GLP-1R activating polypeptide or pharmaceutically acceptable salt thereof and use thereof

Abstract

This application proposes a polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is represented by HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG. This polypeptide or a pharmaceutically acceptable salt thereof can bind to the GLP-1R and effectively activate the GLP-1R. Thus, the polypeptide or a pharmaceutically acceptable salt thereof can effectively treat diseases associated with metabolic disorders (e.g., obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease).

Description

Technical Field

[0001] The present application belongs to the field of biopharmaceutical technology, and specifically relates to a GLP-1R activating polypeptide or a pharmaceutically acceptable salt thereof and uses thereof. Background Art

[0002] GLP-1R (glucagon-like peptide 1 receptor) plays a crucial role in maintaining blood glucose homeostasis. Activation of GLP-1R promotes insulin secretion and inhibits glucagon release, effectively lowering blood glucose levels. Furthermore, GLP-1R is involved in regulating multiple physiological processes, including 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 intestinal L cells that exerts its effects by binding to the GLP-1R. In the pancreas, GLP-1 can reduce the secretion of glucagon and promote the secretion of insulin, thereby lowering blood sugar levels. Current GLP-1R agonists are all developed based on the premise of GLP-1. By simulating the physiological effects of endogenous GLP-1, they can promote the secretion of insulin, inhibit the secretion of glucagon, participate in regulating blood sugar levels, or delay gastric emptying, increase satiety, reduce food intake, help with weight control, and are widely used in clinical practice.

[0004] Therefore, the development of a new GLP-1R agonist is of great use in the treatment of diabetes, obesity and other related diseases. Summary of the Invention

[0005] 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 activating polypeptide or a pharmaceutically acceptable salt thereof.

[0006] This application is based on the following findings of the inventors:

[0007] Most GLP-1R agonists in the prior art are GLP-1 analog peptides. Although they have certain efficacy in regulating blood glucose levels, they still have some significant limitations:

[0008] 1. Stability issues: Longer polypeptide sequences may have a shorter half-life in the body and are easily degraded by enzymes in the body, limiting the duration of their therapeutic effect and the convenience of their application.

[0009] 2. Most existing agonists are GLP-1 analogs, which limits the exploration of peptides with stronger binding ability and greater stability.

[0010] To address the aforementioned shortcomings of existing technologies, this application has developed a novel GLP-1R agonist, offering a novel approach for treating diabetes, obesity, and other related diseases. Through precise computation, deep learning models, and iterative evolutionary model optimization, this application has obtained a novel peptide. Compared to traditional GLP-1 analogs, this peptide has a unique amino acid arrangement and can effectively activate the GLP-1R.

[0011] Therefore, in the first aspect of the present application, the present application proposes a polypeptide or a pharmaceutically acceptable salt thereof. According to the embodiments of the present application, the amino acid sequence of the polypeptide is shown as HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG. The polypeptide of the present application or a pharmaceutically acceptable salt thereof can bind to GLP-1R for effectively activating GLP-1R. Thus, the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof can be used to treat or prevent GLP-1R-related diseases, such as metabolic disorder-related diseases, for example, obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome and non-alcoholic fatty liver disease.

[0012] In a second aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the polypeptide described in the first aspect, or a pharmaceutically acceptable salt thereof. The nucleic acid molecule of the present application may encode the polypeptide described in the first aspect, or a pharmaceutically acceptable salt thereof, which can bind to the GLP-1R for effective activation of the GLP-1R.

[0013] In a third aspect, the present application provides an expression vector. According to embodiments of the present application, the expression vector carries the nucleic acid molecule described in the second aspect. The expression vector of the present application carries the nucleic acid molecule described in the second aspect and can express the polypeptide of the first aspect or a pharmaceutically acceptable salt thereof, which can bind to the GLP-1R to effectively activate the GLP-1R.

[0014] In a fourth aspect, the present application provides a recombinant cell. According to embodiments of the present 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 the present application can express the polypeptide of the first aspect or a pharmaceutically acceptable salt thereof, which can bind to the GLP-1R for effective activation of the GLP-1R.

[0015] In the fifth aspect of the present application, the present application proposes a polypeptide derivative or a pharmaceutically acceptable salt thereof. According to an embodiment of the present 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 and the modifying group are connected. As can be seen from the foregoing, the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof can effectively activate GLP-1R. Therefore, the use of a polypeptide derivative or a pharmaceutically acceptable salt thereof containing the above-mentioned polypeptide can be used to treat or prevent GLP-1R-related diseases, such as metabolic disorder-related diseases, such as obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome and non-alcoholic fatty liver disease.

[0016] In the sixth 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 pharmaceutically acceptable salt thereof described in the first aspect, or the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect. As mentioned above, the above-mentioned polypeptide or pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Thus, the fusion protein containing the above-mentioned polypeptide or pharmaceutically acceptable salt thereof can bind to GLP-1R, be used to detect GLP-1R or be used to activate GLP-1R, and can also be used to treat or prevent GLP-1R-related diseases, such as metabolic disorder-related diseases, such as obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome and non-alcoholic fatty liver disease.

[0017] In a seventh aspect, the present application provides a reagent or kit. According to embodiments of the present application, the reagent or kit includes the polypeptide or pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect, or the fusion protein described in the sixth aspect. As previously mentioned, the above-mentioned polypeptides or pharmaceutically acceptable salts thereof can bind to the GLP-1R and can be used to effectively activate the GLP-1R. Thus, the reagent or kit containing the above-mentioned polypeptides can bind to the GLP-1R and be used to detect the GLP-1R.

[0018] In the eighth 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 pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect, or the fusion protein described in the sixth aspect. As mentioned above, the above-mentioned polypeptide or pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Thus, the pharmaceutical composition containing the above-mentioned polypeptide can be used to treat or prevent GLP-1R-related diseases, such as metabolic disorder-related diseases, such as obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0019] In the ninth aspect of the present application, the present application proposes the use of the polypeptide or pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect, the fusion protein described in the sixth aspect, or the pharmaceutical composition described in the eighth aspect in the preparation of a drug, wherein the drug is used to treat or prevent GLP-1R-related diseases.

[0020] In the tenth aspect of the present application, a method for detecting GLP-1R is provided. According to embodiments of the present application, the method comprises: contacting a sample to be tested with the polypeptide or pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect, the fusion protein described in the sixth aspect, or the reagent or kit described in the seventh aspect; and determining whether the sample to be tested contains GLP-1R based on a signal generated by the contact product. As previously mentioned, the polypeptide or pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Thus, the above method can be used to bind to GLP-1R for detection of GLP-1R.

[0021] 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

[0022] 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:

[0023] Figure 1 Schematic diagram of the peptide design and screening process of this application;

[0024] Figure 2 This is the detection result of the polypeptide activating the GLP-1R target to release cAMP in the examples of this application. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] As used herein, the term "glucagon-like peptide-1 receptor (GLP-1R)" refers to a G protein-coupled receptor (GPCR) that belongs to the class B GPCR family. It plays an important physiological role in the human body, particularly in regulating blood glucose levels. Activation of the GLP-1R promotes insulin secretion and inhibits glucagon release, thereby lowering blood glucose. Furthermore, the GLP-1R is involved in regulating various physiological processes, including gastrointestinal motility, appetite control, and energy balance. Activation of the 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.

[0031] 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.

[0032] In this article, the term "sequence similarity" of amino acids 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.

[0033] .

[0034] 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.

[0035] In this context, the structural formula of the term "αMeK" is .

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

[0037] In this article, the structural formula of the term "N-Me-K" is .

[0038] In this context, the structural formula of the term "Orn" is .

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

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

[0041] 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.

[0042] 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, as described in SM Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19. 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 these salts can be obtained by other methods described in the literature, such as ion exchange.

[0043] As used herein, "pharmaceutical composition" may refer to a composition for use in treating a disease or in vitro cell culture experiments. When used in treating 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.

[0044] 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.

[0045] 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.

[0046] The pharmaceutical compositions of the present application include formulations suitable for parenteral administration. The formulations can conveniently be 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.

[0047] As used herein, the term "agonist" refers to a substance (ligand) that activates the receptor type in question.

[0048] 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 a pharmaceutically acceptable salt thereof, or a drug containing a polypeptide or a pharmaceutically acceptable salt thereof, 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 a pharmaceutically acceptable salt thereof as described herein to an individual in need.

[0049] 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.

[0050] The present application proposes a GLP-1R activating polypeptide or a pharmaceutically acceptable salt thereof and uses thereof, which will be described in detail below.

[0051] Polypeptide or pharmaceutically acceptable salt thereof

[0052] In one aspect of the present application, the present application provides a polypeptide or a pharmaceutically acceptable salt thereof. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown as HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG.

[0053] The GLP-1R activating polypeptide of the present application is obtained through precise calculation, deep learning model and iterative evolution model optimization. It can bind to GLP-1R, exhibits high affinity and high biological activity for GLP-1R, and can effectively activate GLP-1R, providing a new treatment option for patients with metabolic disorder-related diseases (such as diabetes).

[0054] In particular, with the increasing prevalence of diabetes worldwide and the limitations of existing treatment options, the GLP-1R activating polypeptide or a pharmaceutically acceptable salt thereof of the present application can not only provide new treatment options for patients with metabolic disorder-related diseases (such as diabetes), but is also expected to reshape the market landscape for the treatment of metabolic disorder-related diseases (such as diabetes).

[0055] Herein, the term "the amino acid sequence of the polypeptide is as shown in A" includes the amino acid sequence of A, the amino acid sequence of conservatively modified forms of A, or the amino acid sequence with a sequence similarity of more than 90% (for example, 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 A, or the amino acid sequence that has substituted, deleted or added one or more amino acids compared to A and has GLP-1R binding activity, 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 “amino acid sequence of a conservatively modified form of A, or a sequence 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%) with the amino acid sequence shown in A” can be located at any position of the polypeptide of the present application, and can also be located at at least one of positions 2, 4-12, 14, 22, and 25-26, and can also be located at 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 change the structural stability and / or receptor binding activity of the polypeptide containing the amino acid, and are all within the scope of protection of the present application.

[0056] Illustratively, "the amino acid sequence of the polypeptide is as shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG" means that the polypeptide is the amino acid sequence shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG, the amino acid sequence of a conservative modification of HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG, or the sequence similarity with the amino acid sequence shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG is more than 90% (for example, 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%), or the polypeptide is an amino acid sequence in which one or more amino acids are substituted, deleted or added compared to HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG and has GLP-1R binding activity, all of which are within the scope of protection of the present application.

[0057] It should be noted that, as used herein, "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 (e.g., the binding properties of the polypeptides of this application to the GLP-1R). Amino acid substitution refers to the replacement of an amino acid residue in the original peptide chain with an amino acid residue having a similar side chain. Families of amino acid residues 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 nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0058] The present application provides a polypeptide or a pharmaceutically acceptable salt thereof. According to an embodiment of the present application, the polypeptide has an amino acid sequence as shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO: 1), or an amino acid sequence having 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) similarity to HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO: 1); or an amino acid sequence in which one or more amino acids are substituted, deleted, or added to HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO: 1) and has GLP-1R binding activity.

[0059] According to an embodiment of the present application, the above polypeptide or a pharmaceutically acceptable salt thereof may further include at least one of the following technical features:

[0060] According to the embodiments of the present application, compared with the amino acid sequence shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO: 1), 1 to 3 amino acids are substituted, deleted, or added and have GLP-1R binding activity, such as substitution, deletion, or addition of 1, 2, or 3 amino acids.

[0061] 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 pharmaceutically acceptable salt in vivo.

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

[0063] Nucleic acid molecules, expression vectors and recombinant cells

[0064] In a second aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof. The nucleic acid molecule of the present application may encode the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, which may bind to the GLP-1R to effectively activate the GLP-1R.

[0065] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0066] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, nucleic acid molecule sequences in this application include DNA or RNA forms, and disclosure of one form implies disclosure of the other.

[0067] In the third aspect of this application, an expression vector is provided. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the second aspect. When the nucleic acid molecule is linked to the expression vector, the nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling 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, that is, not derived from the expression vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked.

[0068] As used herein, "operably linked" refers to linking an exogenous gene to an expression vector so that the control elements within the expression vector, such as transcriptional control sequences and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used expression vectors include plasmids, bacteriophages, and the like. After the expression vectors according to certain embodiments of the present application are introduced into appropriate recipient cells, they can be mediated by a regulatory system to effectively express the aforementioned polypeptide, thereby enabling the in vitro production of large quantities of the polypeptide.

[0069] In some specific embodiments of the present application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0070] In some specific embodiments of the present application, the expression vector is a lentiviral vector.

[0071] In an optional embodiment of the present application, the expression vector is a plasmid expression vector.

[0072] In a fourth aspect, the present application provides a recombinant cell. According to embodiments of the present 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 or a pharmaceutically acceptable salt thereof. Under suitable conditions, the recombinant cell can effectively express the aforementioned polypeptide or a pharmaceutically acceptable salt thereof within the recombinant cell.

[0073] According to an embodiment of the present application, the recombinant cell is obtained by introducing the expression vector described in the third aspect into a host cell.

[0074] It should be noted that the host cells of the present application are not particularly limited and can be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The aforementioned eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as S. frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0075] According to an embodiment of the present application, the host cell is a eukaryotic cell.

[0076] According to an embodiment of the present 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.

[0077] It should be noted that the "suitable conditions" described in this application refer to conditions suitable for the expression of the polypeptides described herein. Those skilled in the art will readily appreciate that conditions suitable for polypeptide expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cells, suitable cell density, a suitable cell culture environment, and a suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for polypeptide expression based on the specific laboratory environment.

[0078] Polypeptide derivative or pharmaceutically acceptable salt thereof

[0079] In the fifth aspect of the present application, the present application proposes a polypeptide derivative or a pharmaceutically acceptable salt thereof. According to an embodiment of the present 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 connected to the modifying group. As mentioned above, the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof can effectively activate GLP-1R. Thus, the polypeptide derivative or a pharmaceutically acceptable salt thereof containing the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof can be used to treat or prevent GLP-1R-related diseases, such as metabolic disorder-related diseases, such as obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome and non-alcoholic fatty liver disease.

[0080] 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.

[0081] According to an embodiment of the present application, the above-mentioned polypeptide derivative or a pharmaceutically acceptable salt thereof may further include at least one of the following technical features:

[0082] According to an embodiment of the present application, the modifying group is connected to -NH2, -SH, -OH or -COOH of the amino acid side chain in the polypeptide.

[0083] 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.

[0084] According to an embodiment of the present application, the modifying group has at least one of the following structures:

[0085]

[0086] 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 Connected to the -NH- of amino acids.

[0087] Fusion protein, reagent or kit, pharmaceutical composition

[0088] In the sixth 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 pharmaceutically acceptable salt thereof described in the first aspect, or the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect. As can be seen from the foregoing, the above-mentioned polypeptide or pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Thus, the fusion protein containing the above-mentioned polypeptide can bind to GLP-1R and be used to detect GLP-1R or to activate GLP-1R, and can also be used to treat or prevent GLP-1R, such as metabolic disorder-related diseases, such as obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome and non-alcoholic fatty liver disease.

[0089] In an optional embodiment of the present application, the fusion protein further includes a functional fragment.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] In a seventh aspect, the present application provides a reagent or kit. According to embodiments of the present application, the reagent or kit includes the polypeptide or pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect, or the fusion protein described in the sixth aspect. As previously mentioned, the above-mentioned polypeptide or pharmaceutically acceptable salt thereof can bind to the GLP-1R and can be used to effectively activate the GLP-1R. Thus, a reagent or kit containing the above-mentioned polypeptide or pharmaceutically acceptable salt thereof can bind to the GLP-1R and be used to detect the GLP-1R.

[0094] In the eighth aspect of the present application, the present application proposes a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises the polypeptide or pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect, or the fusion protein described in the sixth aspect. As mentioned above, the above-mentioned polypeptide or pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Thus, the pharmaceutical composition containing the above-mentioned polypeptide or pharmaceutically acceptable salt thereof can be used to treat or prevent diseases related to metabolic disorders, such as obesity, diabetes, diseases related to dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0095] According to an embodiment of the present application, the pharmaceutical composition further includes a pharmaceutically acceptable excipient.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] In some optional embodiments of the present application, the pharmaceutical composition of the present application may also contain other active ingredients for treatment.

[0101] 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.

[0102] use

[0103] In the ninth aspect of the present application, the present application proposes the use of the polypeptide or pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect, the fusion protein described in the sixth aspect, or the pharmaceutical composition described in the eighth aspect in the preparation of a drug, wherein the drug is used to treat or prevent GLP-1R-related diseases.

[0104] According to an embodiment of the present application, the GLP-1R-related diseases include metabolic disorder-related diseases.

[0105] According to an embodiment of the present 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.

[0106] method

[0107] In the tenth aspect of the present application, a method for detecting GLP-1R is provided. According to embodiments of the present application, the method comprises: contacting a sample to be tested with the polypeptide or pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or pharmaceutically acceptable salt thereof described in the fifth aspect, the fusion protein described in the sixth aspect, or the reagent or kit described in the seventh aspect to form a contact product; and determining whether the sample to be tested contains GLP-1R based on a signal generated by the contact product. As previously mentioned, the polypeptide or pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Thus, the above method can bind to GLP-1R for detection of GLP-1R.

[0108] According to a specific embodiment of the present application, the signal includes a fluorescent signal.

[0109] According to a specific embodiment of the present application, 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.

[0110] In an eleventh 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 a pharmaceutically acceptable salt thereof described in the first aspect, the polypeptide derivative or a pharmaceutically acceptable salt thereof described in the fifth aspect, the fusion protein described in the sixth aspect, or the pharmaceutical composition described in the eighth aspect.

[0111] In an optional embodiment of the present application, the pharmaceutically acceptable dose can be selected from an effective dose (or effective amount).

[0112] The effective amount of the polypeptide or pharmaceutically acceptable salt thereof herein 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 exigencies of the treatment, several divided doses may be administered daily, or the dose may be proportionally reduced.

[0113] The polypeptides or pharmaceutically acceptable salts thereof, 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 pharmaceutically acceptable salts thereof, 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.

[0114] According to an embodiment of the present 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.

[0115] 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.

[0116] Example 1:

[0117] The schematic diagram of the polypeptide design and screening process of this application is as follows Figure 1 , which covers the steps from GLP-1R sequence generation to screening and optimization. The entire method consists of multiple iterative evolutionary processes, and after each iteration, the bioactivity of the peptides designed by the model is effectively improved. 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.

[0118] First, through a 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 the target protein and the peptide sequence are learned, and specific peptides (i.e., GLP-1R target peptides) are generated for specific target proteins (i.e., GLP-1R). 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:

[0119] 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.

[0120] 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.

[0121] 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:

[0122] 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.

[0123] 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.

[0124] 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 the peptide sequence, implicitly modeling the relationship between the target protein and the 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 a peptide sequence that binds to the given target. Using the trained TPDiffusion and the GLP-1R target sequence as input, a batch of candidate peptide sequences with high affinity for the GLP-1R target is generated.

[0125] Second, affinity maturation is performed on these high-affinity candidate peptide sequences. This approach, a deep reinforcement learning method, guides 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.

[0126] The reward model uses PepAF, which 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 the protein (GLP-1R)-peptide complex; and 2) predicting the affinity of the protein (GLP-1R)-peptide complex. The first task enables the model to learn the complex interactions between the protein (GLP-1R) and peptide, as well as their structural and physicochemical properties, providing a foundation for understanding the binding modes and key features of the protein (GLP-1R)-peptide interaction. 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 the reward model in a deep reinforcement learning method to guide mutations in candidate peptide sequences.

[0127] After obtaining affinity-matured peptides, their biological activity is evaluated, and the results are fed back to the model for the next round of peptide generation and optimization. After one round of iteration, a peptide that effectively activates the GLP-1R target protein is designed, with the sequence shown in SEQ ID NO:1 (i.e., GA-Single.R5.S2). The amino acid sequence shown in SEQ ID NO:1 is as follows:

[0128] HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO: 1).

[0129] Example 2:

[0130] This example aims to evaluate the ability of the candidate polypeptide GA-Single.R5.S2 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:

[0131] Peptide Synthesis and Purification: Based on the amino acid sequence of the designed candidate peptide, the peptide was 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 peptide, free of significant impurities. Furthermore, mass spectrometry (MS) was used to verify the precise molecular weight of the peptide.

[0132] Peptide dissolution conditions: Dissolve the purified candidate peptide in DMSO (dimethyl sulfoxide) solvent to prepare a 1 mg / mL stock solution for subsequent experiments.

[0133] 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.

[0134] 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.

[0135] cAMP activity assay: After cell plating, add accurately diluted candidate peptides 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 receptor. 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.

[0136] 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.

[0137] The experimental results show that the peptide GA-Single.R5.S2 screened in this application has a good function of activating cAMP activity, which can be explained as having high biological activity. Figure 2 and as shown in Table 1.

[0138] Table 1

[0139]

[0140] Example 3

[0141] This example aims to evaluate the in vitro therapeutic effects of the candidate polypeptide in Example 1 (i.e., GA-Single.R5.S2) on obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease. The specific steps are as follows:

[0142] 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 were seeded at a density of 10 cells / cm². When the cell confluence reached 80-95%, adipogenic differentiation medium was added for induction. After 2-3 days of induction, the cells were switched to adipogenic differentiation maintenance medium for 1 day. This induction cycle was repeated 3-5 times until obvious lipid droplets were observed in the cells, indicating that the obesity model was successfully established.

[0143] Then, each candidate peptide was 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 / cm² to ensure normal cell growth and differentiation, and 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 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. The expression levels of adipogenesis-related genes (PPARγ and C / EBPα) were also 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 activity for obesity, possibly by inhibiting adipocyte differentiation and lipid accumulation.

[0144] 2) Diabetes: Pancreatic β-cell line MIN6 cells were cultured in high-glucose DMEM (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 various concentrations (10 nM, 100 nM, 1 μM, and 10 μM) at a cell density of 5 × 10³ cells / cm² and cultured 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 the candidate peptide can increase the secretion of insulin and upregulate the expression levels of GLUT2 and IRS, it means that it may have the effect of improving insulin resistance and promoting insulin secretion, and has potential value in the treatment of diabetes.

[0145] 3) For dyslipidemia-related diseases: A dyslipidemia model was established using a hepatocyte cell line (HepG2) using a culture medium containing high concentrations of fatty acids (Wuhan Shanen Biotechnology Co., Ltd., specifically for HepG2 cells, consisting of EMEM (MEM + NEAA) + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)). Each candidate peptide was added to the cell culture medium at various concentrations (10 nM, 100 nM, 1 μM, and 10 μM) at a cell density of 5 × 10 cells / cm² and cultured for 24–48 hours. Intracellular total cholesterol was measured using an enzymatic assay kit (Sigma-Aldrich Total Cholesterol Assay Kit, Cat. No. MAK043), and triglyceride levels were measured using a Sigma-Aldrich Triglyceride Assay Kit, Cat. No. MAK041. The LDLR kit (Thermo Fisher Scientific, Cat. No. EHLDLR) and the CYP7A1 kit (Proteintech, Cat. No. 18054-1-AP) were also used to measure the expression levels of genes related to cholesterol metabolism, such as the low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1). If the candidate peptide can reduce intracellular total cholesterol and triglyceride levels while simultaneously upregulating LDLR and CYP7A1 expression, it may have a therapeutic effect on dyslipidemia-related diseases by regulating cholesterol synthesis and metabolism.

[0146] 4) For fatty liver disease: HepG2 cells were similarly cultured using a high-lipid medium (Wuhan Shanen Biotechnology Co., Ltd., specifically for HepG2 cells, consisting of EMEM (MEM+NEAA) + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) to induce steatosis and establish a fatty liver disease model. The candidate peptides were added to the cell culture medium at various concentrations (10 nM, 100 nM, 1 μM, and 10 μM) at a cell density of 5 × 10³ cells / cm² and cultured for 24–48 hours. Intracellular lipid droplet content was assessed using Oil Red O staining: the medium was aspirated, the cells were rinsed with 1× PBS, and fixed with 4% neutral formaldehyde for 30 minutes. A working solution of Oil Red O (saturated Oil Red O solution: distilled water = 3:2) was prepared, filtered, and used for staining. 1 mL of the 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 and photographed under a microscope. ELISA kits (Thermo Fisher Scientific TNF-α and IL-6 ELISA kits, Catalog Nos. K1480 and K1501, respectively) were used to measure the secretion levels of inflammatory factors, and colorimetric kits (Cayman Chemical MDA Assay Kit, Catalog No. 700455 and SOD Assay Kit, Catalog No. 705125) were used to measure oxidative stress markers. If the candidate peptide can reduce the accumulation of intracellular lipid droplets, lower the secretion of inflammatory factors, and improve oxidative stress, it may have a therapeutic effect on fatty liver disease, alleviating steatosis and inflammatory damage in hepatocytes.

[0147] 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.

[0148] 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).

[0149] 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.

[0150] Muscle cells: C2C12 muscle cells were cultured in high-glucose DMEM medium to simulate an insulin-resistant environment.

[0151] Candidate peptides were added to cell culture medium at various concentrations (10 nM, 100 nM, 1 μM, and 10 μM) at a cell density of 5 × 10³ cells / cm² and cultured for 24–48 hours. Metabolic syndrome-related markers were then assessed, including the insulin resistance index (HOMA-IR) calculated by measuring insulin and glucose levels, lipid profiles (triglyceride and total cholesterol levels were measured using a Sigma-Aldrich Triglyceride Assay Kit (Cat. No. MAK041) and a Sigma-Aldrich Total Cholesterol Assay Kit (Cat. No. MAK043), and inflammatory cytokine secretion (TNF-α and IL-6 secretion levels were measured using ELISA kits (Thermo Fisher Scientific TNF-α and IL-6 ELISA kits, Cat. Nos. K1480 and K1501, respectively). If the candidate peptide can improve insulin resistance, lower blood lipid levels, and reduce the secretion of inflammatory factors, it indicates that it has potential therapeutic effects on metabolic syndrome and may comprehensively regulate metabolic disorders through a multi-target mechanism of action.

[0152] 6) For non-alcoholic fatty liver disease: HepG2 cells were cultured in a high-fat, high-glucose medium (Wuhan Shanen Biotechnology Co., Ltd., HepG2 cell-specific medium consisting of EMEM (MEM+NEAA) + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) to induce non-alcoholic fatty liver disease-like changes. Each candidate peptide was added to the cell culture medium at various concentrations (10 nM, 100 nM, 1 μM, and 10 μM) at a cell density of 5 × 10³ cells / cm² and 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 reducing fat accumulation and inhibiting inflammation and fibrosis.

[0153] The results showed that the candidate polypeptide in Example 1 (i.e., GA-Single.R5.S2) had the following effects:

[0154] 1) It can significantly reduce the content of lipid droplets in 3T3-L1 adipocytes and reduce the expression of adipogenesis-related genes;

[0155] 2) It can increase the secretion of insulin in pancreatic β-cells (such as MIN6 cells) and upregulate the expression levels of proteins related to the insulin signaling pathway, such as glucose transporter (GLUT2) and insulin receptor substrate (IRS);

[0156] 3) It can 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);

[0157] 4) It can reduce the accumulation of lipid droplets in HepG2 cells, reduce the secretion of inflammatory factors, and improve oxidative stress;

[0158] 5) It can improve indicators related to metabolic syndrome in various cells (such as fat cells, liver cells, muscle cells, etc.);

[0159] 6) It can reduce the fat content in HepG2 cells, reduce the secretion of inflammatory factors, reduce the levels of liver cell damage markers, and inhibit the expression of liver fibrosis-related indicators.

[0160] In summary, it can be shown that the above-mentioned candidate polypeptide GA-Single.R5.S2 has therapeutic effects on obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0161] 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.

[0162] 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 as HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG.

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

3. An expression vector, characterized in that The expression vector carries the nucleic acid molecule according to claim 2.

4. A recombinant cell, characterized in that The recombinant cell carries the nucleic acid molecule according to claim 2 or the expression vector according to claim 3, or the recombinant cell expresses the polypeptide according to claim 1.

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

6. The polypeptide derivative or pharmaceutically acceptable salt thereof according to claim 5, characterized in that: The modifying group is connected to the -NH2 of the amino acid side chain in the polypeptide; The modifying group has at least one of the following structures: 。 7. A fusion protein, characterized in that The invention comprises the polypeptide according to claim 1 or the polypeptide derivative according to any one of claims 5 to 6.

8. A reagent or kit, characterized in that The invention comprises the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, the polypeptide derivative according to any one of claims 5 to 6 or a pharmaceutically acceptable salt thereof, or the fusion protein according to claim 7.

9. A pharmaceutical composition, characterized in that The invention comprises the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, the polypeptide derivative according to any one of claims 5 to 6 or a pharmaceutically acceptable salt thereof, or the fusion protein according to claim 7, and optionally a pharmaceutically acceptable excipient.

10. Use of the polypeptide or pharmaceutically acceptable salt thereof according to claim 1, the polypeptide derivative or pharmaceutically acceptable salt thereof according to any one of claims 5 to 6, the fusion protein according to claim 7, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating or preventing diseases related to metabolic disorders; The metabolic disorder-related disease is at least one of obesity, diabetes, fatty liver disease and metabolic syndrome.

11. The use according to claim 10, characterized in that The metabolic disorder-related disease is non-alcoholic fatty liver disease.

12. A method for detecting GLP-1R for purposes other than disease diagnosis and treatment, characterized in that: include: contacting the sample to be tested with the polypeptide or pharmaceutically acceptable salt thereof according to claim 1, the polypeptide derivative or pharmaceutically acceptable salt thereof according to any one of claims 5 to 6, the fusion protein according to claim 7, or the reagent or kit according to claim 8; Based on the signal generated by the contact product, it is determined whether the sample to be tested contains GLP-1R.

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