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

Abstract

The invention provides a polypeptide or a pharmaceutically acceptable salt of the polypeptide. The amino acid sequence of the polypeptide is as shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG. The invention further provides a preparation method of the polypeptide. The polypeptide or the pharmaceutically acceptable salt thereof can be combined with the GLP-1R and is used for effectively activating the GLP-1R. Therefore, the polypeptide or the pharmaceutically acceptable salt thereof can be used for effectively treating metabolic disorder related diseases (such as obesity, diabetes, dyslipidemia related diseases, fatty liver diseases, metabolic syndromes and non-alcoholic fatty liver diseases).

Description

Technical Field

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

[0002] GLP-1R (i.e., glucagon-like peptide 1 receptor) plays a crucial role in maintaining blood glucose homeostasis. Activation of GLP-1R can promote insulin secretion and inhibit glucagon release, thereby effectively reducing blood glucose levels. In addition, GLP-1R is also 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 intestinal L cells, which exerts its effects by binding to GLP-1R. In the pancreas, GLP-1 can reduce glucagon secretion and promote insulin secretion, thereby lowering blood glucose levels. Current GLP-1R agonists are all developed based on GLP-1. 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, reduce food intake, contribute to weight control, and are widely used clinically.

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

[0005] This application aims to solve at least one of the technical problems existing in the prior art to some extent. To this end, this application provides a GLP-1R activating polypeptide or a pharmaceutically acceptable salt thereof.

[0006] This application is completed based on the following discoveries of the inventors: Most of the GLP-1R agonists in the prior art are GLP-1 analog polypeptides. Although they have certain efficacy in regulating blood glucose levels, there are still some significant limitations: 1. Stability problem: Longer polypeptide sequences may have a shorter half-life in vivo and are easily degraded by enzymes in the body, limiting the duration of their efficacy and the convenience of application.

[0007] 2. Most of the existing agonists are GLP-1 analogs, which limits the exploration of polypeptide spaces with stronger binding ability and greater stability.

[0008] In view of the above-mentioned disadvantages of the prior art, the present application has developed a new GLP-1R agonist, providing a new idea for the treatment of diabetes, obesity and other related diseases. The present application has obtained a brand-new polypeptide through precise calculations, deep learning models and iterative evolution models. Compared with traditional GLP-1 analogs, this polypeptide has a unique amino acid arrangement and can effectively activate GLP-1R.

[0009] Therefore, in the first aspect of the present application, the present application proposes a polypeptide or a pharmaceutically acceptable salt thereof. According to an embodiment of the present application, the amino acid sequence of the polypeptide is as shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG. The polypeptide or the pharmaceutically acceptable salt thereof of the present application can bind to GLP-1R and is used to effectively activate GLP-1R. Thus, the above-mentioned polypeptide or the pharmaceutically acceptable salt thereof can be used to treat or prevent GLP-1R-related diseases, such as diseases related to metabolic disorders, such as obesity, diabetes, diseases related to dyslipidemia, fatty liver disease, metabolic syndrome and non-alcoholic fatty liver disease, etc.

[0010] In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the polypeptide or the pharmaceutically acceptable salt thereof described in the first aspect. The nucleic acid molecule of the present application can encode the polypeptide or the pharmaceutically acceptable salt thereof of the first aspect, and this polypeptide can bind to GLP-1R and is used to effectively activate GLP-1R.

[0011] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment 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 or the pharmaceutically acceptable salt thereof of the first aspect, and this polypeptide can bind to GLP-1R and is used to effectively activate GLP-1R.

[0012] In the fourth aspect of the present application, the present application proposes a recombinant cell. According to an embodiment 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 and obtain the polypeptide or the pharmaceutically acceptable salt thereof of the first aspect, and this polypeptide can bind to GLP-1R and is used to effectively activate GLP-1R.

[0013] In the fifth aspect of the present application, a polypeptide derivative or a pharmaceutically acceptable salt thereof is proposed. According to an embodiment of the present application, the polypeptide derivative or a pharmaceutically acceptable salt thereof includes: 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 can be seen from the foregoing, the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof can effectively activate GLP-1R. Therefore, the polypeptide derivative or a pharmaceutically acceptable salt thereof containing the above polypeptide can be used for treating or preventing GLP-1R-related diseases, such as diseases related to metabolic disorders, for example, obesity, diabetes, diseases related to dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease, etc.

[0014] In the sixth aspect of the present application, a fusion protein is proposed. According to an embodiment of the present application, the fusion protein includes the polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect, or the polypeptide derivative or a pharmaceutically acceptable salt thereof described in the fifth aspect. As can be seen from the foregoing, the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the fusion protein containing the above polypeptide or a pharmaceutically acceptable salt thereof can bind to GLP-1R and is used for detecting GLP-1R or for activating GLP-1R, and can also be used for treating or preventing GLP-1R-related diseases, such as diseases related to metabolic disorders, for example, obesity, diabetes, diseases related to dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease, etc.

[0015] In the seventh aspect of the present application, a reagent or a kit is proposed. According to an embodiment of the present application, the reagent or the kit includes 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, or the fusion protein described in the sixth aspect. As can be seen from the foregoing, the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the reagent or the kit containing the above polypeptide can bind to GLP-1R and is used for detecting GLP-1R.

[0016] In the eighth aspect of the present application, a pharmaceutical composition is proposed. According to an embodiment of the present application, the pharmaceutical composition includes 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, or the fusion protein described in the sixth aspect. As can be seen from the foregoing, the above-mentioned polypeptide or a pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Therefore, the pharmaceutical composition containing the above polypeptide can be used for treating or preventing GLP-1R-related diseases, such as diseases related to metabolic disorders, for example, obesity, diabetes, diseases related to dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease, etc.

[0017] In the ninth aspect of the present application, there is provided 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 drug for treating or preventing GLP-1R-related diseases.

[0018] In the tenth aspect of the present application, there is provided a method for detecting GLP-1R. According to an embodiment of the present application, the method includes: contacting a sample to be detected 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 or kit described in the seventh aspect; and determining whether GLP-1R is contained in the sample to be detected based on the signal generated by the contact product. As can be seen from the foregoing, the above-mentioned polypeptide or a 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 and is used to detect GLP-1R.

[0019] The additional aspects and advantages of the present application will be partly given in the following description, partly will become apparent from the following description, or will be understood through the practice of the present application. Description of the Drawings

[0020] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the description of the embodiments in conjunction with the following drawings, in which: Figure 1 is a schematic diagram of the polypeptide design and screening process of the present application; Figure 2 is the detection result of the polypeptide activating the GLP-1R target to release cAMP in the embodiment of the present application. Detailed Embodiments

[0021] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, the meaning of "plurality" is two or more.

[0022] In this document, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present application, but does not exclude other aspects of the content.

[0023] 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.

[0024] In this article, the term "target protein" refers to a protein that plays a key role in the body and is usually the target of drug development. By binding to the target protein, the drug can regulate its biological activity, thereby achieving the purpose of treating the disease.

[0025] In this article, the term "peptide" refers to a biological macromolecule composed of many amino acids, usually 50 or fewer amino acids linked together by peptide bonds. Peptides have multiple functions in organisms, including participating in various physiological processes of organisms as enzymes, hormones, antibodies, etc. In addition, due to their good biocompatibility, selectivity and high biological activity, peptides are also widely studied for drug discovery and treatment of various diseases, such as metabolic disorder-related diseases. In this article, the term "glucagon-like peptide-1 receptor (GLP-1R)" is a G protein-coupled receptor that belongs to the class B G protein-coupled receptor family. It plays an important physiological role in the human body, especially in regulating blood glucose levels. Activation of GLP-1R can promote the secretion of insulin and inhibit the release of glucagon, thereby lowering blood glucose. In addition, GLP-1R is also involved in regulating gastrointestinal motility, appetite control, and energy balance. Activation of GLP-1R leads to the release of the α subunit of the G protein and activates adenylate cyclase (AC), a membrane-bound enzyme that catalyzes the conversion of ATP (adenosine triphosphate) into cAMP.

[0026] In this article, the term "cyclic adenosine monophosphate (cAMP)" is a small molecule that is ubiquitous in organisms and is converted from ATP (adenosine triphosphate) through the catalytic action of adenylate cyclase. As a key mediator of intracellular signal transduction, cAMP plays a vital role in regulating a variety of cellular functions and physiological processes.

[0027] 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. .

[0028] In this text, amino acids are represented by the 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, capital letters represent amino acids in the L configuration, and lowercase letters represent amino acids in the D configuration.

[0029] In this text, the structural formula of the term "αMeK" is .

[0030] In this text, the structural formula of the term "HoK" is .

[0031] In this text, the structural formula of the term "N-Me-K" is .

[0032] In this text, the structural formula of the term "Orn" is .

[0033] In this text, the structural formula of the term "Dab" is .

[0034] In this text, the structural formula of the term "Dap" is .

[0035] In this text, the term "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of a polypeptide or its derivative and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" as used in this application means approved by a federal regulatory agency or a national government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly in humans.

[0036] In this text, the term "pharmaceutically acceptable salt" refers to organic and inorganic salts of the polypeptides or their derivatives of this application. Pharmaceutically acceptable salts are well known in the art and are described in detail in, for example, S. M. Berge et al., 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 hydrochloride, hydrobromide, phosphate, sulfate, perchlorate) formed by reacting with amino groups, and organic acid salts (such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate), or these salts can be obtained by other methods such as ion exchange as described in the literature.

[0037] As used herein, "pharmaceutical composition" can refer to compositions for the treatment of diseases and can also be used in in vitro cell culture experiments. When used for the treatment of diseases, the term "pharmaceutical composition" generally refers to unit dosage forms and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with excipients that constitute one or more accessory components. Generally, the compositions are prepared by uniformly and sufficiently combining the active polypeptide or its derivative with a liquid excipient, a finely divided solid excipient, or both.

[0038] As used herein, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. Their use is also contemplated in this application, except to the extent that any conventional excipient is incompatible with the polypeptides or their derivatives, pharmaceutical compositions, or drugs containing them of this application, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a detrimental manner.

[0039] Except for any conventional excipient, their use is also contemplated in this application to the extent that they are incompatible with the polypeptides or their derivatives, pharmaceutical compositions, or drugs containing them of this application, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a detrimental manner.

[0040] The pharmaceutical compositions of this application include preparations suitable for parenteral administration. The preparations can conveniently be in unit dosage form and can be prepared by any method known to those skilled in the pharmaceutical art. The amount of the active ingredient that can be combined with the excipient material to prepare a single dosage form is generally the amount of the polypeptide or its derivative that produces a therapeutic effect.

[0041] As used herein, the term "agonist" refers to a substance (ligand) that activates the said receptor type.

[0042] As used herein, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect. The effect can be prophylactic 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 adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as reducing the symptoms associated with the disease. "Treatment" as used herein covers any administration of a polypeptide or a pharmaceutically acceptable salt thereof, or a medicament containing a polypeptide or a pharmaceutically acceptable salt thereof, to an individual to treat, cure, alleviate, improve, reduce or inhibit a disease in the individual, including but not limited to administering a medicament containing the polypeptide or a pharmaceutically acceptable salt thereof described herein to an individual in need thereof.

[0043] As used herein, the term "non-alcoholic fatty liver disease (NAFLD)" generally refers to a clinicopathological syndrome characterized by excessive intrahepatic fat deposition in hepatocytes excluding alcohol and other definite liver-damaging factors, an acquired metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility, including but not limited to simple fatty liver (SFL), non-alcoholic steatohepatitis (NASH) and related cirrhosis.

[0044] The present application provides a GLP-1R activating polypeptide or a pharmaceutically acceptable salt thereof and its uses, which will be described in detail below respectively.

[0045] Polypeptide or a pharmaceutically acceptable salt thereof 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 as shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG.

[0046] The GLP-1R activating polypeptide of the present application is obtained by optimizing through precise calculations, a deep learning model and an iterative evolution model. It can bind to GLP-1R, showing 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).

[0047] In particular, with the continuous increase in the global prevalence of diabetes 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 also is expected to reshape the market pattern of the treatment of metabolic disorder-related diseases (such as diabetes).

[0048] In this text, 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 a conservative modification form of A, or an amino acid sequence having a sequence similarity of more than 90% (such as more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) with the amino acid sequence shown in A, or an amino acid sequence having GLP-1R binding activity after substitution, deletion or addition of one or several amino acids compared with A, and all are within the protection scope of this application. Without special explanation, the conservative modified amino acids or amino acids with similarity differences in "the amino acid sequence of a conservative modification form of A, or an amino acid sequence having a sequence similarity of more than 90% (such as more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) with the amino acid sequence shown in A" can be located at any position of the polypeptide in this application, and can also be located at least one of the 2nd, 4th to 12th, 14th, 22nd, 25th to 26th positions, and can also be located at least one of the 1st, 3rd, 13th, 15th to 21st, 23rd to 24th, 27th to 30th positions. Such amino acid modifications or similarity differences that do not significantly affect or change the structural stability of the polypeptide containing the amino acid and / or its binding activity with the receptor are all within the protection scope of this application.

[0049] Exemplarily, "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 form of HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG, or an amino acid sequence having a sequence similarity of more than 90% (such as more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) with the amino acid sequence shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG, or an amino acid sequence having GLP-1R binding activity after substitution, deletion or addition of one or several amino acids compared with HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG, and all are within the protection scope of this application.

[0050] It should be noted that in this text, "substituting, deleting or adding one or several amino acids" means that after such substitution, deletion or addition of amino acids, it does not significantly affect or change the binding characteristics of the original amino acid sequence (such as the binding characteristics of the polypeptide of the present application to GLP-1R). Amino acid substitution is the substitution of an amino acid residue in the original peptide chain with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been determined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine).

[0051] 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 the polypeptide has an amino acid sequence with more than 90% (such as more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) similarity compared with HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO:1); or the polypeptide has an amino acid sequence that has been substituted, deleted or added with one or several amino acids and has GLP-1R binding activity compared with HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO:1).

[0052] According to an embodiment of the present application, the above-mentioned polypeptide or its pharmaceutically acceptable salt may further include at least one of the following technical features: According to an embodiment of the present application, compared with the amino acid sequence shown in HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO:1), it has been substituted, deleted or added with 1 to 3 amino acids and has GLP-1R binding activity. For example, 1, 2 or 3 amino acids are substituted, deleted or added.

[0053] According to an embodiment of the present application, the amino acid for substitution or addition is selected from amino acid X with an -NH2 in its side chain. Further, more modifiable sites are added to the original polypeptide, and the -NH2 in the side chain of the modifiable site can bind to a modifying group, thereby effectively prolonging the half-life of the polypeptide or its pharmaceutically acceptable salt in vivo.

[0054] 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.

[0055] Nucleic acid molecule, expression vector and recombinant cell In a second aspect of the present application, the present application provides a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the polypeptide or its pharmaceutically acceptable salt described in the first aspect. The nucleic acid molecule of the present application can encode the polypeptide or its pharmaceutically acceptable salt described in the first aspect, and the polypeptide or its pharmaceutically acceptable salt can bind to GLP-1R and is used to effectively activate GLP-1R.

[0056] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0057] It should be noted that for the nucleic acid molecules mentioned herein, those skilled in the art should understand that it actually includes any one of the complementary double strands, or both. For convenience, in this article, although only one strand is given in most cases, the other complementary strand is actually also disclosed. In addition, the nucleic acid molecule sequences in the present application include DNA form or RNA form, and disclosing one means that the other is also disclosed.

[0058] In a third aspect of the present application, the present application provides an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule described in the second aspect. When connecting the above-mentioned nucleic acid molecule to the expression vector, the above-mentioned nucleic acid molecule can be directly or indirectly connected to the control elements on the expression vector, as long as these control elements can control the translation and expression of the above-mentioned nucleic acid molecule, etc. Of course, these control elements can directly come from the expression vector itself or be exogenous, that is, not from the expression vector itself. Of course, the above-mentioned nucleic acid molecule and the control elements can be operably connected.

[0059] As used herein, "operably connected" means connecting an exogenous gene to an expression vector such that the control elements in the expression vector, such as transcriptional control sequences and translational control sequences, etc., can perform their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used expression vectors can be, for example, plasmids, phages, etc. After the expression vector according to some specific embodiments of the present application is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the aforementioned polypeptide can be effectively achieved, and thus a large amount of the polypeptide can be obtained in vitro.

[0060] In some specific embodiments of the present application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a phage.

[0061] In some specific embodiments of the present application, the expression vector is a lentiviral vector.

[0062] In an alternative embodiment of the present application, the expression vector is a plasmid expression vector.

[0063] In a fourth aspect of the present application, the present application provides a recombinant cell. According to the 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 aforementioned polypeptide or a pharmaceutically acceptable salt thereof can be effectively expressed in the recombinant cell.

[0064] According to the embodiments of the present application, the recombinant cell is obtained by introducing the expression vector described in the third aspect into a host cell.

[0065] It should be noted that the host cell of the present application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell or a phage. 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 pombe, Trichoderma, etc., insect cells such as Spodoptera frugiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0066] According to the embodiments of the present application, the host cell is a eukaryotic cell.

[0067] According to the embodiments 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.

[0068] It should be noted that the "suitable conditions" described in the present application refer to the conditions suitable for the expression of the polypeptide described in the present application. It is easily understood by those skilled in the art that the conditions suitable for polypeptide expression include but are not limited to appropriate transformation or transfection methods, appropriate transformation or transfection conditions, healthy cell states, appropriate cell densities, suitable cell culture environments, and appropriate cell culture times. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for polypeptide expression according to the specific environment of the laboratory.

[0069] Polypeptide derivative or a pharmaceutically acceptable salt thereof In the fifth aspect of the present application, a polypeptide derivative or a pharmaceutically acceptable salt thereof is proposed. According to an embodiment of the present application, the polypeptide derivative or a pharmaceutically acceptable salt thereof includes: 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 can be seen from the above, the polypeptide or a pharmaceutically acceptable salt thereof can effectively activate GLP-1R. Therefore, the polypeptide derivative or a pharmaceutically acceptable salt thereof containing the above polypeptide or a pharmaceutically acceptable salt thereof can be used for treating or preventing GLP-1R-related diseases, such as diseases related to metabolic disorders, such as obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease, etc.

[0070] In this context, 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 is within the scope of protection of the present application.

[0071] According to an embodiment of the present application, the above polypeptide derivative or a pharmaceutically acceptable salt thereof may further include at least one of the following technical features: According to an embodiment of the present application, the modifying group is linked to -NH2, -SH, -OH or -COOH on the amino acid side chain in the polypeptide.

[0072] According to an embodiment of the present application, the modifying group is linked to -NH2 on the amino acid side chain in the polypeptide.

[0073] According to an embodiment of the present application, the modifying group has at least one of the following structures: 。

[0075] In this context, " " in the description of the chemical group is used to describe the position of group substitution. That is, the above chemical group is linked to -NH- of the amino acid through 。

[0076] Fusion protein, reagent or kit, pharmaceutical composition In the sixth aspect of the present application, the present application provides a fusion protein. According to an embodiment of the present application, the fusion protein includes 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 can be seen from the above, the above polypeptide or a 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 polypeptide can bind to GLP-1R and can be used to detect GLP-1R or to activate GLP-1R, and can also 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, etc.

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

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

[0079] It should be noted that the above functionally active fragment can be used to act in vivo or in vitro. Exemplarily, when the functionally active fragment is used to act in vivo, it can be used to prevent and / or treat diseases; when the functionally active fragment is used to act in vitro, it is used to specifically bind to a certain substance, and can be used to detect the substance or to diagnose diseases in vitro.

[0080] It should be noted that the above protein tag refers to a short peptide expressed together with the target protein, which is convenient for the expression, detection, tracing, or purification of the polypeptide of the present application. Exemplarily, the protein tag includes at least one of His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

[0081] In the seventh aspect of the present application, the present application provides a reagent or a kit. According to an embodiment of the present application, the reagent or the kit includes 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 can be seen from the above, the above polypeptide or a pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to effectively activate GLP-1R. Thus, the reagent or the kit containing the above polypeptide or a pharmaceutically acceptable salt thereof can bind to GLP-1R and can be used to detect GLP-1R.

[0082] In the eighth aspect of the present application, a pharmaceutical composition is proposed. According to an embodiment of the present 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 can be seen from the foregoing, the above-mentioned polypeptide or a 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 a pharmaceutically acceptable salt thereof can be used to treat or prevent diseases related to metabolic disorders, such as obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease, etc.

[0083] According to an embodiment of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0084] In an alternative embodiment of the present application, the pharmaceutically acceptable excipient refers to conventional pharmaceutical excipients in the pharmaceutical field, such as diluents, buffers, osmotic pressure regulators, pH regulators, etc.

[0085] In an alternative embodiment of the present application, the pharmaceutically acceptable carrier refers to conventional pharmaceutical carriers in the pharmaceutical field, such as protectants, etc.

[0086] In an alternative embodiment of the present application, the pharmaceutically acceptable vehicle refers to conventional pharmaceutical vehicles in the pharmaceutical field, such as solutions (such as water) and liposomes.

[0087] In an alternative embodiment of the present application, examples of suitable pharmaceutically acceptable carriers, excipients, and vehicles are well known in the art. The pharmaceutical composition containing such carriers, excipients, and vehicles can be formulated by well-known conventional methods.

[0088] In some alternative embodiments of the present application, the pharmaceutical composition of the present application may further contain other active ingredients for treatment.

[0089] The pharmaceutical composition of the present application can be administered in different ways, such as enterally, orally (such as a liquid solution), via injection (such as intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally). Preferably, the pharmaceutical composition of the present application is in the form of a freeze-dried preparation or an aqueous solution. The clinical dosing regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any patient depends on many factors, including the patient's physique, body surface area, age, the drug to be administered, gender, administration time and route, general health, and other drugs administered simultaneously. The pharmaceutical composition of the present application can be administered locally or systemically. Preferably, it can be administered intravenously or subcutaneously.

[0090] Use In the ninth aspect of the present application, there is provided 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 treating or preventing GLP-1R-related diseases.

[0091] According to an embodiment of the present application, the GLP-1R-related diseases include diseases related to metabolic disorders.

[0092] According to an embodiment of the present application, the diseases related to metabolic disorders include at least one of obesity, diabetes, diseases related to dyslipidemia, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

[0093] Method In the tenth aspect of the present application, there is provided a method for detecting GLP-1R. According to an embodiment of the present application, the method includes: contacting a sample to be detected 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 or kit described in the seventh aspect to form a contact product; and determining whether the sample to be detected contains GLP-1R based on the signal generated by the contact product. As can be seen from the foregoing, the above-mentioned polypeptide or a 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 detecting GLP-1R.

[0094] According to a specific embodiment of the present application, the signal includes a fluorescence signal.

[0095] According to a specific embodiment of the present application, it further includes determining the content value of GLP-1R in the sample to be detected based on the signal generated by the contact product.

[0096] In the eleventh aspect of the present application, there is provided a method for treating or preventing diseases related to metabolic disorders. According to an embodiment of the present 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.

[0097] In an alternative embodiment of the present application, the pharmaceutically acceptable dose may be selected from an effective dose (or effective amount).

[0098] The effective amount of the polypeptide or its pharmaceutically acceptable salt in the present application may vary with the mode of administration, the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (e.g., through clinical trials). Such factors include, but are not limited to: the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be proportionally reduced.

[0099] The polypeptide or its pharmaceutically acceptable salt, polypeptide derivative or its pharmaceutically acceptable salt, or pharmaceutical composition of the present application can be incorporated into drugs suitable for use in medicine, and these drugs can be prepared in various forms, such as liquid, semi-solid and solid dosage forms, etc., including but not limited to solid dosage forms, semi-solid dosage forms, liquid dosage forms and gas dosage forms, etc. The various ways of the polypeptide or its pharmaceutically acceptable salt, polypeptide derivative or its pharmaceutically acceptable salt, pharmaceutical composition or drug administration of the present application are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, oral, topical, nasal, pulmonary, rectal and smear, but the present application is not limited to these exemplified administration methods.

[0100] According to the embodiments of the present application, the diseases related to metabolic disorders include at least one of obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome and non-alcoholic fatty liver disease.

[0101] The solutions of the present application will be explained below in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those without specific techniques or conditions noted in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0102] Example 1: The schematic diagram of the polypeptide design and screening process of the present application is as Figure 1 , which covers the steps from GLP-1R sequence generation to screening and optimization. The whole method consists of multiple iterative evolution processes. After each iterative evolution, the polypeptides designed by the model can obtain effective improvement in biological activity. Each iterative evolution includes three parts, namely the generation and optimization of brand-new polypeptides based on artificial intelligence, the determination of polypeptide biological activity by wet experiments, and feedback to strengthen the model's perception of biological activity.

[0103] First, through a deep learning method TPDiffusion, a polypeptide sequence that can bind to a target protein can be generated based on the amino acid sequence of the target protein. The process of converting the target protein sequence into a polypeptide sequence can be regarded as a process of giving specific answers to specific questions. By training the conditional diffusion model TPDiffusion, the relationship rules between the target protein and the polypeptide sequence are learned to generate specific polypeptides (i.e., GLP-1R target peptides) for a specific target protein (i.e., GLP-1R). The training of the polypeptide sequence generation model TPDiffusion mainly includes a forward diffusion process and a reverse diffusion process. The forward diffusion process includes the following steps: 1. Encode the amino acid sequence: To model the protein-polypeptide joint feature space, the target protein and polypeptide sequences are spliced together as a whole, and an embedding transformation function is introduced to map each discrete amino acid word or character to a continuous vector encoding space.

[0104] 2. Gradually add noise to the polypeptide sequence: Gaussian noise is gradually added to the polypeptide part in the vector encoding based on the Markov chain until the polypeptide sequence is completely destroyed.

[0105] After the forward diffusion process is completed, a set of clean target protein and noisy polypeptide sequences will be obtained. Then, the polypeptide sequence is restored through the reverse diffusion process, which mainly includes the following steps: 1. Gradually denoise the polypeptide sequence: By constructing a denoising network, the noise distribution added in the forward diffusion process is estimated, and the polypeptide part is gradually denoised until the polypeptide sequence is completely restored.

[0106] 2. Calculate the loss function: The model outputs the probability distribution of the predicted polypeptide sequence. The mean square error loss function is used to calculate the difference between the model prediction result and the real result, and the model parameters are updated through backpropagation.

[0107] The denoising network in TPDiffusion adopts the BERT (Devlin et al. 2018) model. The model architecture of BERT is mainly composed of multiple layers of Transformer (Vaswani et al. 2017) encoders. Each Transformer block contains two parts: self-attention and feed-forward neural network. Through the self-attention mechanism, TPDiffusion will introduce target sequence information during the reverse diffusion process of restoring the polypeptide sequence, thereby implicitly modeling the relationship characteristics between the target protein and the polypeptide sequence, and realizing the relationship mapping from the target protein to the polypeptide sequence. During the generation process, given any target protein sequence, the model will first randomly sample from Gaussian noise and then perform the reverse diffusion process. Guided by the target protein sequence, the noise is gradually eliminated through a fixed number of time steps, and finally, a binding polypeptide sequence for the given target can be generated. By using the trained TPDiffusion, the sequence of the GLP-1R target is used as the input, and a batch of candidate polypeptide sequences that may have high affinity with the GLP-1R target are generated.

[0108] Second, affinity maturation is performed on these high-affinity candidate polypeptide sequences. This method is a deep reinforcement learning method, which guides the evolution of candidate polypeptide sequences by simulating the environment and defining actions. This deep reinforcement learning framework can combine some reward models as prior knowledge to guide the evolution of polypeptides. The reward model can be an affinity prediction model, a solubility prediction model, a toxicity prediction model, etc.

[0109] The reward model uses PepAF, which effectively predicts the binding affinity between the target protein and the polypeptide by comprehensively using structural information, flexibility characteristics, and advanced pre-training strategies. PepAF first learns on two pre-training tasks: 1) estimating the binding free energy of the protein (GLP-1R)-polypeptide complex; 2) predicting the affinity of the protein (GLP-1R)-polypeptide complex. The first task enables the model to learn the complex interactions between the protein (GLP-1R) and the polypeptide, as well as their structures and physicochemical properties, which provides a basis for understanding the binding mode and key characteristics of the protein (GLP-1R)-polypeptide interaction. The second task enables the model to capture a wide range of molecular interactions at the atomic scale. PepAF also improves the prediction accuracy by modeling the target protein structure and the polypeptide flexibility. Taking the core technology PepAF in this patent as the reward model of the deep reinforcement learning method, it guides the mutation of candidate polypeptide sequences.

[0110] After obtaining the affinity matured polypeptide, its biological activity is evaluated, and the results are then fed back to the model to continue the next round of polypeptide generation and optimization. After one round of iteration, a polypeptide that effectively activates the GLP-1R target protein is designed, and the sequence is shown in SEQ ID NO:1 (i.e., GA-Single.R5.S2). Among them, the amino acid sequence shown in SEQ ID NO:1 is as follows: HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG (SEQ ID NO:1).

[0111] Example 2: This example aims to evaluate the ability of the candidate polypeptide GA-Single.R5.S2 obtained in Example 1 to activate cAMP activity on huGLP-1R-FL-CRE-HEK293-A5 cells overexpressing huGLP-1R. By quantitatively measuring the production level of cAMP, this experiment can accurately evaluate the activation effect of the candidate sample on the GLP-1R receptor, providing important biological activity data for the drug development of metabolic diseases such as diabetes. The experimental procedure is as follows: Polypeptide synthesis and purification: According to the amino acid sequence of the designed candidate polypeptide, the polypeptide was synthesized using solid-phase peptide synthesis (Fmoc-SPPS) technology. The purification process used high-performance liquid chromatography (HPLC) to ensure that the synthesized polypeptide had high purity and no obvious impurities. In addition, the exact molecular weight of the polypeptide was further verified by mass spectrometry (MS).

[0112] Polypeptide dissolution conditions: The purified candidate polypeptide was dissolved in DMSO (dimethyl sulfoxide) solvent to prepare a stock solution of 1 mg / mL for subsequent experiments.

[0113] Cell starvation treatment: A huGLP-1R-FL-CRE-HEK293-A5 cell line was constructed, and a huGLP-1R-FL-CRE-HEK293-A5 cell line with high viability was selected for starvation treatment in DMEM medium with 0% fetal bovine serum (FBS) to standardize the experimental conditions and exclude the interference of other factors in the serum.

[0114] Cell digestion, washing and plating: The adherent cells were gently digested with Accutase enzyme and washed with PBS buffer to remove the residual culture medium and enzyme. The cell suspension was accurately plated in a HTRF 96-well low-volume detection plate to ensure the consistency of the number of cells per well, providing a uniform starting condition for subsequent cAMP activity detection.

[0115] cAMP activity detection: After cell plating, accurately diluted candidate polypeptide was added to each well (for specific concentrations, see Figure 2(abscissa), incubated at 37 °C for 20 minutes to simulate the in vivo environment and activate the GLP-1R receptor. Add the cAMP d2 reagent and the working solution of anti-cAMP Eu Cryptate antibody (PerkinElmer, catalog number 62AM4PEB) to each well and incubate at room temperature for 1 hour. This step binds cAMP to the fluorescently labeled antibody to form an energy transfer complex.

[0116] Signal detection: Using the TR-FRET technique, set the excitation wavelength to 340 nm and detect the emission signals at 665 nm and 620 nm respectively. This technique has high sensitivity and anti-interference ability. The FRET signal value is calculated by the ratio of the light intensities at 665 nm and 620 nm, which reflects the concentration change of cAMP, and the change of the ratio is inversely proportional to the cAMP concentration.

[0117] The experimental results show that the polypeptide GA-Single.R5.S2 screened in this application has a good function of activating cAMP activity, indicating its high biological activity. The detection results are as Figure 2 shown in Table 1.

[0118] Table 1

[0119] Example 3 This example aims to evaluate the therapeutic effects of the candidate polypeptide (i.e., GA-Single.R5.S2) in Example 1 on obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease in vitro. The specific steps are as follows: 1) For obesity: Use 3T3-L1 adipocytes. First, induce cell differentiation into adipocytes with high-glucose DMEM medium (containing 4.5 g / L glucose, supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, Thermo Fisher Scientific, catalog number 11995065) to establish an obesity model. When the confluence of 3T3-L1 preadipocytes reaches 80-90%, digest with 0.25% trypsin and inoculate at a density of 2-3×10 4 cells / cm². When the cell confluence reaches 80-95%, add the adipogenic induction differentiation medium for induction. After 2-3 days of induction, change to the adipogenic induction differentiation maintenance medium and maintain for 1 day, and alternate induction 3-5 times until obvious lipid droplets appear in the cells, indicating that the obesity model is successfully constructed.

[0120] Then add each candidate polypeptide to the adipocyte culture medium at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), and control the cell density at 2-3×104 cells / cm² to ensure the normal growth and differentiation of cells, and culture for 24 - 48 hours. Detect the content of lipid droplets in cells by Oil Red O staining method: aspirate the culture medium, rinse with 1×PBS, add 4% neutral formaldehyde solution to fix for 30 min, prepare Oil Red O working solution (saturated Oil Red O solution: distilled water = 3:2), filter and use for staining, add 1 mL of Oil Red O working solution to each well, stain at room temperature for 30 min, aspirate the staining solution, rinse with 1×PBS, observe and take pictures under the microscope. At the same time, use a qPCR kit (Takara RR420A) to detect the expression levels of adipogenesis-related genes (PPARγ, C / EBPα). If the candidate polypeptide can significantly reduce the content of lipid droplets in cells and decrease the expression of adipogenesis-related genes, it indicates that it has potential therapeutic effects on obesity and may play a role by inhibiting the differentiation and lipid accumulation of adipocytes.

[0121] 2) For diabetes: Use pancreatic islet β cell line (MIN6 cells), and culture the cells with high-glucose DMEM medium (containing 4.5 g / L glucose, supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, Thermo Fisher Scientific, catalog number 11995065) to simulate the high-glucose environment of diabetes. Add the candidate polypeptide to cell culture media at different concentrations (10 nM, 100 nM, 1 μM and 10 μM) respectively, control the cell density at 5×10³ cells / cm², and culture for 24 - 48 hours. Use an insulin ELISA kit to detect the secretion amount of insulin in the cell culture supernatant, and at the same time use a GLU2 kit (YUANJU BIO, catalog number YJ24519) and an IRS1 kit (Thermo Fisher Scientific, catalog number KHO0521) to detect the expression levels of proteins related to the insulin signaling pathway such as glucose transporter (GLUT2) and insulin receptor substrate (IRS). If the candidate polypeptide can increase the secretion amount of insulin and up-regulate the expression levels of GLUT2 and IRS, it indicates that it may have the effects of improving insulin resistance and promoting insulin secretion, and has potential value for the treatment of diabetes.

[0122] 3) For dyslipidemia-related diseases: Select the hepatocyte cell line (HepG2 cells), and use a medium containing high concentrations of fatty acids (Wuhan Shang'en Biotechnology Co., Ltd., special medium for HepG2 cells, whose components include EMEM (MEM + NEAA) to treat the cells + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) to establish a dyslipidemia model. Add each candidate polypeptide to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), control the cell density at 5×10³ cells / cm², and culture for 24 - 48 hours. Use an enzymatic assay kit (Total Cholesterol Assay Kit from Sigma-Aldrich, catalog number MAK043) to detect the total cholesterol content in the cells, and use a triglyceride assay kit (Triglyceride Assay Kit from Sigma-Aldrich, catalog number MAK041) to detect the triglyceride content. Meanwhile, use an LDLR kit (Thermo Fisher Scientific, catalog number EHLDLR) and a CYP7A1 kit (proteintech, catalog number 18054-1-AP) to detect the expression levels of genes related to cholesterol metabolism such as low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1). If the candidate polypeptide can reduce the total cholesterol and triglyceride content in the cells and simultaneously upregulate the expression of LDLR and CYP7A1, it indicates that it may have a certain therapeutic effect on dyslipidemia-related diseases by regulating cholesterol synthesis and metabolism.

[0123] 4) For fatty liver disease: Also using HepG2 cells, induced cell steatosis with a high-fat medium (Wuhan Shang'en Biotechnology Co., Ltd., special medium for HepG2 cells, its components include EMEM (MEM + NEAA) + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)) to establish a fatty liver disease model. Add the candidate polypeptides to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM) respectively, control the cell density at 5×10³ cells / cm², and culture for 24 - 48 hours. Use the Oil Red O staining method to detect the content of lipid droplets in cells: aspirate the medium, rinse with 1×PBS, add 4% neutral formaldehyde solution to fix for 30 min, prepare the Oil Red O working solution (saturated Oil Red O solution: distilled water = 3:2), filter and use for staining, add 1 mL of the Oil Red O working solution to each well, stain at room temperature for 30 min, aspirate the staining solution, rinse with 1×PBS, observe and take pictures under a microscope. At the same time, use ELISA kits (TNF-α and IL-6 ELISA kits from Thermo Fisher Scientific, catalog numbers are K1480 and K1501 respectively) to detect the secretion levels of inflammatory factors, and use colorimetric assay kits (MDA Assay Kit from Cayman Chemical, catalog number 700455 and SOD Assay Kit, catalog number 705125) to detect oxidative stress indicators. If the candidate polypeptide can reduce the accumulation of lipid droplets in cells, decrease the secretion of inflammatory factors, and improve the oxidative stress state, it indicates that it may have a therapeutic effect on fatty liver disease and can alleviate steatosis and inflammatory damage of hepatocytes.

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

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

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

[0127] Muscle cells: Use C2C12 muscle cells, cultured with a high-glucose DMEM medium to simulate the insulin resistance environment.

[0128] The candidate polypeptides were separately added to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), and the cell density was controlled at 5×10³ cells / cm². The cells were cultured for 24 - 48 hours. After the culture, the indicators related to metabolic syndrome in each cell were detected, including the insulin resistance index (HOMA-IR, calculated by detecting insulin and glucose levels), blood lipid levels (the levels of triglyceride and total cholesterol were detected using a triglyceride assay kit (Triglyceride Assay Kit from Sigma-Aldrich, catalog number MAK041) and a total cholesterol assay kit (Total Cholesterol Assay Kit from Sigma-Aldrich, catalog number MAK043)), inflammatory factors (the secretion levels of TNF-α and IL-6 were detected using ELISA kits (TNF-α and IL-6 ELISA kits from Thermo Fisher Scientific, catalog numbers K1480 and K1501) respectively). If the candidate polypeptides can improve insulin resistance, reduce blood lipid levels, and decrease the secretion of inflammatory factors, it indicates that they have potential therapeutic effects on metabolic syndrome and may comprehensively regulate metabolic disorders through a multi-target mechanism of action.

[0129] 6) For non-alcoholic fatty liver disease: Using HepG2 cells, cells were induced to have non-alcoholic fatty liver disease-like changes with a high-fat and high-sugar medium (Wuhan Shangen Biotechnology Co., Ltd., a special medium for HepG2 cells, whose components include EMEM (MEM + NEAA) to treat cells + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (P / S)). Each candidate polypeptide was added to cell culture media at different concentrations (10 nM, 100 nM, 1 μM, and 10 μM), and the cell density was controlled at 5×10³ cells / cm², and cultured for 24 - 48 hours. The content of lipid droplets in cells was detected by Oil Red O staining, and at the same time, ELISA kits (IL-1β and IL-8 ELISA kits from Thermo Fisher Scientific, catalog numbers K1480 and K1501 respectively) were used to detect the secretion levels of inflammatory factors, colorimetric assay kits (ALT and AST Assay Kits from Cayman Chemical, catalog numbers 700455 and 705125 respectively) were used to detect the levels of hepatocyte injury markers, and Western blot and qPCR methods were used to detect the expression levels of indicators related to liver fibrosis (collagen I and α-smooth muscle actin). If the candidate polypeptide can reduce the intracellular fat content, decrease the secretion of inflammatory factors, lower the levels of hepatocyte injury markers, and inhibit the expression of indicators related to liver fibrosis, it indicates that it has a certain therapeutic effect on non-alcoholic fatty liver disease and may play a role by reducing fat accumulation, inhibiting inflammation and the fibrosis process.

[0130] The results showed that the candidate polypeptide in Example 1 (i.e., GA-Single.R5.S2) had the following effects: 1) It could significantly reduce the content of lipid droplets in 3T3-L1 adipocytes and decrease the expression of genes related to adipogenesis; 2) It could increase the insulin secretion in pancreatic islet β cell lines (such as MIN6 cells) and up-regulate the expression levels of proteins related to the insulin signaling pathway such as glucose transporter (GLUT2) and insulin receptor substrate (IRS); 3) It could reduce the content of total cholesterol and triglycerides in hepatocyte lines and at the same time up-regulate the expression of genes related to cholesterol metabolism such as low-density lipoprotein receptor (LDLR) and cholesterol 7α-hydroxylase (CYP7A1); 4) It could reduce the accumulation of lipid droplets in HepG2 cells, decrease the secretion of inflammatory factors, and improve the oxidative stress state; 5) It could improve the indicators related to metabolic syndrome in various cells (such as adipocytes, hepatocytes, muscle cells, etc.); 6) It can reduce the fat content in HepG2 cells, decrease the secretion of inflammatory factors, lower the levels of hepatocyte injury markers, and inhibit the expression of liver fibrosis-related indicators.

[0131] In summary, it can be shown that the above-mentioned candidate polypeptide GA-Single.R5.S2 has therapeutic effects on obesity, diabetes, dyslipidemia-related diseases, fatty liver disease, metabolic syndrome, and non-alcoholic fatty liver disease.

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

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

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the polypeptide is shown as HSQGTFTSDYSKYLEEAAAAEFVAWLLAGG.

2. A nucleic acid molecule, an expression vector, or a recombinant cell, characterized in that: The nucleic acid molecule encodes the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof; The expression vector carries the above-mentioned nucleic acid molecule; The recombinant cell carries the aforementioned nucleic acid molecule or the aforementioned expression vector, or the recombinant cell expresses the polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof.

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

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

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

7. 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 3 to 4 or a pharmaceutically acceptable salt thereof, or the fusion protein according to claim 5, and optionally a pharmaceutically acceptable excipient.

8. 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 3 to 4, the fusion protein according to claim 5, or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating or preventing GLP-1R related diseases.

9. The use according to claim 8, characterized in that The GLP-1R-related diseases include metabolic disorder-related diseases.

10. The use according to claim 9, 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.

11. A method for detecting GLP-1R, 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 3 to 4, the fusion protein according to claim 5, or the reagent or kit according to claim 6; Based on the signal generated by the contact product, it is determined whether the sample to be tested contains GLP-1R.

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