Glp-1r-activating polypeptides or pharmaceutically acceptable salts thereof and uses thereof

Abstract

This application discloses a polypeptide or a pharmaceutically acceptable salt thereof. The polypeptide has an amino acid sequence as shown in HSQGTFTSDYSKYLDERAASEFVQWLLAG. The polypeptide or a pharmaceutically acceptable salt thereof can bind to GLP-1R for efficient activation of GLP-1R.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical technology, specifically relating to GLP-1R activating peptides or pharmaceutically acceptable salts thereof and their uses, and more specifically to a GLP-1R activating peptide designed based on an iterative evolutionary frame model. Background Technology

[0002] GLP-1R, or glucagon-like peptide-1 receptor, is an important G protein-coupled receptor (GPCR) that plays a crucial role in maintaining glycemic homeostasis. Activation of GLP-1R promotes insulin secretion and inhibits glucagon release, thereby effectively lowering blood glucose levels. Furthermore, GLP-1R is involved in regulating various physiological processes such as gastrointestinal motility, appetite control, and energy balance, making it an ideal target for the treatment of diabetes and other metabolic diseases.

[0003] GLP-1 is a hormone secreted by L cells in the intestine, which is secreted in large quantities when stimulated by nutrients. GLP-1 exerts its effects by binding to its receptor GLP-1R, which is widely distributed in many tissues of the body, including the pancreas, intestines, brain, and cardiovascular system. In the pancreas, GLP-1 can reduce glucagon secretion and promote insulin secretion, thereby lowering blood sugar levels.

[0004] Current GLP-1R agonists are all developed based on GLP-1, such as exenatide, liraglutide, and semaglutide. By mimicking the physiological effects of endogenous GLP-1, they can promote insulin secretion, inhibit glucagon secretion, participate in regulating blood glucose levels, or delay gastric emptying, increase satiety, and reduce food intake, thus aiding in weight control and finding widespread clinical application. However, these GLP-1R agonists that mimic endogenous GLP-1 may also have problems such as short half-life and easy degradation.

[0005] Therefore, developing a new GLP-1R agonist is of great importance in the treatment of diabetes, obesity and other related diseases. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a GLP-1R activating peptide or a pharmaceutically acceptable salt thereof, which is designed based on an iterative evolutionary frame model, is completely different from endogenous GLP-1, and exhibits excellent activation effects on GLP-1R.

[0007] This application is based on the following discoveries of the inventors:

[0008] Most existing GLP-1R agonists are GLP-1 analog peptides. Although they have some therapeutic effect in regulating blood glucose levels, they still have some significant limitations:

[0009] 1. Stability issues: Longer peptide sequences may have a shorter half-life in vivo and are easily degraded by enzymes in the body, which limits the duration of their therapeutic effect and the convenience of their application.

[0010] 2. Most existing agonists are GLP-1 analogs, which limits the space for exploring peptides with stronger and more stable binding capabilities.

[0011] To address the aforementioned shortcomings of existing technologies, this application develops a novel GLP-1R agonist, providing a new approach for the treatment of diabetes, obesity, and other related diseases. This invention yields a novel peptide through precise calculations and iterative evolutionary model optimization. This peptide possesses a unique amino acid sequence compared to traditional GLP-1 analogs, enabling it to effectively activate the GLP-1R receptor.

[0012] Therefore, in a first aspect of this application, a polypeptide or a pharmaceutically acceptable salt thereof is provided. According to embodiments of this application, the polypeptide has an amino acid sequence as shown in HSQGTFTSDYSKYLDERAASEFVQWLLAG. The polypeptide or a pharmaceutically acceptable salt thereof can bind to GLP-1R to effectively activate GLP-1R. Thus, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can be used to treat or prevent GLP-1R-related diseases, such as metabolic disorders, including obesity, diabetes, dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0013] In a second aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of this application can encode the polypeptide of the first aspect, which can bind to GLP-1R for efficient activation of GLP-1R.

[0014] In a third aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the second aspect. The expression vector of this application, carrying the nucleic acid molecule described in the second aspect, can express the polypeptide of the first aspect, which can bind to GLP-1R for effective activation of GLP-1R.

[0015] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or the recombinant cell expresses the polypeptide described in the first aspect. The recombinant cell of this application can express the polypeptide of the first aspect, which can bind to GLP-1R for effective activation of GLP-1R.

[0016] In a fifth aspect of this application, a polypeptide derivative or a pharmaceutically acceptable salt thereof is provided. According to an embodiment of this application, the polypeptide derivative or a pharmaceutically acceptable salt thereof comprises: the polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect, and a modifying group, wherein the polypeptide or a pharmaceutically acceptable salt thereof is linked to the modifying group. As is known prior, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can effectively activate GLP-1R. Therefore, a polypeptide derivative or a pharmaceutically acceptable salt thereof containing the aforementioned polypeptide can be used to treat or prevent GLP-1R-related diseases, such as metabolic disorder-related diseases, including obesity, diabetes, dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0017] In a sixth aspect of this application, a fusion protein is proposed. According to embodiments of this application, the fusion protein comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, or the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof. As is known prior, the aforementioned polypeptide or its pharmaceutically acceptable salt can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the fusion protein containing the aforementioned polypeptide can bind to GLP-1R for detecting or activating GLP-1R, and can also be used to treat or prevent GLP-1R-related diseases, such as metabolic disorders, including obesity, diabetes, dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0018] In a seventh aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, or the fusion protein described in the sixth aspect. As is known prior, the aforementioned polypeptides can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the reagent or kit containing the aforementioned polypeptides can bind to GLP-1R for the detection of GLP-1R.

[0019] In an eighth aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, or the fusion protein described in the sixth aspect. As is known prior, the aforementioned polypeptides can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the pharmaceutical composition containing the aforementioned polypeptides can be used to treat or prevent GLP-1R-related diseases, such as metabolic disorders, including obesity, diabetes, dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0020] In a ninth aspect of this application, the use of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the sixth aspect, or the pharmaceutical composition described in the fifth aspect in the preparation of a medicament for the treatment or prevention of GLP-1R-related diseases, such as metabolic disorder-related diseases.

[0021] In a tenth aspect of this application, a method for detecting GLP-1R is proposed. According to an embodiment of this application, the method includes: contacting a sample to be tested with the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the sixth aspect, or the reagent described in the seventh aspect; and determining whether the sample to be tested contains GLP-1R based on the signal generated by the contact product. As is previously known, the aforementioned polypeptide can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the above method can be used to bind to GLP-1R for detection.

[0022] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0023] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0024] Figure 1 This is a schematic diagram of the peptide design and screening process in this application;

[0025] Figure 2 The results of cAMP release from the GLP-1R target activated by the peptide in the embodiments of this application are shown.

[0026] Figure 3 This is a three-dimensional structural diagram of the peptide binding to the GLP-1R target in the embodiments of this application. Detailed Implementation

[0027] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0028] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0029] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0030] In this article, the term "target protein" refers to a protein that plays a crucial role in an organism and is often the target of drug development. By binding to a target protein, drugs can regulate its biological activity, thereby achieving the goal of treating diseases.

[0031] In this article, the term "peptide" refers to a biological macromolecule composed of many amino acids, typically 50 or fewer amino acids linked together by peptide bonds. Peptides perform a variety of functions in living organisms, including participating in various physiological processes as enzymes, hormones, and antibodies. Furthermore, due to their excellent biocompatibility, selectivity, and high biological activity, peptides are widely studied for drug discovery and the treatment of various diseases, such as anti-tumor, antiviral, antibacterial, and anti-inflammatory drugs.

[0032] In this article, the term "glucagon-like peptide-1 receptor (GLP-1R)" refers to a G protein-coupled receptor belonging to the class B G protein-coupled receptor family. It plays a crucial physiological role in the human body, particularly in regulating blood glucose levels. Activation of GLP-1R promotes insulin secretion and inhibits glucagon release, thereby lowering blood glucose. Furthermore, GLP-1R is involved in regulating various physiological processes, including gastrointestinal motility, appetite control, and energy balance. Activation of GLP-1R leads to the release of the α subunit of the G protein and activation of adenylate cyclase (AC), a membrane-bound enzyme that catalyzes the conversion of ATP (adenosine triphosphate) to cAMP.

[0033] In this article, the term "cyclic adenosine monophosphate (cAMP)" refers to a small molecule ubiquitous in living organisms, derived 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 cellular functions and physiological processes.

[0034] In this paper, the term "amino acid sequence similarity" is defined using a percentage similarity method, which is calculated by comparing the number of identical or similar amino acids in two protein or polypeptide sequences out of the total number of amino acids.

[0035]

[0036] In this document, amino acids are referred to by their conventional single-letter and three-letter codes for natural amino acids, as well as the generally accepted three-letter codes for other α-amino acids. Unless otherwise specified, in this application, uppercase letters represent amino acids with the L-configuration and lowercase letters represent amino acids with the D-configuration.

[0037] In this paper, the structural formula for the term "αMeK" is:

[0038] In this article, the structural formula for the term "HoK" is:

[0039] In this article, the structural formula for the term "N-Me-K" is:

[0040] In this article, the structural formula for the term "Orn" is:

[0041] In this article, the structural formula for the term "Dab" is:

[0042] In this article, the structural formula for the term "Dap" is:

[0043] In this document, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components comprising a polypeptide or its derivatives and / or with the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0044] In this document, the term "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the polypeptides or their derivatives of the present invention. Pharmaceutically acceptable salts are well-known in the field, as described in the literature: SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. Pharmaceutically acceptable salts formed from non-toxic acids include, but are not limited to, inorganic acid salts (such as hydrochlorides, hydrobromic acids, phosphates, sulfates, and perchlorates) formed by reaction with amino groups, and organic acid salts (such as acetates, oxalates, maleates, tartrates, citrates, succinates, and malonates), or salts obtained by other methods described in the literature, such as ion exchange.

[0045] In this document, "pharmaceutical composition" can refer to a drug for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients that constitute one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining an active polypeptide or its derivative or revitalizer with a liquid excipient, a finely pulverized solid excipient, or both.

[0046] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients is also within the scope of this invention, except for any range of incompatibilities with the polypeptides or derivatives thereof, pharmaceutical compositions, or drugs containing them, such as any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition.

[0047] In addition to any conventional excipients, the use of polypeptides or their derivatives, pharmaceutical compositions or pharmaceuticals containing them that are incompatible with the present invention, such as any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition, is also within the scope of this invention.

[0048] The pharmaceutical compositions disclosed herein include formulations suitable for parenteral administration. The formulations can be conveniently available in unit dosage forms and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient in a single-dose form, which can be prepared in combination with excipients, is generally the amount of the polypeptide or a derivative thereof that produces the therapeutic effect.

[0049] In this paper, the term "agonist" refers to a substance (ligand) that activates the type of receptor.

[0050] In this document, the term "treatment" means used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0051] In this article, the term “non-alcoholic fatty liver disease (NAFLD)” generally refers to a clinicopathological syndrome characterized by excessive fat deposition in hepatocytes excluding alcohol and other clearly defined liver-damaging factors. It is an acquired metabolic stress-induced liver injury closely associated with insulin resistance and genetic susceptibility, including but not limited to simple fatty liver (SFL), non-alcoholic steatohepatitis (NASH), and its associated cirrhosis.

[0052] This application discloses a GLP-1R activating peptide or a pharmaceutically acceptable salt thereof and its uses, which will be described in detail below.

[0053] polypeptides or their pharmaceutically acceptable salts

[0054] In one aspect of this application, a polypeptide or a pharmaceutically acceptable salt thereof is provided. According to embodiments of this application, the polypeptide has an amino acid sequence as shown in HSQGTFTSDYSKYLDERAASEFVQWLLAG (SEQ ID NO:1), or the polypeptide has a sequence similarity of more than 85%, preferably more than 90%, to the amino acid sequence shown in HSQGTFTSDYSKYLDERAASEFVQWLLAG (SEQ ID NO:1); or the polypeptide, compared to the amino acid sequence shown in YSX1GTFTSDYSKYLEX2EAVRX3FIEWLX4, has undergone substitution, deletion, or addition of one or more amino acids and possesses GLP-1R binding activity.

[0055] The GLP-1R activating peptide of this application was obtained through iterative evolutionary model optimization. It can bind to GLP-1R, exhibiting high affinity and bioactivity for the GLP-1R receptor, and can effectively activate GLP-1R, providing a new treatment option for patients with metabolic disorders (such as diabetes). In particular, with the continuous rise in the global prevalence of diabetes and the limitations of existing treatments, the GLP-1R activating peptide of this application, or its pharmaceutically acceptable salt, not only provides a new treatment option for patients with metabolic disorders (such as diabetes), but also has the potential to reshape the market landscape for the treatment of metabolic disorders (such as diabetes).

[0056] It should be noted that in this article, "substitution, deletion, or addition of one or more amino acids" means that the substitution, deletion, or addition of such amino acids does not significantly affect or alter the binding properties of the original amino acid sequence. Amino acid substitution refers to the replacement of amino acid residues in the original peptide chain with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0057] According to embodiments of this application, the above-mentioned polypeptide or its pharmaceutically acceptable salt may further include at least one of the following technical features:

[0058] According to embodiments of this application, the amino acid sequence shown in HSQGTFTSDYSKYLDERAASEFVQWLLAG is modified by substitution, deletion, or addition of 1 to 5 amino acids and possesses GLP-1R binding activity. For example, 1, 2, 3, 4, or 5 amino acids may be substituted, deleted, or added.

[0059] According to embodiments of this application, the amino acid used for substitution or addition is selected from amino acid X containing -NH2 in its side chain. This increases the number of modifiable sites in the original polypeptide, where the -NH2 side chain can bind to the modifying group, thereby effectively prolonging the antibody's half-life in vivo.

[0060] According to embodiments of this application, the amino acid X is selected from K, k, αMeK, HoK, Dap, Dab, Orn, or N-Me-K.

[0061] Nucleic acid molecules, expression vectors and recombinant cells

[0062] In a second aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of this application can encode the polypeptide of the first aspect, which can bind to GLP-1R for efficient activation of GLP-1R.

[0063] According to embodiments of this application, the nucleic acid molecule is DNA.

[0064] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the nucleic acid molecule sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0065] In a third aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the second aspect. When linking the nucleic acid molecule to the expression vector, the nucleic acid molecule can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the connection between the nucleic acid molecule and the control elements only needs to be operably established.

[0066] In this document, "operably ligated" refers to ligating a foreign gene to an expression vector, enabling the control elements within the expression vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the expression vector is introduced into suitable recipient cells, the aforementioned polypeptide can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro production of the polypeptide.

[0067] In some specific embodiments of this application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0068] In some specific embodiments of this application, the expression vector is a lentiviral vector.

[0069] In one optional embodiment of this application, the expression vector is a plasmid expression vector.

[0070] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or the recombinant cell expresses the polypeptide described in the first aspect. Using this recombinant cell under suitable conditions, the aforementioned polypeptide can be effectively expressed within the recombinant cell.

[0071] According to embodiments of this application, the recombinant cells are obtained by introducing the expression vector described in the third aspect into the host cell.

[0072] It should be noted that the host cell in this application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. The prokaryotic cell can be *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*, etc. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*, insect cells such as armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0073] According to an embodiment of this application, the host cell is a eukaryotic cell.

[0074] According to embodiments of this application, the host cell is a mammalian cell, including but not limited to BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0075] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the peptide described in this application. Those skilled in the art will readily understand that suitable conditions for peptide expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for peptide expression based on the specific environment of their laboratory.

[0076] polypeptide derivatives or their pharmaceutically acceptable salts

[0077] In a fifth aspect of this application, a polypeptide derivative or a pharmaceutically acceptable salt thereof is provided. According to an embodiment of this application, the polypeptide derivative or a pharmaceutically acceptable salt thereof comprises: the polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect, and a modifying group, wherein the polypeptide or a pharmaceutically acceptable salt thereof is linked to the modifying group. As is known prior, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can effectively activate GLP-1R. Therefore, a polypeptide derivative or a pharmaceutically acceptable salt thereof containing the aforementioned polypeptide can be used to treat or prevent GLP-1R-related diseases, such as metabolic disorder-related diseases, including obesity, diabetes, dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0078] In this document, the term "modifying group" should be interpreted broadly, and can refer to chemical groups or amino acid fragments. The specific type is not limited, and all are within the scope of protection of this application.

[0079] According to embodiments of this application, the above-mentioned polypeptide derivatives or pharmaceutically acceptable salts thereof may further include at least one of the following technical features:

[0080] According to embodiments of this application, the modifying group is attached to -NH2, -SH, -OH or -COOH of the amino acid side chain in the polypeptide.

[0081] According to embodiments of this application, the modifying group is linked to the -NH2 of the amino acid side chain in the polypeptide.

[0082] According to embodiments of this application, the modifying group has at least one of the following structures:

[0083]

[0084] In this article, the chemical groups described It is used to describe the position of a substituent group. That is, the chemical group mentioned above is substituted by... It is attached to the -NH- group of amino acids.

[0085] Fusion proteins, reagents or kits, pharmaceutical compositions

[0086] In a sixth aspect of this application, a fusion protein is proposed. According to embodiments of this application, the fusion protein comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, or the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof. As is known prior, the aforementioned polypeptide can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the fusion protein containing the aforementioned polypeptide can bind to GLP-1R for detecting or activating GLP-1R, and can also be used to treat or prevent GLP-1R, such as metabolic disorder-related diseases, including obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0087] In an alternative embodiment of this application, the fusion protein further includes a functional fragment.

[0088] In this document, the term "functional fragment" refers to an amino acid fragment, which may be a functionally active fragment or a protein tag. The specific type is not limited and is within the scope of protection of this application.

[0089] It should be noted that the aforementioned functionally active fragments can be used to exert their effects in animals or in vitro. For example, when used in animals, the functionally active fragment can be used to prevent and / or treat diseases; when used in vitro, it can be used to specifically bind to a substance, detect that substance, or diagnose diseases in vitro.

[0090] It should be noted that the aforementioned 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 this application. For example, the protein tag includes at least one of the following: His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

[0091] In a seventh aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, or the fusion protein described in the sixth aspect. As is known prior, the aforementioned polypeptides can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the reagent or kit containing the aforementioned polypeptides can bind to GLP-1R for the detection of GLP-1R.

[0092] In an eighth aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, or the fusion protein described in the sixth aspect. As is known prior, the aforementioned polypeptide can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the pharmaceutical composition containing the aforementioned polypeptide can be used to treat or prevent metabolic disorder-related diseases, such as obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0093] According to embodiments of this application, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0094] In one alternative embodiment of this application, pharmaceutically acceptable excipients refer to pharmaceutical excipients that are conventional in the pharmaceutical field, such as diluents, buffer solutions, osmotic pressure regulators, pH regulators, etc.

[0095] In one alternative embodiment of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field, such as a protectant.

[0096] In one alternative embodiment of this application, pharmaceutically acceptable mediators refer to pharmaceutical mediators conventional in the pharmaceutical field, such as solutions (e.g., water) and liposomes.

[0097] In one alternative embodiment of this application, examples of suitable pharmaceutically acceptable carriers, excipients, and mediators are well known in the art. Pharmaceutical compositions comprising such carriers, excipients, and mediators can be formulated using known conventional methods.

[0098] In some alternative embodiments of this application, the pharmaceutical composition of this application may also contain other active ingredients for treatment.

[0099] The pharmaceutical composition of this application can be administered via various routes, such as enterally, orally (e.g., liquid solution), or by injection (e.g., intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally). Preferably, the pharmaceutical composition of this application is in the form of a lyophilized preparation or an aqueous solution. The clinical dosing regimen will be determined by the attending physician and clinical factors. As is known in the medical field, the dosage for any given patient depends on many factors, including the patient's physique, body surface area, age, the drug to be administered, sex, time and route of administration, general health, and other concurrently administered drugs. The pharmaceutical composition of this application can be administered topically or systemically. Preferably, it can be administered intravenously or subcutaneously.

[0100] use

[0101] In a ninth aspect of this application, the use of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the sixth aspect, or the pharmaceutical composition described in the eighth aspect in the preparation of a medicament for the treatment or prevention of GLP-1R-related diseases is provided.

[0102] According to embodiments of the present invention, the GLP-1R-related diseases include metabolic disorder-related diseases.

[0103] According to embodiments of this application, the metabolic disorder-related diseases include at least one of obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0104] method

[0105] In a tenth aspect of this application, a method for detecting GLP-1R is proposed. According to an embodiment of this application, the method includes: contacting a sample to be tested with the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, a polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, a fusion protein described in the sixth aspect, or a reagent described in the seventh aspect to form a contact product, such as an immune complex; and determining whether the sample to be tested contains GLP-1R based on the signal generated by the contact product. As is previously known, the aforementioned polypeptide can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the above method can be used to bind to GLP-1R for detection.

[0106] According to a specific embodiment of the present invention, the signal includes a fluorescence signal.

[0107] According to a specific embodiment of the present invention, the method further includes determining the content value of GLP-1R in the sample to be tested based on the signal generated by the contact product.

[0108] In the eleventh aspect of this application, a method for treating or preventing metabolic disorder-related diseases is provided. According to embodiments of this application, the method includes administering to a subject a pharmaceutically acceptable dose of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide derivative described in the fifth aspect or a pharmaceutically acceptable salt thereof, the fusion protein described in the sixth aspect, or the pharmaceutical composition described in the eighth aspect.

[0109] In one alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0110] The effective amount of the compound described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0111] The polypeptides, pharmaceutically acceptable salts thereof, polypeptide derivatives thereof, or pharmaceutical compositions of this application may be incorporated into suitable pharmaceuticals, which may be prepared in 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 routes of administration of the polypeptides, pharmaceutically acceptable salts thereof, polypeptide derivatives thereof, pharmaceutical compositions, or pharmaceutical compositions of this application are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, topical, nasal, pulmonary, rectal, and topical administration; however, this application is not limited to these exemplified routes of administration.

[0112] According to embodiments of this application, the metabolic disorder-related diseases include at least one of obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0113] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0114] Example 1:

[0115] The schematic diagram of the peptide design and screening process in this application is shown below. Figure 1 This process encompasses a series of steps, from GLP1R sequence generation to screening and optimization. This workflow is the core of our technical implementation, covering steps from sequence generation to affinity optimization and iterative evolution. The entire framework references the method proposed in patent 2025102889738. The entire method consists of multiple iterative evolutionary processes, after which the model-designed peptides achieve effective enhancements in biological activity. Each iterative evolution includes three parts: novel peptide generation and optimization based on artificial intelligence, wet experimental determination of peptide biological activity, and feedback to strengthen the model's perception of biological activity.

[0116] First, we generated a batch of candidate peptide sequences that may have high affinity for GLP1R, referencing the method proposed in patent 2025102886867. This method is an innovative deep generative model, PepGPT, capable of generating peptide sequences that can bind to the target protein based on its amino acid sequence or structural information. PepGPT is first pre-trained using a next token prediction task. This method involves training the model on a large-scale sequence dataset, aiming to predict the next token in the sequence given the preceding context. Through this learning process, PepGPT can capture the contextual relationships and patterns in a large amount of peptide data. The next step after pre-training is to fine-tune PepGPT using collected protein-peptide pairing data to achieve target protein-guided peptide generation. In this process, the GLP1R protein is used as a conditional input, enabling the model to learn how to generate the corresponding binding peptide sequence. Using the trained PepGPT, we generated a batch of candidate peptide sequences that may have high affinity for the target GLP1R protein as input.

[0117] Next, we perform affinity maturation on these high-affinity candidate peptide sequences. This step primarily references the deep reinforcement learning method for target protein-specific binding peptide generation and optimization proposed in patent 2025101234137. This method is an innovative deep reinforcement learning scheme specifically designed for optimizing peptide sequences, guiding the evolution of candidate peptide sequences by simulating the environment and defining actions. This deep reinforcement learning framework can incorporate reward models as prior knowledge to guide peptide evolution. These reward models can be affinity prediction models, solubility prediction models, or toxicity prediction models, etc.

[0118] For our reward model, we referenced the affinity prediction method proposed in patent 2025101207356. This method is an innovative technical solution called PepAF, which effectively predicts the binding affinity of target proteins and peptides by comprehensively utilizing 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 GLP1R-peptide complex; 2) predicting the affinity of the protein GLP1R-peptide complex. The first task enables the model to learn the complex interactions between proteins and peptides, as well as their structure and physicochemical properties, which provides a foundation for understanding the binding patterns and key features of protein GLP1R-peptide interactions. The second task enables the model to capture a wide range of molecular interactions at the atomic scale. PepAF also improves the accuracy of predictions by modeling the target protein structure and the peptide flexibility. We use the core technology PepAF from this patent as the reward model for a deep reinforcement learning method to guide the mutation of candidate peptide sequences.

[0119] After obtaining affinity-matured peptides, we evaluated their bioactivity and fed the results back to the model to continue the next round of peptide generation and optimization. After two rounds of iteration, we designed a peptide that effectively activates the GLP1R target protein, the sequence of which is shown in SEQ ID NO:1.

[0120] HSQGTFTSDYSKYLDERAASEFVQWLLAG (SEQ ID NO: 1).

[0121] Example 2:

[0122] This embodiment aims to evaluate the cAMP activation ability of a candidate peptide (amino acid sequence HSQGTFTSDYSKYLDERAASEFVQWLLAG) on huGLP1R-FL-CRE-HEK293-A5 cells. By quantitatively measuring cAMP production levels, this experiment can accurately assess the activation effect of the candidate sample on the GLP-1R receptor, providing important bioactivity data for drug development for metabolic diseases such as diabetes. The experimental procedure is as follows:

[0123] Peptide Synthesis and Purification: Based on the designed candidate peptide's amino acid sequence, the peptide was synthesized using solid-phase peptide synthesis (Fmoc-SPPS) technology. High-performance liquid chromatography (HPLC) was employed for purification to ensure the synthesized peptide had high purity and was free of significant impurities. Furthermore, the precise molecular weight of the peptide was verified by mass spectrometry (MS).

[0124] 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.

[0125] Cell starvation treatment: The huGLP1R-FL-CRE-HEK293-A5 cell line was constructed, and highly viable huGLP1R-FL-CRE-HEK293-A5 cells were selected and starved in DMEM medium containing 0% fetal bovine serum (FBS) to standardize experimental conditions and eliminate interference from other factors in serum.

[0126] Cell digestion, washing, and plating: Adherent cells were gently digested with Accutase enzyme and washed with PBS buffer to remove residual culture medium and enzymes. The cell suspension was precisely plated into HTRF 96-well low-volume assay plates to ensure a consistent cell count per well, providing uniform starting conditions for subsequent cAMP activity assays.

[0127] cAMP activity assay: After cell seeding, a precisely diluted candidate peptide was added to each well (see [link to cAMP assay] for specific concentrations). Figure 2 (x-axis) Incubate at 37°C for 20 minutes to simulate the in vivo environment and activate the GLP-1R receptor. Add cAMPd2 reagent and anti-cAMP Eu Cryptate antibody (PerkinElmer, catalog number 62AM4PEB) working solution to each well and incubate at room temperature for 1 hour. This step allows cAMP to bind to the fluorescently labeled antibody, forming an energy transfer complex.

[0128] Signal detection: Using TR-FRET technology, with an excitation wavelength of 340 nm, the emitted signals were detected at 665 nm and 620 nm, respectively. This technology has high sensitivity and anti-interference capability. The FRET signal value was calculated by the ratio of the light intensity at 665 nm to that at 620 nm, reflecting the change in cAMP concentration; the change in the ratio is inversely proportional to the cAMP concentration.

[0129] Test results as follows Figure 2 As shown in Table 1.

[0130] Table 1

[0131] sample <![CDATA[EC 50 (nM)]]> polypeptide 0.04

[0132] Experimental results: The synthesized peptide exhibits good cAMP activation activity, and its EC50... 50 The value is 0.04 nanomolar, indicating that it has high biological activity.

[0133] Furthermore, the crystal structure or molecular dynamics of the target protein-peptide complex was analyzed using AlphaFold and HPEPDOCK methods. The simulation results of the crystal structure or molecular dynamics of the target protein-peptide complex can be found in [link to simulation results]. Figure 3 As shown in the figure. The results demonstrate that the peptide can precisely bind to the active site of the GLP-1R receptor and exert its biological effects.

[0134] Example 3

[0135] This embodiment aims to evaluate the in vitro therapeutic effects of the candidate peptides 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:

[0136] 1) For obesity: Using 3T3-L1 adipocytes, the cells were first induced to differentiate into adipocytes 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 establish an obesity model. When the confluence of 3T3-L1 preadipocytes reached 80-90%, they were digested with 0.25% trypsin at a concentration of 2-3 × 10⁻⁶. 4 cells / cm 2 The cells were seeded at a specific density. When the cell confluence reached 80-95%, adipogenic differentiation-inducing medium was added for induction. After 2-3 days of induction, the medium was replaced with adipogenic differentiation-inducing maintenance medium for 1 day. This alternating induction was repeated 3-5 times until obvious lipid droplets were observed in the cells, indicating that the obesity model had been successfully constructed.

[0137] Then, the candidate peptides were added to adipocyte culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), and the cell density was controlled at 2-3 × 10⁻⁶ cells / mL. 4 cells / cm 2To ensure normal cell growth and differentiation, cells were cultured for 24-48 hours. Intracellular lipid droplet content was detected using Oil Red O staining: the culture medium was aspirated, the cells were washed with 1×PBS, fixed with 4% neutral formaldehyde solution for 30 min, and Oil Red O working solution (saturated Oil Red O solution: distilled water = 3:2) was prepared. After filtration, 1 mL of Oil Red O working solution was added to each well, and staining was performed at room temperature for 30 min. The staining solution was aspirated, the cells were washed with 1×PBS, and observed and photographed under a microscope. Simultaneously, the expression levels of adipogenesis-related genes (PPARγ, C / EBPα) were detected using a qPCR kit (Takara RR420A). If the candidate peptide significantly reduces intracellular lipid droplet content and decreases the expression of adipogenesis-related genes, it indicates a potential therapeutic effect on obesity, possibly by inhibiting adipocyte differentiation and lipid accumulation.

[0138] 2) For diabetes: Pancreatic β-cell line (MIN6 cells) was used and 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. Candidate peptides were added to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), and the cell density was controlled at 5 × 10⁶ cells / mL. 3 cells / cm 2 Cells were cultured for 24-48 hours. Insulin secretion in the cell culture supernatant was detected using an insulin ELISA kit. Simultaneously, the expression levels of proteins related to the insulin signaling pathway, such as glucose transporter 2 (GLUT2) and insulin receptor substrate (IRS), were detected using a GLU2 kit (YUANJU BIO, catalog number YJ24519) and an IRS1 kit (Thermo Fisher Scientific, catalog number KHO0521). If the candidate peptide can increase insulin secretion and upregulate the expression levels of GLUT2 and IRS, it suggests that it may have the effect of improving insulin resistance and promoting insulin secretion, and has potential value for the treatment of diabetes.

[0139] 3) For dyslipidemia-related diseases: HepG2 cells were used as the hepatocyte line. A dyslipidemia model was established using a culture medium containing high concentrations of fatty acids (Wuhan Shangen Biotechnology Co., Ltd., HepG2 cell-specific culture medium, whose components include EMEM (MEM+NEAA) treatment of cells + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)). Candidate peptides were added to cell culture media at different concentrations (10nM, 100nM, 1μM, and 10μM), and the cell density was controlled at 5×10⁶ cells / day.3 cells / cm 2 Cells were cultured for 24-48 hours. Intracellular total cholesterol was detected using an enzymatic assay kit (Sigma-Aldrich Total Cholesterol Assay Kit, MAK043), and triglyceride levels were detected using a triglyceride assay kit (Sigma-Aldrich Triglyceride Assay Kit, MAK041). Simultaneously, the expression levels of genes related to cholesterol metabolism, such as low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1), were detected using an LDLR kit (Thermo Fisher Scientific, EHLDLR) and a CYP7A1 kit (Proteintech, 18054-1-AP). If the candidate peptide can reduce intracellular total cholesterol and triglyceride levels while upregulating LDLR and CYP7A1 expression, it suggests that it may have a therapeutic effect on dyslipidemia-related diseases by regulating cholesterol synthesis and metabolism.

[0140] 4) For fatty liver disease: HepG2 cells were used in the same manner, and a fatty liver disease model was established by inducing steatosis in the cells using high-fat culture medium (Wuhan Shangen Biotechnology Co., Ltd., HepG2 cell-specific culture medium, whose components include EMEM (MEM+NEAA) + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)). Candidate peptides were added to cell culture media at different concentrations (10nM, 100nM, 1μM, and 10μM), and the cell density was controlled at 5×10⁶ cells / year. 3 cells / cm 2Cells were cultured for 24-48 hours. Intracellular lipid droplet content was detected using Oil Red O staining: the culture medium was aspirated, the cells were washed with 1×PBS, fixed with 4% neutral formaldehyde solution for 30 min, and Oil Red O working solution (saturated Oil Red O solution: distilled water = 3:2) was prepared. After filtration, 1 mL of Oil Red O working solution was added to each well, and staining was performed at room temperature for 30 min. The staining solution was aspirated, the cells were washed with 1×PBS, and observed and photographed under a microscope. Simultaneously, ELISA kits (Thermo Fisher Scientific TNF-α and IL-6 ELISA kits, catalog numbers K1480 and K1501, respectively) were used to detect the secretion levels of inflammatory factors, and colorimetric kits (Cayman Chemical MDA Assay Kit, catalog number 700455 and SOD Assay Kit, catalog number 705125) were used to detect oxidative stress indicators. If the candidate peptide can reduce the accumulation of intracellular lipid droplets, decrease the secretion of inflammatory factors, and improve oxidative stress, it indicates that it may have a therapeutic effect on fatty liver disease and can alleviate steatosis and inflammatory damage to hepatocytes.

[0141] 5) For metabolic syndrome: Construct a cell model of metabolic syndrome, which can use a variety of cells (adipocytes, hepatocytes, muscle cells, etc.) to simulate the complex pathological environment of metabolic syndrome.

[0142] Adipocytes: 3T3-L1 adipocytes were induced to differentiate into adipocytes using high-glucose DMEM medium (containing 4.5 g / L glucose, 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin) to establish an obesity model.

[0143] Hepatocytes: HepG2 cells were used and treated with high-fat medium (EMEM + 10% fetal bovine serum + 1% penicillin / streptomycin, with added high concentrations of fatty acids) to simulate the environment of fatty liver disease.

[0144] Muscle cells: C2C12 muscle cells were used and cultured in high-glucose DMEM medium to simulate an insulin-resistant environment.

[0145] The candidate peptides were added to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), and the cell density was controlled at 5 × 10⁶ cells / mL. 3 cells / cm 2Cells were cultured for 24-48 hours. After culture, indicators related to metabolic syndrome were measured in each cell, including the insulin resistance index (HOMA-IR, calculated by detecting insulin and glucose levels), blood lipid levels (triglyceride and total cholesterol levels were detected using a triglyceride assay kit (Sigma-Aldrich Triglyceride Assay Kit, catalog number MAK041) and a total cholesterol assay kit (Sigma-Aldrich Total Cholesterol Assay Kit, catalog number MAK043), and inflammatory factors (TNF-α and IL-6 secretion levels were detected using ELISA kits (Thermo Fisher Scientific TNF-α and IL-6 ELISA kits, catalog numbers K1480 and K1501, respectively)). If the candidate peptide 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, possibly through a multi-target mechanism to comprehensively regulate metabolic disorders.

[0146] 6) For non-alcoholic fatty liver disease: HepG2 cells were induced to exhibit non-alcoholic fatty liver disease-like changes using a high-fat, high-glucose medium (Wuhan Shangen Biotechnology Co., Ltd., HepG2 cell-specific medium, whose components include EMEM (MEM+NEAA) treatment + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)). Candidate peptides were added to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), and the cell density was controlled at 5 × 10⁶ cells / mL. 3 cells / cm 2 Cells were cultured for 24-48 hours. Intracellular lipid droplet content was detected using Oil Red O staining. Simultaneously, ELISA kits (Thermo Fisher Scientific IL-1β and IL-8 ELISA kits, catalog numbers K1480 and K1501, respectively) were used to detect the secretion levels of inflammatory factors. Hepatocyte damage markers were detected using colorimetric assay kits (Cayman Chemical ALT and AST Assay Kits, catalog numbers 700455 and 705125, respectively). Western blot and qPCR were used to detect the expression levels of liver fibrosis-related markers (collagen I and α-smooth muscle actin). If the candidate peptide can reduce intracellular fat content, decrease the secretion of inflammatory factors, reduce the levels of hepatocyte damage markers, and inhibit the expression of liver fibrosis-related markers, it indicates that it has a certain therapeutic effect on non-alcoholic fatty liver disease, possibly by reducing fat accumulation and inhibiting inflammation and fibrosis progression.

[0147] The results showed that the candidate peptide in Example 1 had the following effects:

[0148] 1) It can significantly reduce the content of lipid droplets in 3T3-L1 adipocytes and reduce the expression of adipogenesis-related genes;

[0149] 2) It can increase the amount of insulin secreted 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 2 (GLUT2) and insulin receptor substrate (IRS).

[0150] 3) It can reduce the content 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).

[0151] 4) It can reduce the accumulation of lipid droplets in HepG2 cells, decrease the secretion of inflammatory factors, and improve oxidative stress.

[0152] 5) It can improve indicators related to metabolic syndrome in various cells (such as adipocytes, hepatocytes, muscle cells, etc.);

[0153] 6) It can reduce the intracellular fat content of HepG2 cells, reduce the secretion of inflammatory factors, reduce the level of hepatocyte damage markers, and inhibit the expression of liver fibrosis-related indicators.

[0154] In summary, the above-mentioned candidate peptides have therapeutic effects on obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0155] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0156] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that, It has an amino acid sequence as shown in HSQGTFTSDYSKYLDERAASEFVQWLLAG.

2. A nucleic acid molecule, an expression vector, and a recombinant cell, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1; The expression vector carries the aforementioned nucleic acid molecules; The recombinant cells carry the aforementioned nucleic acid molecules or the aforementioned expression vector, or the recombinant cells express the polypeptide described in claim 1 or 2.

3. A polypeptide derivative or a pharmaceutically acceptable salt thereof, characterized in that, include: The polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, and A modifying group, wherein the polypeptide or a pharmaceutically acceptable salt thereof is linked to the modifying group.

4. The polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that, The modifying group is attached to the -NH2, -SH, -OH or -COOH side chain of the amino acid in the polypeptide; Preferably, the modifying group is linked to the -NH2 group on the side chain of an amino acid in the polypeptide; Optionally, the modifying group has at least one of the following structures:

5. A fusion protein, characterized in that, Includes the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, or the polypeptide derivative of any one of claims 3 to 4 or a pharmaceutically acceptable salt thereof.

6. A reagent or kit, characterized in that, It includes the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the polypeptide derivative of any one of claims 3 to 4 or a pharmaceutically acceptable salt thereof, or the fusion protein of claim 5.

7. A pharmaceutical composition, characterized in that, Includes the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the polypeptide derivative of any one of claims 3 to 4 or a pharmaceutically acceptable salt thereof, or the fusion protein of claim 5; Optionally, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

8. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the polypeptide derivative of any one of claims 3-4 or a pharmaceutically acceptable salt thereof, the fusion protein of claim 5 or the pharmaceutical composition of claim 7 in the preparation of a medicament for the treatment or prevention of GLP-1R-related diseases; Optionally, the GLP-1R-related diseases include metabolic disorder-related diseases.

9. The use according to claim 8, characterized in that, The metabolic disorder-related diseases include at least one of obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

10. A method for detecting GLP-1R, characterized in that, include: The sample to be tested is brought into contact with the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the polypeptide derivative of any one of claims 3 to 4 or a pharmaceutically acceptable salt thereof, the fusion protein of claim 5, or the reagent of claim 6; Based on the signal generated by the contact product, it is determined whether the sample to be tested contains GLP-1R; Optionally, the signal includes a fluorescence signal; Optionally, it further includes determining the content value of GLP-1R in the sample to be tested based on the signal generated by the contact product.

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