sequences. This method is an innovative deep reinforcement learning approach specifically designed for peptide sequence optimization, guiding the evolution of candidate peptide sequences by simulating environments and defining actions. This deep reinforcement learning framework can incorporate reward models as prior knowledge to guide peptide evolution. These reward models can include affinity prediction models, solubility prediction models, or toxicity prediction models.

[0124] PepAF, an innovative reward model, is a technical solution that effectively predicts the binding affinity of target proteins and peptides by comprehensively leveraging structural information, flexibility properties, and advanced pre-training strategies. PepAF first learns on two pre-training tasks: 1) estimating the binding free energy of protein (GLP-1R and GCGR)-peptide complexes; and 2) predicting the affinity of protein (GLP-1R and GCGR)-peptide complexes. The first task enables the model to learn the complex interactions between proteins (GLP-1R and GCGR) and peptides, as well as their structural and physicochemical properties, providing a foundation for understanding the binding modes and key features of protein (GLP-1R and GCGR)-peptide interactions. The second task enables the model to capture a wide range of molecular interactions at the atomic scale. PepAF also improves prediction accuracy by modeling the target protein structure and peptide flexibility. PepAF, the core technology in this patent, is used as a reward model in a deep reinforcement learning method to guide mutations in candidate peptide sequences.

[0125] After obtaining affinity-matured peptides, their biological activity was evaluated, and the results were fed back to the model for the next round of peptide generation and optimization. After one round of iteration, a peptide was designed that effectively activated both GLP-1R and GCGR target proteins, as shown in SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is as follows:

[0126]

[0127] Example 2: Evaluation of the ability of candidate peptides to simultaneously activate GLP-1R and GCGR target proteins

[0128] 1. The purpose of this experiment was to evaluate the ability of the candidate peptide (GA-Dual.R5.S3) obtained in Example 1 to activate cAMP activity in huGLP-1R-FL-CRE-HEK293-A5 cells. By quantitatively measuring cAMP production levels, this experiment can accurately assess the activation effect of candidate samples on the GLP-1R receptor, providing important bioactivity data for drug development for metabolic diseases such as diabetes. The experimental process is as follows:

[0129] Peptide Synthesis and Purification: Based on the designed specific amino acid sequences, candidate peptides were synthesized using solid-phase peptide synthesis (Fmoc-SPPS). High-performance liquid chromatography (HPLC) was used for purification to ensure the high purity of the synthesized peptides, free of significant impurities. Furthermore, mass spectrometry (MS) was used to verify the precise molecular weight of the peptides.

[0130] Peptide dissolution conditions: The purified candidate peptides were dissolved in DMSO (dimethyl sulfoxide) solvent to prepare a 1 mg / mL stock solution for subsequent experiments.

[0131] Cell starvation treatment: The huGLP-1R-FL-CRE-HEK293-A5 cell line was constructed, and the highly viable huGLP-1R-FL-CRE-HEK293-A5 cell line was selected and starved with DMEM medium containing 0% fetal bovine serum (FBS) to standardize experimental conditions and eliminate interference from other factors in the serum.

[0132] Cell Digestion, Washing, and Plating: Adherent cells are gently digested with Accutase and washed with PBS to remove residual culture medium and enzyme. The cell suspension is precisely plated into a 96-well HTRF low-volume assay plate to ensure consistent cell numbers per well, providing uniform starting conditions for subsequent cAMP activity assays.

[0133] cAMP activity assay: After cell plating, add a precisely diluted candidate peptide to each well (for specific concentrations, see Figure 2 The cells were incubated at 37°C for 20 minutes to simulate the in vivo environment and activate the GLP-1R and GCGR receptors. cAMP d2 reagent and anti-cAMP Eu Cryptate antibody (PerkinElmer, Cat. No. 62AM4PEB) working solution were added to each well and incubated at room temperature for 1 hour. This step allows cAMP to bind to the fluorescently labeled antibody, forming an energy transfer complex.

[0134] Signal Detection: Utilizing TR-FRET technology, with an excitation wavelength of 340 nm and emission signals detected at 665 nm and 620 nm, this technology offers high sensitivity and robustness to interference. The FRET signal is calculated as the ratio of the light intensities at 665 nm to 620 nm, reflecting changes in cAMP concentration. Changes in the ratio are inversely proportional to cAMP concentration.

[0135] The results showed that the synthesized candidate polypeptides all had good cAMP activation function, indicating that the candidate polypeptides all had good cAMP activation function, indicating that the candidate polypeptides had high GLP-1R activation biological activity. Figure 2 As shown, the EC of some candidate peptides 50 As shown in Table 1.

[0136] Table 1

[0137]

[0138] 2. The purpose is to evaluate the ability of the candidate peptide (GA-Dual.R5.S3) obtained in Example 1 to activate the GCGR target. The experimental process is as follows:

[0139] First, the candidate peptides were diluted separately using DMEM complete medium to prepare a series of dilutions, and 50 μL of the dilutions were added to the culture plate. Human GCGR-CRE-luci-HEK293-A5 cells were cultured at 1×10 5 At a concentration of 10 cells / well, add 50 μL to the culture plate and mix with the diluted agonist. Incubate the plate in a 37°C incubator for 6 hours to ensure adequate agonist uptake and GCGR activation. Add 50 μL of Bright-Lite (Vazyme, DD1204-04-AA) to each well and read the luciferase signal using a luciferase assay to assess GCGR activation.

[0140] The results showed that the synthesized candidate polypeptide had good luciferase activation function, indicating that the candidate polypeptide had high GCGR activation biological activity. Figure 3 As shown, the EC of some candidate peptides 50 As shown in Table 2.

[0141] Table 2

[0142]

[0143] Example 3:

[0144] This example aims to evaluate the in vitro 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. The specific steps are as follows:

[0145] 1) For obesity: 3T3-L1 adipocytes were used to induce cell differentiation into adipocytes using 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 3T3-L1 preadipocytes reached 80-90% confluency, they were digested with 0.25% trypsin and plated at 2-3 × 10 4 cells / cm 2When the cell confluence reaches 80-95%, add adipogenic differentiation medium for induction. After 2-3 days of induction, switch to adipogenic differentiation maintenance medium for 1 day. Repeat the induction cycle 3-5 times until obvious lipid droplets are observed in the cells, indicating that the obesity model has been successfully established.

[0146] Then, the candidate peptides were added into the adipocyte culture medium at different concentrations (10 nM, 100 nM, 1 μM and 10 μM), and the cell density was controlled at 2-3×10 4 cells / cm 2 To ensure normal cell growth and differentiation, culture for 24-48 hours. Intracellular lipid droplet content was assessed using Oil Red O staining: the culture medium was aspirated, the cells were rinsed with 1× PBS, and fixed with 4% neutral formaldehyde solution for 30 minutes. An Oil Red O working solution (saturated Oil Red O solution: distilled water = 3:2) was prepared, filtered, and used for staining. 1 mL of Oil Red O working solution was added to each well. The cells were stained at room temperature for 30 minutes. The staining solution was aspirated, the cells were rinsed with 1× PBS, and the cells were observed under a microscope and photographed. Simultaneously, the expression levels of adipogenesis-related genes (PPARγ and C / EBPα) were assessed using a qPCR kit (Takara RR420A). If a candidate peptide significantly reduces intracellular lipid droplet content and the expression of adipogenesis-related genes, it indicates potential therapeutic effects for obesity, possibly by inhibiting adipocyte differentiation and lipid accumulation.

[0147] 2) For diabetes: Pancreatic β-cell line (MIN6 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, Cat. No. 11995065) to simulate the high-glucose environment of diabetes. 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 2Culture cells for 24-48 hours. Insulin secretion in the cell culture supernatant was measured using an insulin ELISA kit. The expression levels of proteins associated with the insulin signaling pathway, such as the glucose transporter (GLUT2) and insulin receptor substrate (IRS), were measured using a GLU2 kit (YUANJU BIO, Cat. No. YJ24519) and an IRS1 kit (Thermo Fisher Scientific, Cat. No. KHO0521). If a candidate peptide can increase insulin secretion and upregulate the expression levels of GLUT2 and IRS, it indicates that it may improve insulin resistance and promote insulin secretion, potentially offering therapeutic value for diabetes.

[0148] 3) For dyslipidemia-related diseases: A dyslipidemia model was established using a hepatocyte cell line (HepG2 cells) culture medium containing a high concentration of fatty acids (Wuhan Shanen Biotechnology Co., Ltd., HepG2 cell-specific culture medium, which consists of EMEM (MEM+NEAA) treated cells with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S)). 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 Culture the cells for 24-48 hours. Intracellular total cholesterol levels were measured using an enzymatic assay kit (Sigma-Aldrich Total Cholesterol Assay Kit, Catalog No. MAK043), and triglyceride levels were measured using a triglyceride assay kit (Sigma-Aldrich Triglyceride Assay Kit, Catalog No. MAK041). Furthermore, the expression levels of cholesterol metabolism-related genes, such as the low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1), were measured using an LDLR kit (Thermo Fisher Scientific, Catalog No. EHLDLR) and a CYP7A1 kit (Proteintech, Catalog No. 18054-1-AP). If a candidate peptide can reduce intracellular total cholesterol and triglyceride levels while simultaneously upregulating LDLR and CYP7A1 expression, it suggests that it may have therapeutic effects on dyslipidemia-related diseases by regulating cholesterol synthesis and metabolism.

[0149] 4) For fatty liver disease: HepG2 cells were also used. High-fat culture medium (Wuhan Shanen Biotechnology Co., Ltd., HepG2 cell-specific culture medium, which includes EMEM (MEM+NEAA) + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) was used to induce fatty degeneration in the cells to establish a fatty liver disease model. The candidate peptides were added to the cell culture medium at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM). The cell density was controlled at 5×10 3 cells / cm 2 The cells were cultured for 24-48 hours. Intracellular lipid droplet content was assessed using Oil Red O staining: the culture medium was aspirated, the cells were rinsed with 1× PBS, and fixed with 4% neutral formaldehyde for 30 minutes. An Oil Red O working solution (saturated Oil Red O solution: distilled water = 3:2) was prepared, filtered, and used for staining. 1 mL of Oil Red O working solution was added to each well. The cells were stained at room temperature for 30 minutes. The staining solution was aspirated, the cells were rinsed with 1× PBS, and the cells were observed under a microscope and photographed. Inflammatory cytokine secretion levels were assessed using ELISA kits (TNF-α and IL-6 ELISA kits, Thermo Fisher Scientific, catalog numbers K1480 and K1501, respectively), and oxidative stress markers were assessed using colorimetric assays (MDA Assay Kit, catalog number 700455, and SOD Assay Kit, catalog number 705125, both from Cayman Chemical). If the candidate polypeptide can reduce the accumulation of intracellular lipid droplets, reduce the secretion of inflammatory factors, and improve the oxidative stress state, it means that it may have a therapeutic effect on fatty liver disease and can reduce the fatty degeneration and inflammatory damage of liver cells.

[0150] 5) Targeting metabolic syndrome: Construct a cell model of metabolic syndrome by combining multiple cells (fat cells, liver cells, muscle cells, etc.) to simulate the complex pathological environment of metabolic syndrome.

[0151] Adipocytes: 3T3-L1 adipocytes were used to establish an obesity model by inducing their differentiation into adipocytes using 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).

[0152] Hepatocytes: HepG2 cells were used and treated with high-lipid medium (EMEM + 10% fetal bovine serum + 1% penicillin / streptomycin, supplemented with high concentrations of fatty acids) to simulate the fatty liver disease environment.

[0153] Muscle cells: C2C12 muscle cells were cultured in high-glucose DMEM medium to simulate an insulin-resistant environment.

[0154] 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 The cells are cultured for 24-48 hours. After culture, metabolic syndrome-related markers are measured in each cell type, including the insulin resistance index (HOMA-IR) (calculated by measuring insulin and glucose levels), lipid profiles (using the triglyceride assay kit (Sigma-Aldrich, Cat. No. MAK041) and total cholesterol assay kit (Sigma-Aldrich, Cat. No. MAK043) for triglyceride and total cholesterol levels), and inflammatory cytokine secretion (using ELISA kits (Thermo Fisher Scientific, Cat. No. K1480 and K1501, respectively) for TNF-α and IL-6 secretion). If the candidate peptide can improve insulin resistance, lower lipid levels, and reduce the secretion of inflammatory factors, it indicates potential therapeutic efficacy for metabolic syndrome, potentially regulating metabolic disorders through a multi-target mechanism of action.

[0155] 6) For non-alcoholic fatty liver disease: HepG2 cells were treated with high-fat, high-glucose medium (Wuhan Shanen Biotechnology Co., Ltd., HepG2 cell-specific medium, which includes EMEM (MEM+NEAA) + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) to induce non-alcoholic fatty liver disease-like changes in the 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 2The cells were cultured for 24-48 hours. Intracellular lipid droplet content was measured using Oil Red O staining. Inflammatory cytokine secretion levels were measured using ELISA kits (Thermo Fisher Scientific IL-1β and IL-8 ELISA kits, catalog numbers K1480 and K1501, respectively). Hepatocyte injury marker levels were measured using colorimetric assays (Cayman Chemical ALT and AST Assay Kits, catalog numbers 700455 and 705125, respectively). Western blot and qPCR were used to measure the expression levels of liver fibrosis-related markers (collagen I and α-smooth muscle actin). If a candidate peptide can reduce intracellular fat content, decrease the secretion of inflammatory factors, lower the levels of liver injury markers, and inhibit the expression of liver fibrosis-related markers, it would indicate a therapeutic effect on non-alcoholic fatty liver disease, potentially by alleviating fat accumulation and inhibiting inflammation and fibrosis.

[0156] The results showed that the candidate polypeptide in Example 1 had the following effects:

[0157] 1) Both can significantly reduce the content of lipid droplets in 3T3-L1 adipocytes and reduce the expression of adipogenesis-related genes;

[0158] 2) Both can increase the secretion of insulin in pancreatic β-cell lines (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);

[0159] 3) Reduce the levels of total cholesterol and triglycerides in hepatocytes, while upregulating the expression of genes related to cholesterol metabolism, such as low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1);

[0160] 4) Reduce the accumulation of lipid droplets in HepG2 cells, reduce the secretion of inflammatory factors, and improve oxidative stress;

[0161] 5) Improve indicators related to metabolic syndrome in various cells (such as fat cells, liver cells, muscle cells, etc.);

[0162] 6) Reduce the fat content in HepG2 cells, reduce the secretion of inflammatory factors, reduce the levels of liver cell injury markers, and inhibit the expression of liver fibrosis-related indicators.

[0163] 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.

[0164] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present 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 any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0165] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

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

1.

2. Use of the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a dual-target activator of GLP-1R and GCGR, wherein the activator is used to treat or prevent at least one of the following diseases: obesity, diabetes, fatty liver disease, dyslipidemia and metabolic syndrome.

3. Use of the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a GLP-1R and GCGR dual-target activator for treating or preventing non-alcoholic fatty liver disease.

4. A reagent or kit, characterized in that The invention comprises the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical composition, characterized in that The invention comprises the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 5, characterized in that Including pharmaceutically acceptable excipients.

7. Use of the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 5 or 6, in the preparation of a medicament for treating or preventing at least one of the following diseases: obesity, diabetes, fatty liver disease, dyslipidemia, and metabolic syndrome.

8. Use of the polypeptide or pharmaceutically acceptable salt thereof according to claim 1, or the pharmaceutical composition according to claim 5 or 6, in the preparation of a medicament for treating or preventing non-alcoholic fatty liver disease.

9. A method for detecting GLP-1R and / or GCGR, characterized in that: include: contacting a sample to be tested with the polypeptide or pharmaceutically acceptable salt thereof according to claim 1, or the reagent or kit according to claim 4; Based on the signal generated by the contact product, it is determined whether the sample to be tested contains GLP-1R and / or GCGR.

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