Apelin-13 polypeptide mutant

Abstract

The invention relates to an Apelin-13 polypeptide mutant, a pharmaceutical composition containing the Apelin-13 polypeptide mutant and application of the Apelin-13 polypeptide mutant.

Description

Apelin-13 polypeptide mutants

[0001] Citation of Related Applications

[0002] This disclosure claims all rights and interests in the Chinese invention patent application number 202310638405.7, filed on May 31, 2023 with the State Intellectual Property Office of the People's Republic of China, entitled "A G protein-biased apelin-13 polypeptide mutant and its application," and incorporates the entire contents of this application into this disclosure by reference.

[0003] field

[0004] The present disclosure relates generally to the field of biomedicine, and more particularly to the field of polypeptides.

[0005] background

[0006] Cardiovascular disease is one of the major threats to human physical and mental health, with its prevalence increasing annually. Countries face significant challenges in preventing and treating cardiovascular disease, resulting in a significant social and economic burden. The apelin receptor (APLNR) is a typical class A GPCR with important functions in the cardiovascular system, including promoting cardiac contraction, increasing left ventricular stroke volume, vasodilation, diuresis, and lowering systemic blood pressure.

[0007] Overview

[0008] In one aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0009] (1) A polypeptide represented by the general formula (I):

[0010] a1-Arg-Pro-Arg-Leu-b1-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0011] General formula (I)

[0012] wherein a1 is any natural amino acid, b1 is an amino acid with a negatively charged side chain, and the amino acid residues at positions 1 and 6 form an amide bond; or

[0013] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (I) and having the same function.

[0014] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0015] (1) A polypeptide represented by general formula (II):

[0016] Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0017] General formula (II)

[0018] wherein a2 is selected from amino acids having hydrophobic side chains or amino acids having polar uncharged side chains, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0019] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (II) and having the same function.

[0020] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0021] (1) A polypeptide represented by the general formula (III-1):

[0022] Cys-Arg-a3-Arg-b3-Cys-His-c3-Gly-Pro-d3-e3-f3-g3

[0023] General formula (III-1)

[0024] wherein a3 is selected from an amino acid having a hydrophobic side chain or an amino acid having a polar uncharged side chain, b3 is an amino acid having a hydrophobic side chain, c3 is an amino acid having a positively charged side chain, d3 and e3 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f3 is an amino acid having a hydrophobic side chain or Pro, g3 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0025] (2) A polypeptide represented by the general formula (III-2):

[0026] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3'

[0027] (Disulfidebridge:1-6)

[0028] General formula (III-2)

[0029] wherein a3' is an amino acid with a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0030] (3) A polypeptide having at least 70% homology with the polypeptide represented by general formula (III-1) or general formula (III-2) and having the same function.

[0031] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0032] (1) A polypeptide represented by the general formula (IV):

[0033] Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0034] General formula (IV)

[0035] wherein a4 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0036] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (IV) and having the same function.

[0037] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0038] (1) A polypeptide represented by the general formula (V-1):

[0039] Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5

[0040] General formula (V-1)

[0041] wherein a5 is selected from an amino acid having a hydrophobic side chain or Pro, b5 is an amino acid having a hydrophobic side chain, c5 is an amino acid with a positively charged side chain, d5 and e5 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f5 is an amino acid having a hydrophobic side chain or Pro, g5 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0042] (2) A polypeptide represented by the general formula (V-2):

[0043] Cys-Arg-a5'-Arg-b5'-Cys-His-c5'-Gly-Pro-Nal-Pro-d5'

[0044] General formula (V-2)

[0045] wherein a5' is selected from an amino acid having a hydrophobic side chain or Pro, b5' is an amino acid having a hydrophobic side chain, c5' is an amino acid having a positively charged side chain, d5' is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0046] (3) A polypeptide having at least 70% homology with the polypeptide represented by general formula (III-1) or general formula (III-2) and having the same function.

[0047] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0048] (1) A polypeptide represented by the general formula (VII):

[0049] Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0050] General formula (VII)

[0051] wherein a7 is selected from amino acids having hydrophobic side chains or amino acids having polar uncharged side chains, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0052] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (VII) and having the same function.

[0053] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0054] (1) A polypeptide represented by the general formula (VIII-1):

[0055] Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8

[0056] General formula (VIII-1)

[0057] wherein a8 is selected from an amino acid having a hydrophobic side chain or Pro, b8 is an amino acid having a hydrophobic side chain, c8 is selected from an amino acid having a hydrophobic side chain or an amino acid with a positively charged side chain, d8 is an amino acid having a hydrophobic side chain or Pro, e8 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0058] (2) A polypeptide represented by the general formula (VIII-2):

[0059] Cys-Arg-Pro-Arg-Trp-Cys-His-a8'-Gly-Pro-Nal-Pro-Phe

[0060] General formula (VIII-2)

[0061] wherein a8' is an amino acid with a positively charged side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0062] (3) A polypeptide having at least 70% homology with the polypeptide represented by general formula (III-1) or general formula (III-2) and having the same function.

[0063] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0064] (1) A polypeptide represented by the general formula (IX):

[0065] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe

[0066] General formula (IX)

[0067] wherein a9 is an amino acid with a polar, uncharged side chain; or

[0068] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (IX) and having the same function.

[0069] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0070] (1) A polypeptide represented by the general formula (XI-1):

[0071] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe

[0072] General formula (XI-1)

[0073] wherein a11 is selected from Ala, Phe, Nal, DNal, DMet, Glu or Nle, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond;

[0074] (2) A polypeptide represented by the general formula (XI-2):

[0075] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11'-b11'-Pro-Phe

[0076] General formula (XI-2)

[0077] wherein a11′ is selected from an amino acid having a hydrophobic side chain or an amino acid with a polar uncharged side chain, His or Pro, b11′ is selected from an amino acid having a hydrophobic side chain or Gly, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0078] (3) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XI-1) or general formula (XI-2) and having the same function.

[0079] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0080] (1) A polypeptide represented by the general formula (XII):

[0081] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe

[0082] General formula (XII)

[0083] wherein a12 is selected from an amino acid with a positively charged side chain, an amino acid with a hydrophobic side chain, an amino acid with a polar uncharged side chain, or an amino acid with a negatively charged side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0084] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XII) and having the same function.

[0085] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0086] (1) A polypeptide represented by the general formula (XIII):

[0087] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe

[0088] General formula (XIII)

[0089] wherein a12 is selected from an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, or His, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0090] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XIII) and having the same function.

[0091] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0092] (1) A polypeptide represented by the general formula (XIV):

[0093] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14

[0094] General formula (XIV)

[0095] wherein a14 is selected from an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, His or Nal, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0096] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XIV) and having the same function.

[0097] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0098] (1) A polypeptide represented by the general formula (XV):

[0099] Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe

[0100] General formula (XV)

[0101] wherein a15 and b15 are each independently selected from an amino acid with a positively charged side chain or Pro, c15 is selected from an amino acid with a hydrophobic side chain or an amino acid with a positively charged side chain, and the cysteine ​​residues at positions 1 and 5 form a disulfide bond; or

[0102] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XV) and having the same function.

[0103] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0104] (1) A polypeptide represented by the general formula (XVI):

[0105] Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe

[0106] General formula (XVI)

[0107] wherein a16 is selected from an amino acid with a positive side chain or Pro, c15 is selected from an amino acid with a hydrophobic side chain, an amino acid with a positive side chain or Pro, and the cysteine ​​residues at positions 1 and 4 form a disulfide bond; or

[0108] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XVI) and having the same function.

[0109] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0110] (1) A polypeptide represented by the general formula (XVII):

[0111] *-Arg-a17-b17-Leu-*-His-C17-Gly-Pro-DMet-Gly-Pro-Phe

[0112] General formula (XVII)

[0113] wherein a17 and b17 are each independently selected from amino acids with positively charged side chains, or a17 and / or b17 are absent, c17 is an amino acid with a positively charged side chain, * is a non-natural amino acid, and a ring structure is formed between *; or

[0114] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XVII) and having the same function.

[0115] In another aspect, the present disclosure relates to a pharmaceutical composition comprising the Apelin-13 polypeptide mutant described in the present disclosure and a pharmaceutically acceptable carrier, diluent or excipient.

[0116] In another aspect, the present disclosure relates to a method for treating or preventing a disease or condition responsive to agonism of an APLNR receptor, comprising administering to an individual in need thereof a therapeutically or prophylactically effective amount of the Apelin-13 polypeptide mutant of the present disclosure or the pharmaceutical composition of the present disclosure.

[0117] In yet another aspect, the present disclosure relates to the Apelin-13 polypeptide mutants of the present disclosure for use in treating or preventing a disease or condition responsive to agonism of an APLNR receptor.

[0118] In another aspect, the present disclosure relates to use of the Apelin-13 polypeptide mutants described herein in the preparation of a medicament for treating or preventing a disease or condition responsive to agonism of an APLNR receptor.

[0119] BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 shows the structure of the apelin-APLNR-Gi1 complex and the results of the interaction analysis between the ligand and the receptor, among which: A: cryo-electron microscopy density map and molecular model of the apelin-APLNR-Gi1 complex, B: cryo-electron microscopy density map and molecular model of the polypeptide ligands apelin and MM07, C: dose-dependence curves of apelin and MM07 inducing Gi protein dissociation (left) and β-arrestin recruitment (right).

[0121] Figure 2 shows the analysis results of the APLNR bias signal transduction mechanism, where A: density map of the interaction between apelin and the key amino acid residues affecting bias in APLNR, B: density map of the interaction between MM07 and the key amino acid residues affecting bias in APLNR, CF: G protein activation and β-arrestin recruitment detection of key amino acid mutations interacting with M11 and F13, G: interaction pattern diagram of the "twin hotspots" on the receptor with M11 and F13 of peptide agonists.

[0122] Figure 3 shows the dose-dependence curves of the positive control MM07 and the designed peptides WN370, WN353, and WN123 for inducing Gi protein dissociation and β-arrestin recruitment.

[0123] FIG4 is a structural analysis of the interaction between the polypeptides with the mutation of M11 site to A, Nal, V, P, G, and the mutation of P3 to W and L5 to F based on Nal and the receptor.

[0124] FIG5 shows the dose-dependence curves of the positive control MM07 and the designed peptides WN680, WN386, and WN648 for inducing Gi protein dissociation and β-arrestin recruitment.

[0125] Figure 6 shows the structural analysis of the interaction between the peptide with G and A inserted between P10 and M11, and the peptide with P3 mutated to W and L5 mutated to F based on the insertion of G between P10 and M11, and the receptor.

[0126] FIG7 shows the dose-dependence curves of the positive control MM07 and the designed peptide WN561 inducing Gi protein dissociation and β-arrestin recruitment.

[0127] FIG8 shows the structural analysis of the interaction between a polypeptide in which the mutation M11 is DMet and G or A is inserted between DMet at position 11 and P at position 12 and the receptor.

[0128] FIG9 shows the dose-dependence curves of the positive control MM07 and WN561, WN562, and WN563 inducing Gi protein dissociation and β-arrestin recruitment.

[0129] Figure 10 shows the interaction between R2 mutated to A, S, K, C, and T based on WN561 and the receptor.

[0130] FIG11 shows the dose-dependent curves of WN561, WN564, WN565 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0131] Figure 12 shows the interaction between P3 mutated to Y, S, T, and C based on WN561 and the receptor.

[0132] FIG13 shows the dose-dependent curves of WN561, WN566 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0133] FIG14 shows the interaction between R4 mutated to A and K and the receptor based on WN561.

[0134] FIG15 shows the dose-dependent curves of WN561, WN567 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0135] Figure 16 shows the interaction between L5 mutated to A, I, and V based on WN561 and the receptor.

[0136] FIG17 shows the dose-dependent curves of WN561, WN571, WN572 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0137] Figure 18 shows the interaction between H7 mutated to A, S, R, K, G, T, and C based on WN561 and the receptor.

[0138] FIG19 shows the dose-dependent curves of WN561, WN573, WN574 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0139] Figure 20 shows the interaction between K8 mutated to A and R and the receptor based on WN561.

[0140] FIG21 shows the dose-dependent curves of WN561, WN575 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0141] Figure 22 shows the interaction between G9 mutated to S, C, and T based on WN561 and the receptor.

[0142] FIG23 shows the dose-dependent curves of WN561, WN580, WN581, WN582 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0143] Figure 24 shows the interaction between G12 mutated to F, S, W, C, and Y on the basis of WN561 and the receptor.

[0144] FIG25 shows the dose-dependent curves of WN561, WN584 and MM07 inducing Gi protein dissociation and recruitment of β-arrestin.

[0145] Figure 26 shows the interaction between the F14 mutation to L, I, P, V, and W on the basis of WN561 and the receptor.

[0146] Figure 27 shows all the key amino acid residues that affect the binding of ligands to receptors.

[0147] Details

[0148] In the following description, certain specific details are included to provide a thorough understanding of each disclosed embodiment. However, one skilled in the relevant art will recognize that the embodiments can be implemented without one or more of these specific details and with other methods, components, materials, etc.

[0149] Unless otherwise required by this disclosure, throughout this specification and the claims that follow, the words "include" and "comprising" should be construed in an open, inclusive sense, ie, "including, but not limited to."

[0150] Reference throughout this specification to "one embodiment" or "another embodiment" or "an embodiment" or "certain embodiments" means that the particular referenced elements, structures, or features described in connection with that embodiment are included in at least one embodiment. Thus, appearances of the phrases "one embodiment" or "an embodiment" or "another embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or features may be combined in any suitable manner in one or more embodiments.

[0151] It should be understood that the singular articles "a," "an," and "the" used in the specification and appended claims of this disclosure include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a pharmaceutical composition comprising a "nucleoside compound" includes one nucleoside compound, or two or more nucleoside compounds.

[0152] definition

[0153] Accordingly, unless otherwise indicated to the contrary, the following terms used in the specification and appended claims shall have the following meanings:

[0154] In the present disclosure, the term "pharmaceutically acceptable carrier, diluent or excipient" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration for use in humans or animals, and various forms of carriers that have no side effects on the composition of the pharmaceutical composition.

[0155] In this disclosure, the term "therapeutically effective amount" refers to an amount of a compound or combination of compounds that ameliorates, reduces, or eliminates a particular disease or condition and symptoms of a particular disease or condition, or prevents or delays the onset of a particular disease or condition or symptoms of a particular disease or condition. The amount of a compound described in this disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, and the age, weight, etc. of the mammal to be treated, but the amount of a compound described in this disclosure can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0156] As used herein, "treating" or "treatment" encompasses treating a relevant disease or condition in a mammal, such as a human, suffering from the relevant disease or condition, and includes:

[0157] (i) preventing a disease or disease state from occurring in a mammal, particularly where the mammal is susceptible to said disease state but has not yet been diagnosed with such disease state;

[0158] (ii) inhibiting the disease or disease state, i.e., preventing its occurrence; or

[0159] (iii) ameliorating the disease or condition, even if the disease or condition regresses or does not progress.

[0160] As used in this disclosure, the terms "disease" and "disease state" may be used interchangeably, or may be distinct in that a particular disease or disease state may have no known causative agent (and therefore cannot be explained etiologically) and therefore is not recognized as a disease, but rather is considered an undesirable disease state or condition in which clinicians have identified a more or less specific constellation of symptoms. DETAILED DESCRIPTION

[0161] In one aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0162] (1) A polypeptide represented by the general formula (I):

[0163] a1-Arg-Pro-Arg-Leu-b1-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0164] General formula (I)

[0165] wherein a1 is any natural amino acid, b1 is an amino acid with a negatively charged side chain, and the amino acid residues at positions 1 and 6 form an amide bond; or

[0166] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (I) and having the same function.

[0167] In certain embodiments, illustrative examples of a1 that can be used in the present disclosure include, but are not limited to, Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0168] In certain embodiments, illustrative examples of b1 that can be used in the present disclosure include, but are not limited to, Glu and Asp.

[0169] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0170] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0171] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by general formula (I).

[0172] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are:

[0173] Lys-Arg-Pro-Arg-Leu-Glu-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0174] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0175] (1) A polypeptide represented by general formula (II):

[0176] Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0177] General formula (II)

[0178] wherein a2 is selected from amino acids having hydrophobic side chains or amino acids having polar uncharged side chains, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0179] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (II) and having the same function.

[0180] In certain embodiments, illustrative examples of a2 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gln.

[0181] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0182] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0183] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (II).

[0184] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0185] Cys-Ala-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0186] Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0187] Cys-Lys-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0188] Cys-Cys-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); and

[0189] Cys-Thr-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0190] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0191] (1) A polypeptide represented by the general formula (III-1):

[0192] Cys-Arg-a3-Arg-b3-Cys-His-c3-Gly-Pro-d3-e3-f3-g3

[0193] General formula (III-1)

[0194] wherein a3 is selected from an amino acid having a hydrophobic side chain or an amino acid having a polar uncharged side chain, b3 is an amino acid having a hydrophobic side chain, c3 is an amino acid having a positively charged side chain, d3 and e3 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f3 is an amino acid having a hydrophobic side chain or Pro, g3 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0195] (2) A polypeptide represented by the general formula (III-2):

[0196] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3'

[0197] General formula (III-2)

[0198] wherein a3' is an amino acid with a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0199] (3) A polypeptide having at least 70% homology with the polypeptide represented by general formula (III-1) or general formula (III-2) and having the same function.

[0200] In certain embodiments, illustrative examples of a3 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gin.

[0201] In certain embodiments, illustrative examples of b3 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0202] In certain embodiments, illustrative examples of c3 that can be used in the present disclosure include, but are not limited to, Lys and Arg.

[0203] In certain embodiments, illustrative examples of d3 and e3 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, DMet, Phe, Tyr, Trp, and Gly.

[0204] In certain embodiments, illustrative examples of f3 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.

[0205] In certain embodiments, illustrative examples of g3 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0206] In certain embodiments, illustrative examples of a3' that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0207] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P2927.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0208] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0209] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (III-1) or a polypeptide represented by formula (III-2).

[0210] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0211] Cys-Arg-Tyr-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0212] Cys-Arg-Ser-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0213] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0214] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6);

[0215] Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0216] Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfide bridge:1-6); and

[0217] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe (Disulfide bridge: 1-6).

[0218] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0219] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6); and

[0220] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Trp (Disulfide bridge: 1-6).

[0221] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0222] (1) A polypeptide represented by the general formula (IV):

[0223] Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0224] General formula (IV)

[0225] wherein a4 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0226] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (IV) and having the same function.

[0227] In certain embodiments, illustrative examples of a2 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0228] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y2646.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 , 180 ECL2 or 182 ECL2 combination.

[0229] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0230] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (IV).

[0231] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0232] Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); and

[0233] Cys-Arg-Pro-Lys-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0234] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0235] (1) A polypeptide represented by the general formula (V-1):

[0236] Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5

[0237] General formula (V-1)

[0238] wherein a5 is selected from an amino acid having a hydrophobic side chain or Pro, b5 is an amino acid having a hydrophobic side chain, c5 is an amino acid with a positively charged side chain, d5 and e5 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f5 is an amino acid having a hydrophobic side chain or Pro, g5 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0239] (2) A polypeptide represented by the general formula (V-2):

[0240] Cys-Arg-a5'-Arg-b5'-Cys-His-c5'-Gly-Pro-Nal-Pro-d5'

[0241] General formula (V-2)

[0242] wherein a5' is selected from an amino acid having a hydrophobic side chain or Pro, b5' is an amino acid having a hydrophobic side chain, c5' is an amino acid having a positively charged side chain, d5' is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0243] (3) A polypeptide having at least 70% homology with the polypeptide represented by general formula (III-1) or general formula (III-2) and having the same function.

[0244] In certain embodiments, illustrative examples of a5 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.

[0245] In certain embodiments, illustrative examples of b5 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0246] In certain embodiments, illustrative examples of c5 that can be used in the present disclosure include, but are not limited to, Lys and Arg.

[0247] In certain embodiments, illustrative examples of d5 and e5 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, DMet, Phe, Tyr, Trp, and Gly.

[0248] In certain embodiments, illustrative examples of f5 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.

[0249] In certain embodiments, illustrative examples of g5 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0250] In certain embodiments, illustrative examples of a5' that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.

[0251] In certain embodiments, illustrative examples of b5' that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0252] In certain embodiments, illustrative examples of c5' that can be used in the present disclosure include, but are not limited to, Lys and Arg.

[0253] In certain embodiments, illustrative examples of d5' that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0254] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E1985.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0255] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0256] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (V-1) or a polypeptide represented by formula (V-2).

[0257] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0258] Cys-Arg-Pro-Arg-Ala-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0259] Cys-Arg-Pro-Arg-Tyr-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0260] Cys-Arg-Pro-Arg-Trp-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0261] Cys-Arg-Pro-Arg-Arg-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0262] Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0263] Cys-Arg-Pro-Arg-Trp-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu (Disulfide bridge: 1-6);

[0264] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu (Disulfide bridge: 1-6);

[0265] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0266] Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);

[0267] Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr-Phe (Disulfide bridge: 1-6);

[0268] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe (Disulfide bridge: 1-6);

[0269] Cys-Arg-Pro-Arg-Ile-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and

[0270] Cys-Arg-Pro-Arg-Val-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0271] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0272] Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6);

[0273] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6); and

[0274] Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Trp (Disulfide bridge: 1-6).

[0275] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0276] (1) A polypeptide represented by the general formula (VII):

[0277] Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0278] General formula (VII)

[0279] wherein a7 is selected from amino acids having hydrophobic side chains or amino acids having polar uncharged side chains, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0280] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (VII) and having the same function.

[0281] In certain embodiments, illustrative examples of a2 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gln.

[0282] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I1093.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0283] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0284] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (VII).

[0285] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0286] Cys-Arg-Pro-Arg-Leu-Cys-Ala-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0287] Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0288] Cys-Arg-Pro-Arg-Leu-Cys-Arg-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge:1-6);

[0289] Cys-Arg-Pro-Arg-Leu-Cys-Lys-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0290] Cys-Arg-Pro-Arg-Leu-Cys-Thr-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); and

[0291] Cys-Arg-Pro-Arg-Leu-Cys-Cys-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0292] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0293] (1) A polypeptide represented by the general formula (VIII-1):

[0294] Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8

[0295] General formula (VIII-1)

[0296] wherein a8 is selected from an amino acid having a hydrophobic side chain or Pro, b8 is an amino acid having a hydrophobic side chain, c8 is selected from an amino acid having a hydrophobic side chain or an amino acid with a positively charged side chain, d8 is an amino acid having a hydrophobic side chain or Pro, e8 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0297] (2) A polypeptide represented by the general formula (VIII-2):

[0298] ys-Arg-Pro-Arg-Trp-Cys-His-a8'-Gly-Pro-Nal-Pro-Phe

[0299] General formula (VIII-2)

[0300] wherein a8' is an amino acid with a positively charged side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0301] (3) A polypeptide having at least 70% homology with the polypeptide represented by general formula (III-1) or general formula (III-2) and having the same function.

[0302] In certain embodiments, illustrative examples of a8 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.

[0303] In certain embodiments, illustrative examples of b8 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0304] In certain embodiments, illustrative examples of c8 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, and Lys.

[0305] In certain embodiments, illustrative examples of d8 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.

[0306] In certain embodiments, illustrative examples of e8 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.

[0307] In certain embodiments, illustrative examples of a8' that can be used in the present disclosure include, but are not limited to, Arg and Lys.

[0308] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R1684.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0309] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0310] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (VIII-1) or a polypeptide represented by formula (VIII-2).

[0311] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0312] Cys-Arg-Pro-Arg-Leu-Cys-His-Ala-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0313] Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0314] Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0315] Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6);

[0316] Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfidebridge:1-6);

[0317] Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge:1-6);

[0318] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Cys-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0319] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Thr-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0320] Ac-Lys-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:2-7);

[0321] Ac-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:2-7);

[0322] Ac-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:4-9);

[0323] Ac-Lys-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:4-9);

[0324] Ac-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:3-8);

[0325] Ac-Lys-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:3-8);

[0326] Ac-Arg-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:5-10);

[0327] Ac-Lys-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:5-10); and

[0328] Ac-Leu-Arg-Gly-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10).

[0329] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are:

[0330] Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6).

[0331] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0332] (1) A polypeptide represented by the general formula (IX):

[0333] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe

[0334] General formula (IX)

[0335] wherein a9 is an amino acid with a polar, uncharged side chain; or

[0336] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (IX) and having the same function.

[0337] In certain embodiments, illustrative examples of a9 that can be used in the present disclosure include, but are not limited to, Ser, Thr, Asn, and Gln.

[0338] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0339] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0340] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (IX).

[0341] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are:

[0342] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

[0343] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0344] (1) A polypeptide represented by the general formula (XI-1):

[0345] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe

[0346] General formula (XI-1)

[0347] wherein a11 is selected from Ala, Phe, Nal, DNal, DMet, Glu or Nle, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond;

[0348] (2) A polypeptide represented by the general formula (XI-2):

[0349] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11'-b11'-Pro-Phe

[0350] General formula (XI-2)

[0351] wherein a11′ is selected from an amino acid having a hydrophobic side chain or an amino acid with a polar uncharged side chain, His or Pro, b11′ is selected from an amino acid having a hydrophobic side chain or Gly, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0352] (3) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XI-1) or general formula (XI-2) and having the same function.

[0353] In certain embodiments, illustrative examples of a11′ that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, His, and Pro.

[0354] In certain embodiments, illustrative examples of b11' that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Gly.

[0355] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0356] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0357] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (XI-1) or a polypeptide represented by formula (XI-2).

[0358] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0359] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Ala-Pro-Phe(Disulfide bridge:1-6);

[0360] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Phe-Pro-Phe(Disulfide bridge:1-6);

[0361] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6);

[0362] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DNal-Pro-Phe(Disulfide bridge:1-6);

[0363] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Pro-Phe(Disulfide bridge:1-6);

[0364] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Glu-Pro-Phe(Disulfide bridge:1-6); and

[0365] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nle-Pro-Phe (Disulfide bridge: 1-6).

[0366] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0367] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe(Disulfide bridge:1-6);

[0368] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Met-Gly-Pro-Phe(Disulfide bridge:1-6);

[0369] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6);

[0370] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);

[0371] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DAla-Gly-Pro-Phe(Disulfide bridge:1-6);

[0372] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DLeu-Gly-Pro-Phe(Disulfide bridge:1-6);

[0373] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DIle-Gly-Pro-Phe(Disulfide bridge:1-6);

[0374] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DVal-Gly-Pro-Phe(Disulfide bridge:1-6);

[0375] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DGlu-Gly-Pro-Phe(Disulfide bridge:1-6);

[0376] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DTyr-Gly-Pro-Phe(Disulfide bridge:1-6);

[0377] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DHis-Gly-Pro-Phe (Disulfide bridge:1-6);

[0378] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DPhe-Gly-Pro-Phe(Disulfide bridge:1-6);

[0379] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DTrp-Gly-Pro-Phe(Disulfide bridge:1-6);

[0380] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DPro-Gly-Pro-Phe(Disulfide bridge:1-6);

[0381] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DGln-Gly-Pro-Phe(Disulfide bridge:1-6); and

[0382] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DAsn-Gly-Pro-Phe(Disulfide bridge:1-6).

[0383] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0384] (1) A polypeptide represented by the general formula (XII):

[0385] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe

[0386] General formula (XII)

[0387] wherein a12 is selected from an amino acid with a positively charged side chain, an amino acid with a hydrophobic side chain, an amino acid with a polar uncharged side chain, or an amino acid with a negatively charged side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0388] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XII) and having the same function.

[0389] In certain embodiments, illustrative examples of a12 that can be used in the present disclosure include, but are not limited to, Arg, Lys, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp, and Glu.

[0390] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0391] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0392] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (XII).

[0393] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0394] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Phe-Pro-Phe(Disulfide bridge:1-6);

[0395] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Ser-Pro-Phe(Disulfide bridge:1-6);

[0396] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Asp-Pro-Phe(Disulfide bridge:1-6);

[0397] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Trp-Pro-Phe(Disulfide bridge:1-6);

[0398] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DArg-Pro-Phe(Disulfide bridge:1-6);

[0399] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DPhe-Pro-Phe(Disulfide bridge:1-6);

[0400] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DLeu-Pro-Phe(Disulfide bridge:1-6);

[0401] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DTrp-Pro-Phe(Disulfide bridge:1-6);

[0402] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DMet-Pro-Phe(Disulfide bridge:1-6);

[0403] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DGln-Pro-Phe(Disulfide bridge:1-6);

[0404] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DTyr-Pro-Phe(Disulfide bridge:1-6); and

[0405] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DSer-Pro-Phe (Disulfide bridge: 1-6).

[0406] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0407] (1) A polypeptide represented by the general formula (XIII):

[0408] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe

[0409] General formula (XIII)

[0410] wherein a12 is selected from an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, or His, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0411] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XIII) and having the same function.

[0412] In certain embodiments, illustrative examples of a13 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp, Glu, and His.

[0413] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y2997.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0414] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0415] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (XIII).

[0416] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0417] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfide bridge:1-6);

[0418] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Ser-Phe(Disulfide bridge:1-6);

[0419] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DTyr-Phe (Disulfide bridge: 1-6);

[0420] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DHis-Phe (Disulfide bridge: 1-6);

[0421] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DPhe-Phe (Disulfide bridge: 1-6);

[0422] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DTrp-Phe (Disulfide bridge: 1-6);

[0423] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DLeu-Phe (Disulfide bridge: 1-6);

[0424] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DAla-Phe (Disulfide bridge: 1-6);

[0425] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DSer-Phe (Disulfide bridge: 1-6);

[0426] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DGln-Phe (Disulfide bridge: 1-6); and

[0427] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DGlu-Phe (Disulfide bridge: 1-6).

[0428] In another aspect, the present disclosure relates to Apelin-13 polypeptide mutants, which comprise

[0429] (1) A polypeptide represented by the general formula (XIV):

[0430] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14

[0431] General formula (XIV)

[0432] wherein a14 is selected from an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, His or Nal, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or

[0433] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XIV) and having the same function.

[0434] In certain embodiments, illustrative examples of a14 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp, Glu, His, and Nal.

[0435] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2combination.

[0436] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 or Y299 7.49 Binding, thereby affecting the bias of G protein.

[0437] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (XIV).

[0438] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0439] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Ala(Disulfide bridge:1-6);

[0440] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6);

[0441] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Nal(Disulfide bridge:1-6);

[0442] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr(Disulfidebridge:1-6);

[0443] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DHis(Disulfidebridge:1-6);

[0444] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DAsn(Disulfide bridge:1-6);

[0445] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Dglu(Disulfide bridge:1-6);

[0446] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Ile(Disulfide bridge:1-6);

[0447] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Pro(Disulfide bridge:1-6);

[0448] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Val(Disulfide bridge:1-6); and

[0449] Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Trp (Disulfidebridge: 1-6).

[0450] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0451] (1) A polypeptide represented by the general formula (XV):

[0452] Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe

[0453] General formula (XV)

[0454] wherein a15 and b15 are each independently selected from an amino acid with a positively charged side chain or Pro, c15 is selected from an amino acid with a hydrophobic side chain or an amino acid with a positively charged side chain, and the cysteine ​​residues at positions 1 and 5 form a disulfide bond; or

[0455] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XV) and having the same function.

[0456] In certain embodiments, illustrative examples of a15 that can be used in the present disclosure include, but are not limited to, Arg, Lys, and Pro.

[0457] In certain embodiments, illustrative examples of b15 that can be used in the present disclosure include, but are not limited to, Arg, Lys, and Pro.

[0458] In certain embodiments, illustrative examples of c15 that can be used in the present disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, and Lys.

[0459] In certain embodiments, the Apelin-13 polypeptide mutant is ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0460] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (XV).

[0461] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0462] Cys-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge:1-5);

[0463] Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-5);

[0464] Cys-Arg-Pro-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-5); and

[0465] Cys-Arg-Pro-Arg-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-5).

[0466] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0467] (1) A polypeptide represented by the general formula (XVI):

[0468] Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe

[0469] General formula (XVI)

[0470] wherein a16 is selected from an amino acid with a positive side chain or Pro, c15 is selected from an amino acid with a hydrophobic side chain, an amino acid with a positive side chain or Pro, and the cysteine ​​residues at positions 1 and 4 form a disulfide bond; or

[0471] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XVI) and having the same function.

[0472] In certain embodiments, illustrative examples of a16 that can be used in the present disclosure include, but are not limited to, Arg, Lys, and Pro.

[0473] In certain embodiments, b16 useful in the present disclosure is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, Lys, and Pro.

[0474] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T2957.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0475] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (XVI).

[0476] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0477] Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-4);

[0478] Cys-Pro-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-4);

[0479] Cys-Arg-Pro-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-4); and

[0480] Cys-Pro-Arg-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-4).

[0481] In another aspect, the present disclosure relates to an Apelin-13 polypeptide mutant comprising

[0482] (1) A polypeptide represented by the general formula (XVII):

[0483] *-Arg-a17-b17-Leu-*-His-C17-Gly-Pro-DMet-Gly-Pro-Phe

[0484] General formula (XVII)

[0485] wherein a17 and b17 are each independently selected from amino acids with positively charged side chains, or a17 and / or b17 are absent, c17 is an amino acid with a positively charged side chain, * is a non-natural amino acid, and a ring structure is formed between *; or

[0486] (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XVII) and having the same function.

[0487] In certain embodiments, illustrative examples of a17 and b17 that can be used in the present disclosure include, but are not limited to, Arg, Lys, and Pro.

[0488] In certain embodiments, both a17 and b17 are absent for use in the present disclosure.

[0489] In certain embodiments, illustrative examples of b16 that can be used in the present disclosure include, but are not limited to, Arg and Lys.

[0490] In certain embodiments, illustrative examples of * that can be used in the present disclosure include, but are not limited to:

[0491] In certain embodiments, illustrative examples of ring structures formed between * that can be used in the present disclosure include, but are not limited to, staple structures and triazole-containing structures.

[0492] In certain embodiments, exemplary examples of ring structures formed between * that can be used in the present disclosure include but are not limited to

[0493] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M1133.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 combination.

[0494] In certain embodiments, the Apelin-13 polypeptide mutant disclosed herein consists of a polypeptide represented by formula (XVII).

[0495] In certain embodiments, the Apelin-13 polypeptide mutants disclosed herein are selected from:

[0496] as well as

[0497]

[0498] In another aspect, the present disclosure relates to a pharmaceutical composition comprising the Apelin-13 polypeptide mutant described in the present disclosure and a pharmaceutically acceptable carrier, diluent or excipient.

[0499] In another aspect, the present disclosure relates to a method for treating or preventing a disease or condition responsive to agonism of an APLNR receptor, comprising administering to an individual in need thereof a therapeutically or prophylactically effective amount of the Apelin-13 polypeptide mutant of the present disclosure or the pharmaceutical composition of the present disclosure.

[0500] In certain embodiments, illustrative examples of diseases or disease states that can be used in the present disclosure include, but are not limited to, acute decompensated heart failure, acute heart failure, chronic heart failure, pulmonary hypertension, atrial fibrillation, kidney disease, hypertrophy, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes, obesity, peripheral arterial disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burns, preeclampsia, anti-aging, analgesia, sleep apnea syndrome, pulmonary embolism, asthma, stress ulcers, liver fibrosis, lung injury, brain injury, ischemic stroke, cancer, Alzheimer's disease, Parkinson's disease, cerebral infarction, renal hypertension and neurodegenerative diseases.

[0501] In certain embodiments, illustrative examples of individuals that can be used with the present disclosure include, but are not limited to, mammals.

[0502] In certain embodiments, the present disclosure can be used in humans as individuals.

[0503] In yet another aspect, the present disclosure relates to the Apelin-13 polypeptide mutants of the present disclosure for use in treating or preventing a disease or condition responsive to agonism of an APLNR receptor.

[0504] In another aspect, the present disclosure relates to use of the Apelin-13 polypeptide mutants described herein in the preparation of a medicament for treating or preventing a disease or condition responsive to agonism of an APLNR receptor.

[0505] On the other hand, the present disclosure relates to a G protein-biased apelin-13 polypeptide mutant, wherein the G protein-biased agonist MM07 is further mutated, wherein the amino acid sequence of MM07 is CRPRLCHKGPMPF, wherein a disulfide bond is formed between the cysteine ​​residues at positions 1 and 6,

[0506] The mutant is any of the following:

[0507] (1) Mutate M11 to D-amino acid based on MM07;

[0508] (2) Mutate M11 based on MM07 to any of the following: A, G, Nal, V, P;

[0509] (3) Insert A or G between P10 and M11 based on MM07;

[0510] (4) Based on MM07, P3 was mutated to W, L5 was mutated to F, and M11 was mutated to Nal;

[0511] (5) Based on MM07, P3 was mutated to W, L5 was mutated to F, and G was inserted between P10 and M11.

[0512] In certain embodiments, the mutant MM07 is further mutated, and the mutant is obtained by mutating M11 to a D-type amino acid on the basis of MM07, specifically any one of the following: D-type M, D-type Nal.

[0513] In certain embodiments, the mutant MM07 is further mutated to obtain a mutant in which M11 is mutated to a D-type amino acid on the basis of MM07, and an amino acid is inserted between amino acids 11 and 12. The inserted amino acid is specifically any one of the following: G, A.

[0514] In certain embodiments, the mutant MM07 is further mutated to obtain any one of the following:

[0515] (1) WN561: Based on MM07, M11 is mutated to D-type M, and G is inserted between D-type M11 and P12. The sequence is: CRPRLCHKGPMDGPF, where MD represents D-type M and a disulfide bond is formed between the cysteine ​​residues at positions 1 and 6;

[0516] (2) Based on MM07, M11 was mutated into D-type M, and A was inserted between D-type M11 and P12.

[0517] In certain embodiments, the mutant WN561 is further mutated to obtain any one of the following:

[0518] (1) Mutation of R2 to A, S, K, C, or T based on WN561;

[0519] (2) P3 was mutated to Y, S, T, or C based on WN561;

[0520] (3) Mutating R4 to A or K based on WN561;

[0521] (4) L5 was mutated to A, I, or V based on WN561;

[0522] (5) Mutate H7 to A, S, R, K, T, C, or G based on WN561;

[0523] (6) Mutate K8 to A or R based on WN561;

[0524] (7) Mutate G9 to S, C, or T based on WN561;

[0525] (8) Mutation of G12 to F, S, W, C, or Y based on WN561;

[0526] (9) Mutate F14 to L, I, P, V, or W based on WN561.

[0527] In another aspect, the present disclosure relates to the use of the G protein-biased apelin-13 polypeptide mutant or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating or preventing a disease or condition responsive to agonism of an APLNR (APJ) receptor.

[0528] In certain embodiments, the disease or condition is selected from the group consisting of acute decompensated heart failure, chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes, obesity, peripheral arterial disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burns, preeclampsia, cancer, Alzheimer's disease, Parkinson's disease, stroke, and neurodegenerative disease.

[0529] In another aspect, the present disclosure relates to a drug for treating or preventing a disease or condition responsive to agonism of an APLNR receptor, wherein the active substance is the G protein-biased apelin-13 polypeptide mutant described in the present disclosure or a pharmaceutically acceptable salt thereof.

[0530] Hereinafter, the present disclosure will be explained in detail through the following examples in order to better understand the various aspects and advantages of the present application. However, it should be understood that the following examples are non-limiting and are only used to illustrate certain embodiments of the present disclosure.

[0531] The reagents and equipment used in the examples of this disclosure are conventional and commercially available.

[0532] General preparation process of peptides

[0533] Peptide sequence: If the sequence is ARSS

[0534] 1. Synthesis of Peptides

[0535] Synthesis order: from the C-terminus to the N-terminus of the peptide chain

[0536] a. Resin swelling: Weigh a calculated amount of fmoc-Ser(tbu)-OH resin and pour it into the reaction column. Add DMF and soak for 30 minutes, then drain.

[0537] b. Deprotection:

[0538] Add an appropriate amount of deprotection solution (piperidine = 20% hexahydropyridine + 80% DMF) to the reaction column, stir and agitate with nitrogen for 30 minutes, drain, add an appropriate amount of DMF to the reaction column, agitate with nitrogen for 2 minutes, drain, and repeat the operation 6 times.

[0539] c. Weighing:

[0540] Weigh 3 times the molar amount of the protected amino acid and 2.85 times the molar amount of HBTU (condensing agent) for later use.

[0541] d. Feeding:

[0542] The prepared protected amino acid and HBTU were added to the reaction column, and then 6 times the molar amount of NMM (organic base) of the resin was added, and the mixture was stirred with nitrogen for 30 minutes.

[0543] e. Post-reaction washing:

[0544] The solution in the reaction column was drained, and an appropriate amount of DMF was added for washing. Nitrogen was agitated for 2 minutes, and the column was drained. The operation was repeated 3 times.

[0545] f. Testing:

[0546] Place an appropriate amount (10-20 pellets) of resin in a small test tube and add two drops each of Solution A, Solution B, and Solution C. Heat in a dry-well heater for 3 minutes (110°C). If the solution appears blue and the resin is opaque and variegated upon removal, the reaction is incomplete and needs to be repeated. If the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete and the next amino acid can be attached. Repeat steps b through f until the last amino acid is attached. This results in the sequence ADSS. Finally, remove the Fmoc protecting group on the FmocAla-OH residue, exposing the N-terminus.

[0547] g. Washing and drying after synthesis:

[0548] After the last amino acid is attached and deprotection and washing are complete, drain the column, add an appropriate amount of methanol, aerate with nitrogen for 2 minutes, drain, then add an appropriate amount of DCM, aerate with nitrogen for 2 minutes, and drain three times. Finally, add an appropriate amount of methanol to the reactor, aerate with nitrogen for 2 minutes, drain, and repeat this process two more times. Place the resin in a suitable container and dry it in a vacuum desiccator for 12 hours before cleavage.

[0549] 2. Peptide cleavage (cleavage)

[0550] Cutting: Place the dried resin into a suitable round-bottom flask, add an appropriate amount of cutting solution (1g / 10ml), and place it in a constant temperature shaker at 25℃ for 2 hours.

[0551] Filtration: Use a 50mL sand core funnel to filter out the resin particles, then pour the filtrate into a glass container of a centrifuge tube, add 6-8 times the volume of anhydrous ether, and stir while adding. The precipitated white solid is the desired crude polypeptide.

[0552] Washing: Divide the white solid into centrifuge tubes, seal the tubes and centrifuge them at 4000 r / min for 3 minutes. Remove the tubes, discard the supernatant, add ether, stir evenly with a glass rod, and centrifuge again. Repeat this washing process 5 times.

[0553] Liquid phase ring closure synthesis:

[0554] The crude linear peptide was diluted to 10 -3 mol / L, place in an ice bath and fully dissolve, then add 1.5 times the amount of HOAt, 1.5 times the amount of HATU, and 10 times the amount of DIEA, continue stirring in the ice bath for 2 hours, remove the ice bath, continue stirring at room temperature, and distill under reduced pressure to obtain a solid residue.

[0555] Drying: After washing five times, place the peptide in a vacuum desiccator and dry it for 24 hours. The resulting white powder is the crude product of the desired peptide, which is weighed and purified.

[0556] Note: Amino acids and reagents used

[0557] A. Amino acids

[0558] Fmoc-Arg(pbf)-OH

[0559] Fmoc-Ala-OH

[0560] Fmoc-Ser(tbu)-OH

[0561] B. Condensation agent and organic base

[0562] HBTU

[0563] NMM

[0564] C. Solvent and nitrogen:

[0565] DMF

[0566] DCM

[0567] Methanol

[0568] Hexahydropyridine

[0569] 98% N2

[0570] D.Test fluid

[0571] Solution A: 80% phenol + 20% anhydrous ethanol

[0572] Solution B: redistilled pyridine

[0573] Solution C: 5g ninhydrin + 100mL anhydrous ethanol

[0574] E. Preparation of Deprotection Solution

[0575] 20% piperidine + 80% DMF

[0576] G. Cutting lysate

[0577] 87.5% TFA + 5% thioanisole + 2.5% phenol + 2.5% EDT + 2.5% H2O

[0578] 3. Purification Method Steps

[0579] 1. First perform 10-100% analysis of the crude product, determine the retention time of the main peak, and then perform linear gradient analysis.

[0580] 2. Solubility of the sample Water and acetonitrile (4:1) can fully dissolve the peptide.

[0581] 3. Dissolve 200 mg of polypeptide in a 10 mL dissolution bottle. After ultrasonic dissolution, filter with a 0.45 μm organic phase filter to obtain a clear solution.

[0582] 4. Prepare the linear gradient to collect the peak and determine whether the MS is correct.

[0583] 5. Track and provide feedback on the purity of the collected liquid to obtain the amount of liquid that meets the purity requirements.

[0584] 6. The collected qualified liquid is concentrated at low temperature at 38°C to a liquid volume of 50-100mL (in special cases, it is generally not concentrated and directly frozen).

[0585] 7. Place the concentrated liquid in a 100mL beaker and freeze it into a solid state with liquid nitrogen or dry ice.

[0586] 8. Drying

[0587] 9. Weighing

[0588] General Characterization Methods for Peptides

[0589] 1. Ability to confirm whether the target peak is the prepared peptide sample by MS during the purification process:

[0590] 1. Prepare the sample to be tested and ensure that it is suitable for mass spectrometry analysis, which usually requires dissolution and extraction.

[0591] 2. Adjust instrument parameters

[0592] Select an ion source: Choose an appropriate ion source based on the sample properties, such as an electrospray ionization source (ESI) or a chemical ionization source (CI) for gas chromatography-mass spectrometry.

[0593] 3. Set the mass spectrometry analysis mode: Select the mass spectrometry analysis mode as ion monitoring mode.

[0594] 4. Perform Mass Spectrometry Analysis

[0595] After starting the ion source and waiting for it to stabilize, inject the peptide sample into the mass spectrometer.

[0596] 5. Data Collection

[0597] Start mass spectrometry analysis and collect mass spectrometry data in real time, including mass spectrum and relative abundance.

[0598] 6. Analyze Mass Spectrometry Data

[0599] Mass spectrometry software is used to analyze the mass spectrum and identify proton peaks or molecular ion peaks. Based on the mass spectrometry data, it is determined whether the target peptide is present in the sample.

[0600] 2. Then test the purity of the peptide by HPLC:

[0601] 1. Turn on the HPLC workstation, condense the mobile phase pipeline, and connect the detection system.

[0602] 2. Adjust the flow rate. When using a new mobile phase for the first time, you can test the pressure first. The higher the flow rate, the higher the pressure. Generally, do not exceed 2000. Click injure, select an appropriate flow rate, click on, run the baseline, and observe the baseline.

[0603] 3. Set the sampling method, steady the flow for 2-5 minutes, return the baseline to zero, switch the injection valve from loading to injection, and set the sampling time.

[0604] Filter and load the resulting peptide sample. After the method is complete, click postrun to record data and create markers. After all samples have been run, run a baseline again using the above method to wash away any residue.

[0605] 4. Calculate the ratio of the main peak to all peaks, which is the purity of the peptide sample.

[0606] Preparation Example 1

[0607] Lys-Arg-Pro-Arg-Leu-Glu-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0608] Preparation of (Disulfide bridge: 1-6)

[0609] The synthetic raw materials are: lysine, arginine, proline, leucine, glutamic acid, histidine, glycine, D-methionine, and alanine. The reagents used include: condensing agent and organic base HBTU and NMM, DMF, DCM, methanol, hexahydropyridine, 98% N2; detection solution (liquid A: 80% phenol + 20% anhydrous ethanol; liquid B: redistilled pyridine; liquid C: 5g ninhydrin + 100mL anhydrous ethanol); deprotection solution: 20% hexahydropyridine + 80% DMF; cleavage solution: 87.5% TFA + 5% thioanisole + 2.5% phenol + 2.5% EDT + 2.5% H2O.

[0610] The synthesis steps are as follows:

[0611] a. Resin swelling: Weigh 1 g of fmoc-Phe(tbu)-OH resin and pour it into the reaction column. Add 15 mL of DMF and soak for 30 minutes, then drain (the first amino acid Phe is connected to the resin).

[0612] b. Deprotection: 25 ml of deprotection solution (piperidine = 20% hexahydropyridine + 80% DMF) was added to the reaction column, stirred with nitrogen for 30 minutes, drained, 25 mL of DMF was added to the reaction column, nitrogen was stirred for 2 minutes, drained, and the operation was repeated 6 times.

[0613] c. Weigh the materials: Weigh 4.8g of protected amino acid and 0.562g of HBTU (contraction agent) for later use.

[0614] d. Feeding: Add the prepared protected amino acid and HBTU to the reaction column, then add 0.608 mL of NMM (organic base) to the resin, and stir with nitrogen for 30 minutes.

[0615] e. Post-reaction washing: drain the solution in the reaction column, add 25 mL of DMF for washing, agitate with nitrogen for 2 minutes, drain, and repeat the operation three times.

[0616] f. Test: Place 20 resin pellets in a small test tube and add two drops each of Solution A, Solution B, and Solution C. Heat in a dry-block heater for 3 minutes (110 degrees Celsius).

[0617] If the solution appears blue after removal and the resin is variegated and opaque, the reaction is incomplete and needs to be repeated. If the solution is slightly yellow and the resin is colorless and transparent, the reaction is complete and the next amino acid, Pro, is ligated. Repeat steps b through f to ligate Gly, DMet, Pro, Gly, Lys, His, Lys, Glu, Leu, Arg, Pro, Arg, and Lys, respectively. This yields the title sequence. Finally, remove the Fmoc protecting group on FmocAla-OH to expose the N-terminus.

[0618] g. Washing and drying after synthesis:

[0619] After the last amino acid is connected and deprotected and washed, drain and add an appropriate amount of methanol to the reaction column, agitate with nitrogen for 2 minutes, drain and add an appropriate amount of DCM, agitate with nitrogen for 2 minutes, drain and repeat the operation 3 times. Finally, add an appropriate amount of methanol to the reactor, agitate with nitrogen for 2 minutes, drain and repeat the operation 2 times. Put the resin into a suitable container and place it in a vacuum dryer to dry under vacuum for 12 hours before cutting.

[0620] 2. Peptide cleavage (cleavage)

[0621] Cutting: Place the dried resin into a suitable round-bottom flask, add an appropriate amount of cutting solution (1 g / 10 mL), and place it in a constant temperature shaker at 25°C for 2 hours.

[0622] Filtration: Use a 50mL sand core funnel to filter out the resin particles, then pour the filtrate into a glass container of a centrifuge tube, add 6-8 times the volume of anhydrous ether, and stir while adding. The precipitated white solid is the desired crude polypeptide.

[0623] Washing: Divide the white solid into centrifuge tubes, seal the tubes and centrifuge them at 4000 r / min for 3 minutes. Remove the tubes, discard the supernatant, add ether, stir evenly with a glass rod, and centrifuge again. Repeat this washing process 5 times.

[0624] Liquid phase ring closure synthesis:

[0625] The crude linear peptide was diluted to 10 -3 mol / L, place in an ice bath and fully dissolve, then add 1.5 times the amount of HOAt, 1.5 times the amount of HATU, and 10 times the amount of DIEA, continue stirring in the ice bath for 2 hours, remove the ice bath, continue stirring at room temperature, and distill under reduced pressure to obtain a solid residue.

[0626] Drying: The polypeptide after washing 5 times was placed in a vacuum desiccator and vacuum dried for 24 hours.

[0627] The final white powder is the crude product of the desired polypeptide, which is weighed and purified.

[0628] 3. Purification Method Steps

[0629] 1. First perform 10-100% analysis of the crude product, determine the retention time of the main peak, and then perform linear gradient analysis.

[0630] 2. Solubility of the sample Water and acetonitrile (4:1) can fully dissolve the peptide.

[0631] 3. Dissolve 200 mg of polypeptide in a 10 ml dissolution bottle. After ultrasonic dissolution, filter with a 0.45 μm organic phase filter to obtain a clear solution.

[0632] 4. Prepare the linear gradient to collect the peak and determine whether the MS is correct.

[0633] 5. Track and provide feedback on the purity of the collected liquid to obtain the amount of liquid that meets the purity requirements.

[0634] 6. Concentrate the collected qualified liquid at low temperature, concentration temperature: 38 ° C, and concentrate to 50-100 mL of liquid (in special cases, it is generally not concentrated and directly frozen)

[0635] 7. Place the concentrated liquid in a 100mL beaker and freeze it into a solid state with liquid nitrogen or dry ice.

[0636] 8. Drying

[0637] MS characterization: Mass spectrometry shows the mass-to-charge ratio of the peptide, with four target peaks: [M+Na+H] 2+ =810.35, [M+2H] 2+ =799.4, [M+3H] 3+ =533.35, [M+4H] 4+ =400.35. Target peaks were detected at these positions, thus the purified peptide was indeed the title polypeptide sequence.

[0638] HPLC results showed that the purity of the title polypeptide was 96.37%.

[0639] Preparation Example 2

[0640] Cys-Ala-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe

[0641] Preparation of (Disulfide bridge: 1-6)

[0642] The preparation process is as described in Preparation Example 1, using the amino acid raw materials cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, and phenylalanine. Alternative Method Preparation Example 1: After the first amino acid is completely reacted, the next amino acid, Pro, is attached. The above five steps (b-f) are then repeated to attach Gly, DMet, Pro, Gly, Lys, His, Cys, Leu, Arg, Pro, Ala, and Cys, respectively. The title sequence is obtained. Finally, the Fmoc protecting group on FmocAla-OH is removed to expose the N-terminus.

[0643] MS characterization: Mass spectrometry shows the mass-to-charge ratio of the peptide, with four target peaks: [M+Na+H] 2+ =810.35, [M+2H] 2+ =799.4, [M+3H] 3+ =533.35, [M+4H] 4+ = 400.35. Target peaks were detected at all four positions, thus confirming that the purified peptide was indeed the title polypeptide sequence.

[0644] HPLC results showed that the purity of the title polypeptide was 97.52%.

[0645] The amino acid sequence of the wild-type apelin-13 polypeptide is Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe (represented by the single letters QRPRLSHKGPMPF).

[0646] The amino acid sequence of MM07 is Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Met-Pro-Phe (disulfide bridge 1-6, i.e., Cys at positions 1 and 6 form a disulfide bond) (represented by single letters as CRPRLCHKGPMPF (disulfide bridge 1-6)).

[0647] The remaining mutant polypeptide sequences used in this application are shown in Table 1.

[0648] Table 1

[0649] 1. Nanobit experiment:

[0650] Experimental principle: Binary Technology (NanoBiT) is a reporter gene technology for detecting protein interactions using structural complementation. The system consists of two subunits: Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 11 amino acids), which can be fused to target proteins. The LgBiT and SmBiT subunits have been optimized for excellent stability and minimized self-association. When the target protein fused with these two subunits interacts, the two subunits bind to form an active enzyme, generating a bright luminescent signal. We linked the Gα protein to LgBiT and Gγ to SmBiT, and cloned the receptor genes into the pcDNA3.1 vector. The dissociation of Gα and Gγ was measured to assess the ability of ligands to activate the receptor-coupled G protein.

[0651] Experimental Methods: The above plasmids were co-transfected into HEK293T cells cultured in a 6-well plate for expression. 24 hours after transfection, the culture medium was replaced with complete medium containing 10% FBS overnight. 24-48 hours after transfection, the transfected 6-well plate was removed, washed once with PBS, and trypsinized with 1 ml of trypsin for 1 minute. The cells were then resuspended in 3 ml of DEMEM complete medium and seeded into a 96-well plate for overnight culture. The next day, the 96-well plate was removed, washed once with D-PBS, and coelenterazine 400a substrate prepared in HBSS was added and incubated for 50 minutes. The basal signal was measured, and then the ligand at various concentrations was added, mixed, and the signal after ligand stimulation was measured. The ligand-induced signal was normalized to the baseline signal to demonstrate the G protein dissociation response.

[0652] 2. BRET assay:

[0653] Principle of the experiment: BRET assay is to study the interaction between proteins by labeling bioluminescent donors such as Renilla luciferase (Rluc) or fluorescent receptors such as enhanced green or yellow fluorescent proteins (EGFP or EYFP). When the interacting parts are co-expressed in cells, in the presence of substrate coelenterazine, the proteins are close enough (in the presence of Energy transfer from Rluc to EYFP occurs within a certain distance (< 100 nm). The strict distance between the donor and acceptor molecules makes BRET an ideal tool for studying receptor-protein interactions involved in all GPCR functions and their regulation by agonists and antagonists.

[0654] Experimental methods:

[0655] The BRET method was used to detect the recruitment of β-arrestin. The APLNR receptor C-terminus was fused with the Rluc8 fragment, and the Venus fragment was inserted into the N-terminus of β-arrestin2 and cloned into the pBiT1.1 plasmid. The above plasmids were co-transfected into a 6-well plate in which HEK293T cells had been cultured for expression. 24 hours after cell transfection, the culture medium was replaced with a complete culture medium containing 10% FBS overnight. 24h-48h after the cells were transfected, the transfected 6-well plate was removed, washed with PBS, digested with 1ml of trypsin for 1min, and resuspended with 3ml of DMEM complete culture medium. The resuspended cells were inoculated into a 96-well plate and cultured overnight. The next day, the 96-well plate was removed, washed with D-PBS, and 40μl of HBSS was added. The transfected cells were then stimulated with different concentrations of ligand (5μl) for 40min. Then, the luciferase substrate luminal luciferin was added for 400 h, and the BRET signals were detected: Venus (520-560 nm) and RLuc8 (460-485 nm).

[0656] 3. Protein Purification

[0657] Cryo-electron microscopy samples of the APLNR-Gi1 complex bound to apelin and MM07 were prepared. Human wild-type (WT) APLNR, Gi1-Gβγ, and scFv16 genes were cloned into the pFastBac1 vector (Invitrogen) and expressed in Sf9 insect cells at a 1:1:1 ratio. To purify the APLNR-Gi1 complex, the cell pellet was suspended in a buffer containing 20 mM HEPES pH 7.5, 2 mM MgCl2, 100 mM NaCl, and a protease inhibitor cocktail (Bimake). Then, 1.2 mg of scFv16 antibody, 12 mU / ml apyrase (Sigma), and agonists (50 μM apelin and 50 μM MM07) were added. The mixture was incubated at room temperature for 1 hour to allow complex formation. 0.5% (w / v) dodecylmaltodextrin neopentyl glycol and 0.1% (w / v) cholesterol hemisuccinate were then added and incubated at 4°C for 2 hours. After incubation with MBP (NEB), proteins were eluted with 20 mM maltose. TEV protease was then added for 1 hour to remove MBP. The eluate was concentrated using a 100 kDa concentrator and injected onto a Superose 6increase 10 / 300 GL column (GE Healthcare) in a running buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% dodecyl glutamyl neopentyl glycol, 0.0002% (w / v) hemiacetyl cholesterol, and 0.00025% (w / v) glycosapogenin, along with agonists (50 μM apelin and 50 μM MM07). Fractions of interest were collected and concentrated to 10-15 mg / ml for electron microscopy.

[0658] 4. Data collection and structural analysis

[0659] For the APLNR-Gi1 complex, cryo-EM data were acquired on a Titan Krios at an accelerating voltage of 300 kV. Micrographs were recorded in super-resolution mode using SerialEM software (Schorb, Haberbosch et al. 2019) with a Gatan K2 detector. The pixel size of the apelin complex was Image stacks were acquired at a dose rate of approximately 8.0 electrons / Ų / s, with defocus values ​​ranging from -1.0 to -2.5 μm. The total exposure time was 8 seconds, and 40 frames were recorded for each micrograph. A total of 2520 images of apelin-bound complexes were collected. For the MM07 complex, A total of 4454 micrographs were collected at a pixel size of 21.3 electrons / Ų / s, with defocus values ​​ranging from -0.8 to -1.8 μm. The total exposure time was 2.35 seconds, and 36 frames were recorded per micrograph. The collected data were processed using RELION 3.1 and CryoSPARC v3.1. The model was docked to the cryo-EM density map using UCSF Chimera (Pettersen, Goddard et al. 2004). The final model was refined using the "Integrated Validation (cryo-EM)" module in Phenix. The structure was drawn using UCSF Chimera and UCSF Chimera X software.

[0660] Example 1

[0661] Structural analysis of the APLNR-G protein complex

[0662] The amino acid sequence of human APLNR is shown in SEQ ID No. 1 (NCBI sequence number: NP_005152.1), and the encoding gene sequence is shown in SEQ ID No. 2 (NCBI sequence number: NM_005161.6). The G protein is human Gi1 (Gi1 is a subtype of Gi protein), with an amino acid sequence shown in SEQ ID No. 3 (NCBI sequence number: NP_001027619.1), and the encoding gene sequence is shown in SEQ ID No. 4 (NCBI sequence number: NM_001032447.1).

[0663] To investigate the ligand recognition mechanism of APLNR, we resolved the cryo-EM structures of the APLNR-Gi complex activated by the balanced agonist apelin and the G protein-biased agonist MM07 (CAS No. 1876450-21-3) at resolutions of and

[0664] First, stable apelin-13-APLNR-Gi1 (apelin-13 complex with APLNR and Gi1) and MM07-APLNR-Gi1 (MM07 complex with APLNR and Gi1) protein complexes were purified using a baculovirus-based insect expression system and stabilized with the scFv16 antibody (laboratory expression and purification, amino acid sequence shown in SEQ ID No. 5). Subsequently, data were collected using a 300 kV cryo-electron microscope. Data processing and 3D reconstruction were performed using RELION software, and modeling and structural refinement were performed using WINCOOT and PHENIX software to obtain a high-resolution ternary complex structure.

[0665] The experimental results are shown in Figure 1. The experimental results show that we have resolved the high-resolution cryo-electron microscopy structure of the apelin-APLNR-Gi1 complex (A and B in Figure 1), which allows us to well analyze the interaction between the ligand and the receptor. We overexpressed the APLNR receptor in HEK293 cells, and used nanobit experiments to detect the ability of apelin and MM07 to activate APLNR to recruit G proteins, and bret experiments to detect the ability of ligands to activate arrestin. The experimental results are shown in Figure 1C. The EC50 of apelin to activate G protein signaling is 9.793×10 -10 M, ECs recruiting β-arrestin 50 is 8.688×10 -9 MM07 activates G protein signaling in ECs 50 is 1.377×10 -8 M, ECs recruiting β-arrestin 50 5.495×10 -6 Compared with apelin, MM07's ability to activate arrestin was nearly 1000-fold weaker, while its G protein activity was about 10-fold weaker than that of apelin. These results indicate that MM07 is a G protein-biased agonist but still retains considerable arrestin activity.

[0666] Example 2

[0667] Analysis of the APLNR-biased signal transduction mechanism

[0668] By comparing the differences in the binding pockets of APLNR activated by two ligands, the specific amino acid sites where different agonists bind to APLNR were identified, and the recognition and activation mechanisms of the endogenous peptide apelin and the G protein-biased agonist MM07 were elucidated. Key amino acid sites involved in ligand recognition in APLNR were mutated, and ligand-activated downstream G protein and β-arrestin activities were measured using nanobit and BRET assays, respectively, to determine the effects of point mutations on the ability of APLNR to bind to downstream G proteins and β-arrestin under the action of different ligands, thus elucidating the mechanism of biased signal transduction.

[0669] The results are shown in Figure 2. The residues of the ligand-binding pockets of the APLNR structures bound by apelin (A) and MM07 (B) are shown. The density of the peptide ligands M11 and F13, as well as the residues on the receptor that interact with these two amino acids, is shown as a surface plot. Functional assay results indicate that mutations in key amino acids located in the "twin hotspots" affect G protein or β-arrestin activity (Figure 2C and E show amino acid mutations that reduce β-arrestin activity more than G protein, indicating that the receptor prefers G protein binding; Figure 2D and F show amino acid mutations that reduce G protein activity more than β-arrestin, indicating that the receptor prefers β-arrestin binding). This suggests that the two "twin hotspots" on the receptor are key to influencing the selectivity of APLNR for downstream signaling molecules. M11 and F13 of apelin and MM07, respectively, insert into the two pockets and interact with key amino acid residues in the "twin hotspots." Therefore, altering the Met 11 and Phe 13 positions, which interact with the receptor, may facilitate the design of G protein-biased peptide agonists.

[0670] Example 3

[0671] (1) Based on the above structural analysis, we conducted mutation studies on MM07, mutated M11 to A (WN123), Nal (WN353), and DNal (WN370) to synthesize three peptides, and conducted functional experiments. The results are shown in Figure 3. The functional experiments showed that the three peptides WN123, WN353, and WN370 almost lost the ability to recruit β-arrestin, but retained the ability to activate G protein.

[0672] We performed structural analysis of the corresponding amino acid mutations in WinCoot. As shown in Figure 4, we found that mutations to small residues such as A significantly weakened the interaction with the first hotspot, while mutations to F (a large residue) did not affect its insertion into the hotspot pocket. Simulating mutations of other small residues in WinCoot revealed that mutations to V (WN148), P (WN149), and G (WN150), similar to the M11A mutation, weakened the interaction with the first hotspot and, therefore, also resulted in loss of β-arrestin activity of the receptor. We resolved the structure of the mutation of M11 to Nal (WN353) and found that Nal was completely deflected in the opposite direction of M11, with enhanced interactions with Y35 and T295 in the first hotspot and weakened interactions with I109, F110, and Y264, consistent with G protein bias. Although the structure of the M11 mutant to DNal (WN370) has not been resolved, our functional data indicate that this mutation also results in a loss of the receptor's ability to recruit β-arrestin without affecting G protein activity. Functional experiments also demonstrate that the Nal11 mutant, with simultaneous mutations of P3 to W and L5 to F (WN386), is a fully G protein-biased agonist.

[0673] (2) As shown in Figure 5, we also designed a peptide (WN680) with Gly inserted between P10 and M11. We found that the first hotspot could not be inserted into G11, which greatly weakened the interaction with the key residues and weakened the activity of β-arrestin, which was consistent with the functional experiments.

[0674] As shown in Figure 6, structural analysis indicates that inserting a small residue (A) between P10 and M11 (WN682) also weakens the interaction with the first hotspot, thus achieving the same effect. WN648, formed by simultaneously mutating the third and fifth amino acid residues of WN386 to W and F, also failed to recruit β-arrestin, but retained G protein activity.

[0675] (3) In addition, when M11 was mutated to DMet and G was inserted between DMet at position 11 and P at position 12 (WN561), functional data showed that β-arrestin activity was completely lost, as shown in Figure 7. Structural data analysis showed that the main effect was on the interaction between the ligand and the two hotspots. This indicates that weakening the interaction between amino acid 13 and the second hotspot will weaken β-arrestin activity. Therefore, inserting A between DMet and P at position 12 can also achieve the same effect (DMet CA) (Figure 8).

[0676] Example 4

[0677] (1) Based on WN561, we mutated R2: WN562 (R2A) and WN563 (R2S). The functional experimental results are shown in Figure 9. β-arrestin activity was lost, while G protein activity was retained, indicating that the mutation of R2 to small residues A and S is also consistent.

[0678] Structural analysis reveals that K and R are polar, positively charged amino acids that can form electrostatic interactions with the surrounding E residues. C, T, and S, like polar, uncharged amino acids, can also form polar interactions with the surrounding E residues. Structural analysis indicates that mutations R2K (WN586), R2C (WN587), and R2T (WN588) also result in loss of β-arrestin activity. The structural data are shown in Figure 10.

[0679] (2) Based on WN561, mutations were made on P3: WN564 (P3Y) and WN565 (P3S). The functional experimental results are shown in Figure 11. β-arrestin activity was lost, while G protein activity was retained, indicating that the P3 mutation to Y and S is also consistent.

[0680] Structural analysis reveals that P3Y forms polar interactions with surrounding Ys, P3S forms polar interactions with surrounding Es, and C and T, like S, are polar, uncharged amino acids and also form polar interactions with surrounding Es. Therefore, structural analysis suggests that mutations in P3T (WN589) and P3C (WN590) also result in loss of β-arrestin activity. The structural data are shown in Figure 12.

[0681] (3) Based on WN561, a mutation was made on R4: WN566 (R4A). The functional experimental results are shown in Figure 13. β-arrestin activity was lost, while G protein activity was retained, indicating that the R4 mutation to A is also consistent.

[0682] Structural analysis reveals that both K and R are polar, positively charged amino acids, forming polar interactions with the surrounding Y. Therefore, structural analysis suggests that the R4K(WN591) mutation also results in loss of β-arrestin activity. The structural data are shown in Figure 14.

[0683] (4) Based on WN561, a mutation was made on L5: WN567 (L5A). ​​The functional experimental results are shown in Figure 15. β-arrestin activity was lost, while G protein activity was retained, indicating that the L5 mutation to A is also consistent.

[0684] Structural analysis reveals that V and I, like L, are nonpolar amino acids and can form nonpolar interactions with the surrounding F and Y groups. Therefore, structural analysis suggests that mutations L5I (WN592) and L5V (WN593) also result in loss of β-arrestin activity. The structural data are shown in Figure 16.

[0685] (5) Based on WN561, mutations were made on H7: WN571 (H7A) and WN572 (H7S). The functional experimental results are shown in Figure 17. β-arrestin activity was lost, while G protein activity was retained, indicating that the H7 mutation to A and S also met the requirements.

[0686] According to structural analysis, R and K, like H, are polar, positively charged amino acids that can form polar interactions with the surrounding N and Y groups. Therefore, structural analysis indicates that mutations in H7R (WN594) and H7K (WN595) will also lead to loss of β-arrestin activity. Mutations in H7A to small side chain residues also meet the criteria, so structural analysis indicates that H7G (WN606) will also lead to loss of β-arrestin activity. C and T, like S, are polar, uncharged amino acids that can form polar interactions with the surrounding Y groups. Therefore, structural analysis indicates that mutations in H7T (WN596) and H7C (WN597) will also lead to loss of β-arrestin activity. The structural data are shown in Figure 18.

[0687] (6) Based on WN561, mutations were made on K8: WN573 (K8A) and WN574 (K8R). The functional experimental results are shown in Figure 19. β-arrestin activity was lost, while G protein activity was retained, indicating that the K8 mutation to A and R is also consistent.

[0688] Structural analysis indicates that mutations to small side chain residues in H7A are also consistent with this finding. Like K, R is a polar, positively charged amino acid, capable of forming electrostatic interactions with the surrounding E residues and polar interactions with the surrounding Y residues. Therefore, structural analysis suggests that the K8R mutation also results in loss of β-arrestin activity. The structural data are shown in Figure 20.

[0689] (7) Based on WN561, a mutation was made on G9: WN575 (G9S). The functional experimental results are shown in Figure 21. β-arrestin activity was lost, while G protein activity was retained, indicating that the mutation of G9 to S is also consistent.

[0690] Structural analysis reveals that C and T, like S, are polar, uncharged amino acids and can form polar interactions with surrounding Y, S, and T groups. Therefore, structural analysis suggests that mutations in G9C (WN598) and G9T (WN599) also result in loss of β-arrestin activity. The structural data are shown in Figure 22.

[0691] (8) Based on WN561, mutations were made on G12: WN580 (G12F), WN581 (G12S), and WN582 (G12W). The functional experimental results are shown in Figure 23. β-arrestin activity was lost, while G protein activity was retained, indicating that the mutation of G12 to S and the large side chain F and W were also consistent.

[0692] Structural analysis indicates that mutations to amino acids with large side chains, such as F and W, enhance nonpolar interactions with M183 on the receptor, shifting the receptor toward the G protein. Like F and W, Y is a large side chain amino acid. Therefore, structural analysis suggests that the G12Y (WN601) mutation also results in loss of β-arrestin activity. Like S, C is a polar, uncharged amino acid that can form polar interactions with the surrounding S residues. Therefore, structural analysis suggests that the G12C (WN600) mutation also results in loss of β-arrestin activity. The structural data are shown in Figure 24.

[0693] (9) Based on WN561, a mutation was made at F14 (because the C-terminus of WN561 was extended, the original F13 position became F14 position): WN584 (F14L). The functional experimental results are shown in Figure 25. β-arrestin activity was lost, while G protein activity was retained, indicating that the mutation of F14 to the non-polar amino acid L was also consistent.

[0694] Structural analysis indicates that amino acid 14 is close to the phospholipid bilayer. Like amino acids F and L, amino acids I, P, V, and W are non-polar, increasing receptor stability and forming non-polar interactions with the surrounding V residues. Therefore, structural analysis indicates that mutations F14I (WN602), F14P (WN603), F14V (WN604), and F14W (WN605) also result in loss of β-arrestin activity. The structural data are shown in Figure 26.

[0695] Example 5

[0696] As described in Example 1, we analyzed the structure of the agonist-activated receptor-bound G protein complex and analyzed the ligand binding pocket. We determined that all the key amino acid residues that affect the binding of ligand to receptor as shown in Figure 27 include: Y35 1.39 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、I109 3.32 、F110 3.33 、M113 3.36 、R168 4.64 、M183 ECL2、Y185 ECL2 、E198 5.35 、Y264 6.51 、K268 6.55 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、T295 7.39 、Y299 7.43 、M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Q180 ECL2 and P292 7.36 .

[0697] We used the same signal detection method as in Example 2 to detect the dissociation of Gα and Gβγ by nanobit to determine the effect of mutations in all amino acid sites that interact with apelin and MM07 on receptor activity. We found that M183 ECL2 、F110 3.33 、I109 3.32 、K265 6.55 、Y35 1.39 、Y299 7.49 、W85 2.60 、Y88 2.63 、Y93 ECL1 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、M288 7.32 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 、Y182 ECL2 It is the key amino acid residue that affects receptor interaction, as shown in the following table △pEC 50The results of Emax and Emax showed that alanine mutation at these amino acid sites significantly weakened the G protein signal mediated by APLNR. 50 Compared with the wild type (WT), these amino acids are decreased, so these amino acids are key amino acids for the interaction between receptor and ligand.

[0698] Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, compared with the control group by one-way ANOVA, followed by Dunnett's multiple comparison test. ND, complete loss of activity, EC cannot be determined. 50 .

[0699] Table 2. Ligand-induced Gα-Gγ dissociation of wild-type (WT) and APLNR mutants

[0700] We also identified the key amino acid residues that affect receptor bias, as shown in Figure 2 and Table 3. ECL2 、F110 3.33 、I109 3.32 、K268 6.55 、Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 These are the key amino acids that affect the bias. Differences in the interactions with these amino acids will directly affect the receptor's bias towards G protein (Table 2) and β-arrestin (Table 3), which is also the basis for our peptide design.

[0701] Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant, compared with the control group by one-way ANOVA, followed by Dunnett's multiple comparison test.

[0702] Table 3. Ligand-induced β-arrestin recruitment in wild-type (WT) and APLNR mutants

[0703] Example 6

[0704] Referring to the methods of Examples 3 and 4, key amino acid residues were replaced on the basis of WN561, and the N-terminus and C-terminus were extended. The functional experimental results are shown in Table 4. β-arrestin activity was lost, while G protein activity was retained, with a complete bias towards G protein.

[0705] Table 4. G protein and β-arrestin activity and bias statistics of peptide sequences

[0706] In this disclosure, relational terms such as first and second, etc. are used merely to distinguish one entity or operation from another entity or operation, but do not necessarily require or imply any actual relationship or order between these entities or operations.

[0707] It will be appreciated from the foregoing that, although specific embodiments of the present disclosure have been described for illustrative purposes, various modifications or variations may be made by those skilled in the art without departing from the spirit and scope of the present disclosure. Such modifications or variations are intended to fall within the scope of the appended claims of the present disclosure.

Claims

1. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (I): a1-Arg-Pro-Arg-Leu-b1-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe General formula (I) in, a1 is any natural amino acid, b1 is an amino acid with a negatively charged side chain, and the amino acid residues at positions 1 and 6 form an amide bond; or (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (I) and having the same function.

2. The Apelin-13 polypeptide mutant according to claim 1, wherein the a1 is selected from Arg, His, Lys, Asp, Glu, Ser, Thr, Asn, Gln, Cys, Sec, Gly, Pro, Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

3. The Apelin-13 polypeptide mutant according to claim 1 or 2, wherein the b1 is selected from Glu and Asp.

4. The Apelin-13 polypeptide mutant according to any one of claims 1 to 3, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

5. The Apelin-13 polypeptide mutant according to any one of claims 1 to 4, which consists of a polypeptide represented by general formula (I).

6. The Apelin-13 polypeptide mutant according to any one of claims 1 to 5, wherein the polypeptide represented by general formula (I) is: Lys-Arg-Pro-Arg-Leu-Glu-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (amide bonds at positions 1, 6).

7. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (II): Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe General formula (II) in, a2 is selected from amino acids with hydrophobic side chains or amino acids with polar uncharged side chains, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (II) and having the same function.

8. The Apelin-13 polypeptide mutant according to claim 7, wherein the a2 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn and Gln.

9. The Apelin-13 polypeptide mutant according to claim 7 or 8, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

10. The Apelin-13 polypeptide mutant according to any one of claims 7 to 9, which consists of a polypeptide represented by general formula (II).

11. The Apelin-13 polypeptide mutant according to any one of claims 7 to 10, wherein the polypeptide represented by general formula (II) is selected from: Cys-Ala-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Lys-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Cys-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); and Cys-Thr-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

12. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (III-1): Cys-Arg-a3-Arg-b3-Cys-His-c3-Gly-Pro-d3-e3-f3-g3 General formula (III-1) in, a3 is selected from an amino acid having a hydrophobic side chain or an amino acid having a polar uncharged side chain, b3 is an amino acid having a hydrophobic side chain, c3 is an amino acid having a positively charged side chain, d3 and e3 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f3 is an amino acid having a hydrophobic side chain or Pro, g3 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) A polypeptide represented by the general formula (III-2): Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3' General formula (III-2) wherein a3' is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (3) having at least 70% homology with the polypeptide represented by general formula (III-1) or general formula (III-2) And polypeptides with the same function.

13. The Apelin-13 polypeptide mutant according to claim 12, wherein the a3 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn and Gln.

14. The Apelin-13 polypeptide mutant according to claim 12 or 13, wherein the b3 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

15. The Apelin-13 polypeptide mutant according to any one of claims 12 to 14, wherein the c3 is selected from Lys and Arg.

16. The Apelin-13 polypeptide mutant according to any one of claims 12 to 15, wherein said d3 and e3 are each independently selected from Ala, Val, Ile, Leu, DMet, Phe, Tyr, Trp and Gly.

17. The Apelin-13 polypeptide mutant according to any one of claims 12 to 16, wherein the f3 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro.

18. The Apelin-13 polypeptide mutant according to any one of claims 12 to 17, wherein the g3 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

19. The Apelin-13 polypeptide mutant according to any one of claims 12 to 18, wherein the a3' is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

20. The Apelin-13 polypeptide mutant according to any one of claims 12 to 19, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 , 180 ECL2 or 182 ECL2 Combine.

21. The Apelin-13 polypeptide mutant according to any one of claims 12 to 20, which consists of the polypeptide represented by the general formula (III-1) or the polypeptide represented by the general formula (III-2).

22. The Apelin-13 polypeptide mutant according to any one of claims 12 to 21, wherein the polypeptide represented by general formula (III-1) is selected from: Cys-Arg-Tyr-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Ser-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfide bridge:1-6); and Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe (Disulfide bridge: 1-6).

23. The Apelin-13 polypeptide mutant according to any one of claims 12 to 21, wherein the polypeptide represented by general formula (III-2) is selected from: Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6); and Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Trp (Disulfide bridge: 1-6).

24. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (IV): Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe General formula (IV) in, a4 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (IV) and having the same function.

25. The Apelin-13 polypeptide mutant according to claim 24, wherein the a4 is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr or Trp.

26. The Apelin-13 polypeptide mutant according to claim 24 or 25, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 , 180 ECL2 or 182 ECL2 Combine.

27. The Apelin-13 polypeptide mutant according to any one of claims 24 to 26, which consists of a polypeptide represented by general formula (IV).

28. The Apelin-13 polypeptide mutant according to any one of claims 24 to 27, wherein the polypeptide represented by general formula (IV) is selected from: Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); and Cys-Arg-Pro-Lys-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

29. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (V-1): Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5 General formula (V-1) in, a5 is selected from an amino acid having a hydrophobic side chain or Pro, b5 is an amino acid having a hydrophobic side chain, c5 is an amino acid having a positively charged side chain, d5 and e5 are each independently selected from an amino acid having a hydrophobic side chain or Gly, f5 is an amino acid having a hydrophobic side chain or Pro, g5 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) A polypeptide represented by the general formula (V-2): Cys-Arg-a5'-Arg-b5'-Cys-His-c5'-Gly-Pro-Nal-Pro-d5' General formula (V-2) wherein a5' is selected from an amino acid having a hydrophobic side chain or Pro, b5' is an amino acid having a hydrophobic side chain, c5' is an amino acid having a positively charged side chain, d5' is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (3) A polypeptide having at least 70% homology with the polypeptide represented by the general formula (III-1) or the general formula (III-2) and having the same function.

30. The Apelin-13 polypeptide mutant according to claim 29, wherein the a5 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro.

31. The Apelin-13 polypeptide mutant according to claim 29 or 30, wherein the b5 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

32. The Apelin-13 polypeptide mutant according to any one of claims 29 to 31, wherein the c5 is selected from Lys and Arg.

33. The Apelin-13 polypeptide mutant according to any one of claims 29 to 32, wherein said d5 and e5 are each independently selected from Ala, Val, Ile, Leu, DMet, Phe, Tyr, Trp and Gly.

34. The Apelin-13 polypeptide mutant according to any one of claims 29 to 33, wherein the f5 is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro.

35. The Apelin-13 polypeptide mutant according to any one of claims 29 to 34, wherein the g5 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

36. The Apelin-13 polypeptide mutant according to any one of claims 29 to 35, wherein the a5' is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro.

37. The Apelin-13 polypeptide mutant according to any one of claims 29 to 36, wherein the b5' is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

38. The Apelin-13 polypeptide mutant according to any one of claims 29 to 37, wherein the c5' is selected from Lys and Arg.

39. The Apelin-13 polypeptide mutant according to any one of claims 29 to 38, wherein the d5' is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

40. The Apelin-13 polypeptide mutant according to any one of claims 29 to 39, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

41. The Apelin-13 polypeptide mutant according to any one of claims 29 to 40, which consists of the polypeptide represented by general formula (V-1) or the polypeptide represented by general formula (V-2).

42. The Apelin-13 polypeptide mutant according to any one of claims 29 to 41, wherein the polypeptide represented by general formula (V-1) is selected from: Cys-Arg-Pro-Arg-Ala-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Tyr-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Trp-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Arg-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Trp-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Ile-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); and Cys-Arg-Pro-Arg-Val-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

43. The Apelin-13 polypeptide mutant according to any one of claims 29 to 41, wherein the polypeptide represented by general formula (V-2) is selected from: Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6); and Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Trp (Disulfide bridge: 1-6).

44. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (VII): Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe General formula (VII) in, a7 is selected from amino acids with hydrophobic side chains or amino acids with polar uncharged side chains, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (VII) and having the same function.

45. The Apelin-13 polypeptide mutant of claim 44, wherein the a7 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn and Gln.

46. ​​The Apelin-13 polypeptide mutant according to claim 44 or 45, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

47. The Apelin-13 polypeptide mutant according to any one of claims 44 to 46, which consists of a polypeptide represented by general formula (VII).

48. The Apelin-13 polypeptide mutant according to any one of claims 44 to 47, wherein the polypeptide represented by general formula (VII) is selected from: Cys-Arg-Pro-Arg-Leu-Cys-Ala-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-Arg-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-Lys-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-Thr-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); and Cys-Arg-Pro-Arg-Leu-Cys-Cys-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

49. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (VIII-1): Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8 General formula (VIII-1) in, a8 is selected from amino acids with hydrophobic side chains or Pro, b8 is an amino acid with a hydrophobic side chain, and c8 is selected from amino acids with hydrophobic side chains or amino groups with positively charged side chains. Acid, d8 is an amino acid having a hydrophobic side chain or Pro, e8 is an amino acid having a hydrophobic side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) A polypeptide represented by the general formula (VIII-2): Cys-Arg-Pro-Arg-Trp-Cys-His-a8'-Gly-Pro-Nal-Pro-Phe General formula (VIII-2) wherein a8' is an amino acid with a positively charged side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (3) A polypeptide having at least 70% homology with the polypeptide represented by the general formula (III-1) or the general formula (III-2) and having the same function.

50. The Apelin-13 polypeptide mutant according to claim 49, wherein the a8 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro.

51. The Apelin-13 polypeptide mutant according to claim 49 or 50, wherein b8 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

52. The Apelin-13 polypeptide mutant according to any one of claims 49 to 51, wherein the c8 is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg and Lys.

53. The Apelin-13 polypeptide mutant according to any one of claims 49 to 52, wherein the d8 is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Pro.

54. The Apelin-13 polypeptide mutant according to any one of claims 49 to 53, wherein the e8 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr and Trp.

55. The Apelin-13 polypeptide mutant according to any one of claims 49 to 54, wherein a8' is selected from Arg, Lys, XXXX.

56. The Apelin-13 polypeptide mutant according to any one of claims 49 to 55, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

57. The Apelin-13 polypeptide mutant according to any one of claims 49 to 56, which consists of the polypeptide represented by general formula (VIII-1) or the polypeptide represented by general formula (VIII-2).

58. The Apelin-13 polypeptide mutant according to any one of claims 49 to 57, wherein the polypeptide represented by general formula (VIII-1) is selected from: Cys-Arg-Pro-Arg-Leu-Cys-His-Ala-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr-Phe (Disulfidebridge:1-6); Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Cys-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Thr-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6); Ac-Lys-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 2-7); Ac-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 2-7); Ac-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 4-9); Ac-Lys-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 4-9); Ac-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8); Ac-Lys-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 3-8); Ac-Arg-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10); Ac-Lys-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10); and Ac-Leu-Arg-Gly-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 5-10).

59. The Apelin-13 polypeptide mutant according to any one of claims 49 to 57, wherein the polypeptide represented by general formula (VIII-2) is: Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe (Disulfide bridge: 1-6).

60. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (IX): Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe General formula (IX) in, a9 is an amino acid with a polar, uncharged side chain; or (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (IX) and having the same function.

61. The Apelin-13 polypeptide mutant according to claim 60, wherein said a9 is selected from the group consisting of Ser, Thr, Asn and Gln.

62. The Apelin-13 polypeptide mutant according to claim 60 or 61, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

63. The Apelin-13 polypeptide mutant according to any one of claims 60 to 62, which consists of a polypeptide represented by general formula (IX).

64. The Apelin-13 polypeptide mutant according to any one of claims 60 to 63, wherein the polypeptide represented by general formula (II) is: Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6).

65. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (XI-1): Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe General formula (XI-1) in, a11 is selected from Ala, Phe, Nal, DNal, DMet, Glu or Nle, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; (2) A polypeptide represented by the general formula (XI-2): Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11'-b11'-Pro-Phe General formula (XI-2) wherein a11' is selected from an amino acid with a hydrophobic side chain or an amino acid with a polar uncharged side chain, His or Pro, b11' is selected from an amino acid with a hydrophobic side chain or Gly, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (3) A polypeptide having at least 70% homology with the polypeptide represented by the general formula (XI-1) or the general formula (XI-2) and having the same function.

66. The Apelin-13 polypeptide mutant according to claim 65, wherein the a11' is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, His and Pro.

67. The Apelin-13 polypeptide mutant according to claim 65 or 66, wherein the b11' is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp and Gly.

68. The Apelin-13 polypeptide mutant according to any one of claims 65 to 67, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

69. The Apelin-13 polypeptide mutant according to any one of claims 65 to 68, which consists of a polypeptide represented by general formula (XI-1) or a polypeptide represented by general formula (XI-2).

70. The Apelin-13 polypeptide mutant according to any one of claims 65 to 69, wherein the polypeptide represented by general formula (XI-1) is selected from: Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Ala-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Phe-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DNal-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Glu-Pro-Phe(Disulfide bridge:1-6); and Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nle-Pro-Phe (Disulfide bridge: 1-6).

71. The Apelin-13 polypeptide mutant according to any one of claims 65 to 69, wherein the polypeptide represented by general formula (XI-2) is selected from: Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Met-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DAla-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DLeu-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DIle-Gly-Pro-Phe (Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DVal-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DGlu-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DTyr-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DHis-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DPhe-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DTrp-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DPro-Gly-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DGln-Gly-Pro-Phe(Disulfide bridge:1-6); and Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DAsn-Gly-Pro-Phe(Disulfide bridge:1-6).

72. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (XII): Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe General formula (XII) in, a12 is selected from an amino acid with a positively charged side chain, an amino acid with a hydrophobic side chain, an amino acid with a polar uncharged side chain, or an amino acid with a negatively charged side chain, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) having at least 70% homology with the polypeptide represented by general formula (XII) and having the same function Energy polypeptide.

73. The Apelin-13 polypeptide mutant of claim 72, wherein the a12 is selected from Arg, Lys, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp and Glu.

74. The Apelin-13 polypeptide mutant according to claim 72 or 73, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

75. The Apelin-13 polypeptide mutant according to any one of claims 72 to 74, which consists of a polypeptide represented by general formula (XII).

76. The Apelin-13 polypeptide mutant according to any one of claims 72 to 75, wherein the polypeptide represented by general formula (XII) is selected from: Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Phe-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Ser-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Asp-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Trp-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DArg-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DPhe-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DLeu-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DTrp-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DMet-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DGln-Pro-Phe(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DTyr-Pro-Phe(Disulfide bridge:1-6); and Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-DSer-Pro-Phe (Disulfide bridge: 1-6).

77. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (XIII): Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe General formula (XIII) in, a12 is selected from an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, or His, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) A polypeptide having at least 70% homology with the polypeptide represented by the general formula (XIII) and having the same function.

78. The Apelin-13 polypeptide mutant of claim 77, wherein the a13 is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp, Glu and His.

79. The Apelin-13 polypeptide mutant according to claim 77 or 78, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

80. The Apelin-13 polypeptide mutant according to any one of claims 77 to 79, which consists of a polypeptide represented by general formula (XIII).

81. The Apelin-13 polypeptide mutant according to any one of claims 77 to 80, wherein the polypeptide represented by general formula (XIII) is selected from: Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Ser-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DTyr-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DHis-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DPhe-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DTrp-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DLeu-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DAla-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DSer-Phe (Disulfide bridge: 1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DGln-Phe (Disulfide bridge: 1-6); and Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-DGlu-Phe (Disulfide bridge: 1-6).

82. Apelin-13 polypeptide mutant, which comprises (1) a polypeptide represented by the general formula (XIV): Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14 General formula (XIV) Wherein, a14 is selected from an amino acid having a hydrophobic side chain, an amino acid having a polar uncharged side chain, an amino acid having a negatively charged side chain, His or NaI, and the cysteine ​​residues at positions 1 and 6 form a disulfide bond; or (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XIV) and having the same function.

83. The Apelin-13 polypeptide mutant of claim 82, wherein a14 is selected from the group consisting of Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp, Glu, His and Nal.

84. The Apelin-13 polypeptide mutant according to claim 82 or 83, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

85. The Apelin-13 polypeptide mutant according to any one of claims 82 to 84, which consists of a polypeptide represented by general formula (XIV).

86. The Apelin-13 polypeptide mutant according to any one of claims 82 to 85, wherein the polypeptide represented by general formula (XIV) is selected from: Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Ala(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Nal(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr(Disulfidebridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DHis(Disulfidebridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DAsn(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Dglu(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Ile(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Pro(Disulfide bridge:1-6); Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Val(Disulfide bridge:1-6); and Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Trp (Disulfidebridge: 1-6).

87. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (XV): Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe General formula (XV) in, a15 and b15 are each independently selected from an amino acid with a positively charged side chain or Pro, c15 is selected from an amino acid with a hydrophobic side chain or an amino acid with a positively charged side chain, and the cysteine ​​residues at positions 1 and 5 form a disulfide bond; or (2) A polypeptide having at least 70% homology with the polypeptide represented by general formula (XV) and having the same function.

88. The Apelin-13 polypeptide mutant according to claim 87, wherein a15 and b15 are each independently selected from Arg, Lys and Pro.

89. The Apelin-13 polypeptide mutant of claim 87 or 88, wherein c15 is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg and Lys.

90. The Apelin-13 polypeptide mutant according to any one of claims 87 to 89, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

91. The Apelin-13 polypeptide mutant according to any one of claims 87 to 90, which consists of a polypeptide represented by general formula (XV).

92. The Apelin-13 polypeptide mutant according to any one of claims 87 to 91, wherein the polypeptide represented by general formula (XV) is selected from: Cys-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-5); Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-5); Cys-Arg-Pro-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-5); and Cys-Arg-Pro-Arg-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-5).

93. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (XVI): Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe General formula (XVI) in, a16 is selected from an amino acid with a positive side chain or Pro, c15 is selected from an amino acid with a hydrophobic side chain, an amino acid with a positive side chain or Pro, and the cysteine ​​residues at positions 1 and 4 form a disulfide bond; or (2) A polypeptide having at least 70% homology with the polypeptide represented by the general formula (XVI) and having the same function.

94. The Apelin-13 polypeptide mutant according to claim 93, wherein the a16 is selected from the group consisting of Arg, Lys and Pro.

95. The Apelin-13 polypeptide mutant of claim 93 or 94, wherein b16 is selected from Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, Lys and Pro.

96. The Apelin-13 polypeptide mutant according to any one of claims 93 to 95, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

97. The Apelin-13 polypeptide mutant according to any one of claims 93 to 96, which consists of a polypeptide represented by general formula (XVI).

98. The Apelin-13 polypeptide mutant according to any one of claims 93 to 97, wherein the polypeptide represented by general formula (XVI) is selected from: Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-4); Cys-Pro-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-4); Cys-Arg-Pro-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-4); and Cys-Pro-Arg-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-4).

99. Apelin-13 polypeptide mutant, comprising (1) A polypeptide represented by the general formula (XVII): *-Arg-a17-b17-Leu-*-His-C17-Gly-Pro-DMet-Gly-Pro-Phe General formula (XVII) in, a17 and b17 are each independently selected from amino acids with positively charged side chains, or a17 and / or b17 are absent, c17 is an amino acid with a positively charged side chain, * is a non-natural amino acid, and * forms a cyclic structure; or (2) A polypeptide having at least 70% homology with the polypeptide represented by the general formula (XVII) and having the same function.

100. The Apelin-13 polypeptide mutant according to claim 99, wherein a17 and b17 are each independently selected from Arg, Lys and Pro.

101. The Apelin-13 polypeptide mutant according to claim 99, wherein both a17 and b17 are absent.

102. The Apelin-13 polypeptide mutant according to any one of claims 99 to 101, wherein b16 is selected from Arg and Lys.

103. The Apelin-13 polypeptide mutant according to any one of claims 99 to 102, wherein 104. The Apelin-13 polypeptide mutant according to any one of claims 99 to 103, wherein the ring structure formed between * is a staple structure or a structure containing triazole.

105. The Apelin-13 polypeptide mutant according to any one of claims 99 to 104, wherein the ring structure formed between * is 106. The Apelin-13 polypeptide mutant according to any one of claims 99 to 105, wherein the Apelin-13 polypeptide mutant is identical to M183 ECL2 、F110 3.33 、I109 3.32 、K268 6.55 , Y264 6.51 、K265 6.55 、Y35 1.39 、T295 7.39 、Y299 7.49 、F78 2.53 、W85 2.60 、Y88 2.63 、Y93 ECL1 、M113 3.36 、R168 4.64 、Y185 ECL2 、E198 5.35 、Y271 6.58 、S275 6.62 、M288 7.32 、F291 7.35 、P163 4.59 、L201 5.38 、V164 4.60 、S205 5.42 、P292 7.36 、M288 7.32 、Q180 ECL2 or Y182 ECL2 Combine.

107. The Apelin-13 polypeptide mutant according to any one of claims 99 to 106, which consists of a polypeptide represented by general formula (XVII).

108. The Apelin-13 polypeptide mutant according to any one of claims 99 to 107, wherein the polypeptide represented by general formula (XVII) is selected from: as well as 109. A pharmaceutical composition comprising the Apelin-13 polypeptide mutant according to any one of claims 1 to 108, and a pharmaceutically acceptable carrier, diluent or excipient.

110. A method for treating or preventing a disease or condition responsive to agonism of an APLNR receptor, comprising administering to an individual in need thereof a therapeutically or prophylactically effective amount of the Apelin-13 polypeptide mutant of any one of claims 1 to 108 or the pharmaceutical composition of claim 109.

111. The method of claim 110, wherein the disease or condition is selected from the group consisting of acute decompensated heart failure, acute heart failure, chronic heart failure, pulmonary hypertension, atrial fibrillation, kidney disease, hypertrophy, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes, obesity, peripheral arterial disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burns, preeclampsia, anti-aging, analgesia, sleep apnea syndrome, pulmonary embolism, asthma, stress ulcer, liver fibrosis, lung injury, brain injury, ischemic stroke, cancer, Alzheimer's disease, Parkinson's disease, cerebral infarction, renal hypertension and neurodegenerative diseases.

112. The method of claim 110 or 111, wherein the individual is a mammal, preferably a human.

113. G protein-biased apelin-13 peptide mutants, in, The amino acid sequence of MM07 is CRPRLCHKGPMPF, which is obtained by further mutation based on the G protein-biased agonist MM07. The cysteine ​​residues at positions 1 and 6 form a disulfide bond. The mutant is any of the following: (1) Mutate M11 to D-amino acid based on MM07; (2) Mutate M11 to any of the following based on MM07: A, G, Nal, V, P; (3) Insert A or G between P10 and M11 based on MM07; (4) Based on MM07, P3 was mutated to W, L5 was mutated to F, and M11 was mutated to Nal; (5) Based on MM07, P3 was mutated to W, L5 was mutated to F, and G was inserted between P10 and M11.

114. The G protein-biased apelin-13 polypeptide mutant according to claim 113, in, The mutant is obtained by further mutation on the basis of mutant MM07, when M11 is mutated to D-type amino acid on the basis of MM07, specifically any one of the following: D-type M, D-type Nal.

115. The G protein-biased apelin-13 polypeptide mutant of claim 114, in, The mutant is obtained by further mutation based on mutant MM07, in which M11 is mutated to D-type amino acid based on MM07, and an amino acid is inserted between amino acids at positions 11 and 12, and the inserted amino acid is specifically any one of the following: G, A.

116. The G protein-biased apelin-13 polypeptide mutant of claim 115, in, The mutant is obtained by further mutation based on mutant MM07. The mutant is any of the following: (1) WN561: Based on MM07, M11 was mutated to D-type M, and G was inserted between D-type M11 and P12. The sequence is: CRPRLCHKGPMDGPF, where MD represents D-type M, and a disulfide bond is formed between the cysteine ​​residues at positions 1 and 6; (2) Based on MM07, M11 was mutated to D-type M, and A was inserted between D-type M11 and P12.

117. The G protein-biased apelin-13 polypeptide mutant according to claim 116, in, The mutant WN561 is further mutated and is any of the following: (1) Mutation of R2 to A, S, K, C, or T based on WN561; (2) P3 was mutated to Y, S, T, or C based on WN561; (3) Mutation of R4 to A or K based on WN561; (4) L5 was mutated to A, I, or V based on WN561; (5) Mutation of H7 to A, S, R, K, T, C, or G based on WN561; (6) Mutation of K8 to A or R based on WN561; (7) Mutation of G9 to S, C, or T based on WN561; (8) Mutation of G12 to F, S, W, C, or Y based on WN561; (9) Mutate F14 to L, I, P, V or W based on WN561.

118. Use of the G protein-biased apelin-13 polypeptide mutant or a pharmaceutically acceptable salt thereof according to any one of claims 113 to 117 in the preparation of a medicament for treating or preventing a disease or condition responsive to agonism of an APLNR (APJ) receptor.

119. The use according to claim 118, in, The disease or condition is selected from the group consisting of acute decompensated heart failure, chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes, obesity, peripheral arterial disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burns, preeclampsia, cancer, Alzheimer's disease, Parkinson's disease, stroke, and neurodegenerative diseases.

120. A medicament for treating or preventing a disease or condition responsive to agonism of an APLNR receptor, in, The active substance is the G protein-biased apelin-13 polypeptide mutant or a pharmaceutically acceptable salt thereof as described in any one of claims 113 to 117.

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