WO2026007693A1 - Apelin-13多肽突变体 - Google Patents

Apelin-13多肽突变体

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Publication number
WO2026007693A1
WO2026007693A1 PCT/CN2025/101655 CN2025101655W WO2026007693A1 WO 2026007693 A1 WO2026007693 A1 WO 2026007693A1 CN 2025101655 W CN2025101655 W CN 2025101655W WO 2026007693 A1 WO2026007693 A1 WO 2026007693A1
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Prior art keywords
pro
arg
cys
gly
γglu
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PCT/CN2025/101655
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English (en)
French (fr)
Inventor
张岩
王伟伟
戢素玉
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Liangzhu Laboratory
Zhejiang University
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Liangzhu Laboratory
Zhejiang University
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Publication of WO2026007693A1 publication Critical patent/WO2026007693A1/zh
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • This disclosure generally pertains to the biomedical field, and more specifically, to the field of peptides.
  • Apelin receptor is a typical class A GPCR that plays important functions in the cardiovascular system, including promoting cardiac contraction, increasing left ventricular stroke volume, vasodilation, diuresis, and lowering systemic blood pressure.
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • general formula (I) aa1-Arg-Pro-Arg-Leu-b1-His-cc1-Gly-Pro-DMet-Gly-Pro-Phe
  • b1 is an amino acid with a negatively charged side chain
  • aa1 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG
  • cc1 is selected from C11-25 hydrocarbon acyl- ⁇ Glu-Lys, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-Lys, C11-25 hydrocarbon acyl-m ⁇ OEG- ⁇ Glu-Lys, carboxyl-C 10-24 hydrocarbon acyl-m ⁇ OEG- ⁇ Glu-Lys or PEG-Lys, and the amino acid residues at positions 1 and 6 form an amide bond, where m is an integer from 1 to
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • general formula (II) aa2-Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe
  • a2 is selected from amino acids with hydrophobic side chains or amino acids with polar, uncharged side chains, and aa2 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • a3 is selected from amino acids with hydrophobic side chains or amino acids with polar, uncharged side chains; b3 is an amino acid with hydrophobic side chains; c3 is an amino acid with positively charged side chains; d3 and e3 are each independently selected from amino acids with hydrophobic side chains or Gly; f3 is an amino acid with hydrophobic side chains or Pro; g3 is an amino acid with hydrophobic side chains; aa3 is selected from C11-25 alkylacyl- ⁇ Glu, carboxyl- C10-24 alkylacyl- ⁇ Glu, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form disulfide bonds, where m is an integer from 1 to 5
  • a3' is an amino acid with a hydrophobic side chain
  • aa3' is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a4 is an amino acid with a hydrophobic side chain, and aa4 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a5 is selected from an amino acid with a hydrophobic side chain or Pro
  • b5 is an amino acid with 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 with a hydrophobic side chain or Gly
  • f5 is an amino acid with a hydrophobic side chain or Pro
  • g5 is an amino acid with a hydrophobic side chain
  • aa5 is selected from C11-25 alkylacyl- ⁇ Glu, carboxyl- C10-24 alkylacyl- ⁇ Glu, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond,
  • a5' is selected from an amino acid with a hydrophobic side chain or Pro
  • b5' is an amino acid with a hydrophobic side chain
  • c5' is an amino acid with a positively charged side chain
  • d5' is an amino acid with a hydrophobic side chain
  • aa5' is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG
  • cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • a7 is selected from amino acids with hydrophobic side chains or amino acids with polar, uncharged side chains, and aa7 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a8 is selected from amino acids with hydrophobic side chains or Pro
  • b8 is an amino acid with hydrophobic side chains
  • c8 is selected from amino acids with hydrophobic side chains or amino acids with positively charged side chains
  • d8 is an amino acid with hydrophobic side chains or Pro
  • e8 is an amino acid with hydrophobic side chains
  • aa8 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, n is an integer from 1 to 8, or
  • a8' is an amino acid with a positively charged side chain, and aa8' is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a9 is an amino acid with a polar, uncharged side chain, and aa9 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • polypeptide represented by general formula (XI-1) aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe
  • XI-1 aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe
  • a11 is selected from Ala, Phe, Nal, DNal, DMet, Glu or Nle, and aa11 is selected from C 11-25 hydrocarbon acyl- ⁇ Glu, carboxyl-C 10-24 hydrocarbon acyl- ⁇ Glu, C 11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl-C 10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C 11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl-C 10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8;
  • a11' is selected from amino acids with hydrophobic side chains or amino acids with polar, uncharged side chains, His, or Pro; b11' is selected from amino acids with hydrophobic side chains or Gly; aa11' is selected from C11-25 alkylacyl- ⁇ Glu, carboxyl- C10-24 alkylacyl- ⁇ Glu, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a12 is selected from amino acids with positively charged side chains, amino acids with hydrophobic side chains, amino acids with polar and uncharged side chains, or amino acids with negatively charged side chains; aa12 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a14 is selected from amino acids with hydrophobic side chains, amino acids with polar, uncharged side chains, amino acids with negatively charged side chains, His, or Nal; aa14 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a15 and b15 are each independently selected from amino acids with positively charged side chains or Pro
  • c15 is selected from amino acids with hydrophobic side chains or amino acids with positively charged side chains
  • aa15 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG or PEG, and the cysteine residues at positions 1 and 5 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a16 is selected from amino acids with positively charged side chains or Pro
  • b16 is selected from amino acids with hydrophobic side chains, amino acids with positively charged side chains or Pro
  • aa16 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG or PEG, and the cysteine residues at positions 1 and 4 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • a17 and b17 are each independently selected from proline or arginine, or a17 and/or b17 are absent; c17 is an amino acid with a positively charged side chain; * represents a non-natural amino acid; and * forms a cyclic structure between the amino acids.
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a18 is selected from:
  • b18 is selected from amino acids with hydrophobic side chains or amino acids with negatively charged side chains, and the cysteine residues at positions 1 and 6 form a disulfide bond; or
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a19 is selected from:
  • b19 is selected from amino acids with hydrophobic side chains or amino acids with negatively charged side chains, c19 is selected from amino acids with hydrophobic side chains, and the cysteine residues at positions 1 and 6 form a disulfide bond; or
  • compositions comprising the Apelin-13 polypeptide mutant described herein, and pharmaceutically acceptable carriers, diluents, or excipients.
  • the pharmaceutical composition described in this disclosure further comprises at least one other active ingredient.
  • exemplary examples of at least one other active ingredient that can be used in the pharmaceutical compositions of this disclosure include, but are not limited to, active ingredients that interact with GLP1R, GLP2R, GIPR, GCGR, FGF21R, NPY2R, or GDF15.
  • exemplary examples of at least one other active ingredient that can be used in the pharmaceutical compositions of this disclosure include, but are not limited to, Vurolenatide, exenatide, ROSE-010, GX-G6, orforglipron, FNP-120, UTMD-GLP-1, GLP-1mimetics, VTC-G15, curaglutide, exendin-4, P-11, CTRS-101, HD-7671, HTL-097, PGN-OB2, GL-0034, and others.
  • this disclosure relates to a method for treating or preventing a disease or disease state that responds to an agonistic response to the APLNR receptor, comprising administering to an individual in need of the method a therapeutically or preventively effective amount of the Apelin-13 polypeptide mutant or the pharmaceutical composition described in this disclosure.
  • this disclosure relates to the Apelin-13 polypeptide mutant described herein for the treatment or prevention of diseases or disease states that respond to an agonistic response to the APLNR receptor.
  • this disclosure relates to the use of the Apelin-13 peptide mutant described herein in the preparation of a medicament for treating or preventing diseases or disease states that respond to an agonistic response to the APLNR receptor.
  • FIG. 1 shows the structural diagram of the apelin-APLNR-Gi1 complex and the results of the interaction analysis between the ligand and the receptor.
  • 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 peptide ligands apelin and MM07
  • C dose-dependent curves of apelin and MM07 inducing Gi protein dissociation (left) and ⁇ -arrestin recruitment (right).
  • FIG. 2 shows the results of the APLNR bias signal transduction mechanism analysis.
  • A density map of the interaction between apelin and key amino acid residues in APLNR that affect bias
  • B density map of the interaction between MM07 and key amino acid residues in APLNR that affect bias
  • C-F detection of G protein activation and ⁇ -arrestin recruitment by key amino acid mutations interacting with M11 and F13
  • G interaction pattern of "twin hotspots" on the receptor with peptide agonists M11 and F13.
  • Figure 3 shows the SDS-page characterization results of the fusion protein of this disclosure.
  • Figure 4 shows the effects of different drug administration groups on body weight and food intake of DIO mice.
  • A weight loss during drug administration
  • B weight loss as a percentage of original body weight
  • C Q2D food intake during drug administration
  • D cumulative food intake.
  • Figure 5 shows the effects of different drug administration groups on changes in muscle content in DIO mice.
  • A changes in muscle content during the administration period in different drug administration groups
  • B changes in muscle content as a percentage of initial muscle content
  • C statistical analysis of significant differences in muscle content changes.
  • FIG. 6 shows the effects of different drug administration groups on changes in fat content in DIO mice.
  • A represents changes in fat content during the drug administration period in different drug administration groups
  • B represents changes in the proportion of fat content changes to initial fat content
  • C represents statistical analysis of significant differences in fat content changes.
  • FIG. 7 shows the effects of different drug administration groups on changes in fat mass in DIO mice.
  • A changes in fat mass during the administration period in different drug administration groups
  • B changes in the proportion of fat mass change to initial fat mass
  • C statistical analysis of significant differences in fat mass change.
  • FIG 8 shows the effects of different drug administration groups on muscle mass changes in DIO mice.
  • A represents the changes in muscle mass during the drug administration period in different drug administration groups
  • B represents the changes in the proportion of muscle mass change to initial fat mass.
  • FIG. 9 shows the effects of different drug administration groups on changes in muscle/fat mass in DIO mice.
  • A represents changes in muscle/fat mass during drug administration in different drug administration groups
  • B represents statistical analysis of the significance of changes in muscle/fat mass.
  • Figure 10 shows the effect of sequence 216 on the changes in muscle and adipose tissue weight after dissection in DIO mice.
  • A significance analysis of muscle tissue weight (g) after dissection
  • B significance analysis of adipose tissue weight (g) after dissection
  • C significance analysis of changes in muscle tissue mass/adipose tissue mass after dissection.
  • Figure 11 shows the effect of sequence 216 on body weight and food intake in DIO mice, where A: weight loss (g) and percentage (%) during administration, and B: cumulative food intake during administration.
  • Figure 12 shows the effect of sequence 216 on the weight of different adipose tissues in DIO mice, where A: perirenal white adipose tissue rpWAT after 28 days of drug administration, B: epididymal white adipose tissue eWAT, C: inguinal white adipose tissue iWAT, and D: brown adipose tissue BAT.
  • Figure 13 shows the TAC mouse model and the administration regimen.
  • Figure 14 shows the immunofluorescence of cardiomyocytes.
  • Figure 15 shows the echocardiogram results of the TAC model.
  • Figure 16 shows the results of the cardiomyocyte hypertrophy detection.
  • Figure 17 shows the results of the cardiac hypertrophy test.
  • Figure 18 shows the results of the detection of cardiac hypertrophy-related factors.
  • Figure 19 shows the results of myocardial fibrosis detection.
  • an embodiment refers to including, in at least one embodiment, a specific reference element, structure, or feature related to that embodiment. Therefore, the phrases “an embodiment,” “an embodiment,” or “another embodiment” appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.
  • C1-4 alkyl describes alkyl groups having a total of 1 to 4 carbon atoms as defined below
  • C3 - C10 cycloalkyl describes cycloalkyl groups having a total of 3 to 10 carbon atoms as defined below.
  • the total number of carbons in the abbreviations does not include carbons that may be present in substituents of the group.
  • alkyl group refers to an aliphatic hydrocarbon group.
  • the alkyl group can be a "saturated alkyl group,” meaning it does not contain any alkene or alkyne portion, i.e., an alkyl group.
  • the alkyl group can also be an "unsaturated alkyl group,” meaning it contains at least one alkene or alkyne portion.
  • alkene portion i.e., an alkenyl group
  • alkenyl group refers to a straight-chain or branched hydrocarbon chain group consisting of 12 to 24 carbon atoms and at least one carbon-carbon double bond, connected to the rest of the molecule by single bonds, such as vinyl, propenyl, butenyl, pentenyl, pent-1,4-dienyl, etc.
  • alkynyl portion refers to a straight-chain or branched hydrocarbon chain group consisting of 12 to 24 carbon atoms and at least one carbon-carbon triple bond, connected to the rest of the molecule by single bonds.
  • the alkyl group whether saturated or unsaturated, can be branched or straight-chain.
  • the hydrocarbon group may have 12 to 24 carbon atoms (whenever it appears in this disclosure, a numerical range such as “12 to 14” refers to every integer in the given range; such as “12 to 24” means that the hydrocarbon group may have 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, etc. up to and including 24 carbon atoms, although this definition also covers the occurrence of the term “hydrocarbon” where no numerical range is specified).
  • C 11-25 hydrocarbon acyl- ⁇ Glu refers to a group having the following structure:
  • carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu refers to a group having the following structure:
  • C 11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG refers to a group having the following structure:
  • carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG refers to a group having the following structure:
  • C 11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG refers to a group having the following structure:
  • carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG refers to a group having the following structure:
  • C 11-25 hydrocarbon acyl- ⁇ Glu-Lys refers to a group having the following structure:
  • carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-Lys refers to a group having the following structure:
  • PEG refers to a group having the following structure:
  • wild-type Apelin-13 polypeptide refers to a polypeptide having the following amino acid sequence: Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe, which is represented by the single letter QRPRLSHKGPMPF.
  • cyste residues at positions 1 and 6 forming disulfide bonds refers to the formation of disulfide bonds at positions 1 and 6 of the cysteine residues in the corresponding wild-type Apelin-13 polypeptide of the Apelin-13 polypeptide mutant of this disclosure.
  • Disulfide bridge:a-b refers to the formation of disulfide bonds at the a and b positions of the corresponding wild-type Apelin-13 polypeptide in the mutant of this disclosure.
  • Disulfide bridge:1-6 refers to the formation of a disulfide bond at positions 1 and 6 in the Apelin-13 polypeptide mutant of this disclosure, corresponding to the wild-type Apelin-13 polypeptide.
  • drug-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 that has been approved by the U.S. Food and Drug Administration for use in humans or animals and has no adverse effects on the composition of the pharmaceutical composition.
  • the term "therapeuticly effective amount” refers to the amount of a compound or combination of compounds that improves, reduces, or eliminates a particular disease or condition and its symptoms, or avoids or delays the onset of a particular disease or condition or its symptoms.
  • the amount of the compound constituting a "therapeuticly effective amount” described in this disclosure will vary depending on the compound, the disease state and its severity, and the age, weight, etc., of the mammal to be treated; however, those skilled in the art can conventionally determine the amount of the compound described in this disclosure based on their own knowledge and this disclosure.
  • to treat encompasses the treatment of a related disease or condition in mammals, such as humans, suffering from the related disease or ailment, and includes:
  • disease and “disease state” may be used interchangeably or may be different, because a particular disease or disease state may not have a known causative agent (and therefore cannot be explained by etiology), and thus is not recognized as a disease, but rather as an undesirable disease state or symptom in which a clinician has identified a more or less specific set of symptoms.
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • general formula (I) aa1-Arg-Pro-Arg-Leu-b1-His-cc1-Gly-Pro-DMet-Gly-Pro-Phe
  • b1 is an amino acid with a negatively charged side chain
  • aa1 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG or PEG
  • cc1 is selected from C11-25 hydrocarbon acyl- ⁇ Glu-Lys, C11-25 hydrocarbon acyl- ⁇ Glu-Lys, C11-25 hydrocarbon acyl-m ⁇ OEG- ⁇ Glu-Lys, C 11-25 alkyl acyl-m ⁇ OEG- ⁇ Glu-Lys or PEG-Lys, and the amino acid residues at positions 1 and 6 form an amide bond, where m is an integer from 1 to 5 and n is an integer from
  • exemplary instances of a1 that can be used in this 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.
  • exemplary examples that can be used for b1 of this disclosure include, but are not limited to, Glu and Asp.
  • exemplary examples that can be used for aa1 in this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • exemplary examples that can be used for cc1 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (I).
  • the Apelin-13 peptide mutant of this disclosure is:
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • general formula (II) aa2-Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe
  • a2 is selected from amino acids with hydrophobic side chains or amino acids with polar, uncharged side chains, and aa2 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • exemplary instances that can be used for a2 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gln.
  • exemplary examples that can be used for aa2 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (II).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • COOHC 22Alkyl CO- ⁇ Glu-2 ⁇ OEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • exemplary instances that can be used for b3 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary instances that can be used for c3 of this disclosure include, but are not limited to, Lys and Arg.
  • exemplary instances that can be used for d3 and e3 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, DMet, Phe, Tyr, Trp, and Gly.
  • exemplary instances that can be used with g3 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary examples that can be used for aa3 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by formula (III-1) or a polypeptide represented by formula (III-2).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a4 is an amino acid with a hydrophobic side chain
  • aa3 is selected from C11-25 alkylacyl- ⁇ Glu, carboxyl- C10-24 alkylacyl- ⁇ Glu, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 acyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-n ⁇ PEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • exemplary instances that can be used for a4 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary examples that can be used for aa4 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , 180 ECL2 or 182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (IV).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a5 is selected from an amino acid with a hydrophobic side chain or Pro
  • b5 is an amino acid with 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 with a hydrophobic side chain or Gly
  • f5 is an amino acid with a hydrophobic side chain or Pro
  • g5 is an amino acid with a hydrophobic side chain
  • aa1 is selected from C11-25 alkylacyl- ⁇ Glu, carboxyl- C10-24 alkylacyl- ⁇ Glu, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond,
  • a5' is selected from an amino acid with a hydrophobic side chain or Pro
  • b5' is an amino acid with a hydrophobic side chain
  • c5' is an amino acid with a positively charged side chain
  • d5' is an amino acid with a hydrophobic side chain
  • aa5' is selected from C11-25 alkylacyl- ⁇ Glu, carboxyl- C10-24 alkylacyl- ⁇ Glu, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-n ⁇ PEG, or PEG
  • cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • exemplary instances that can be used with a5 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.
  • exemplary instances that can be used for b5 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary instances that can be used for c5 of this disclosure include, but are not limited to, Lys and Arg.
  • exemplary instances that can be used for d5 and e5 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, DMet, Phe, Tyr, Trp, and Gly.
  • exemplary instances that can be used with f5 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.
  • exemplary instances that can be used with g5 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary examples that can be used for aa5 of this disclosure include, but are not limited to, C11-25 hydrocarbon- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • exemplary instances that can be used for a5’ of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.
  • exemplary instances that can be used for b5’ of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary instances that can be used for c5’ of this disclosure include, but are not limited to, Lys and Arg.
  • exemplary instances that can be used for d5’ of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary examples that can be used for aa5' of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (V-1) or a polypeptide represented by general formula (V-2).
  • the Apelin-13 peptide mutants disclosed herein are selected from:
  • COOHC 18Alkyl CO- ⁇ Glu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • a7 is selected from amino acids with hydrophobic side chains or amino acids with polar, uncharged side chains, and aa7 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • exemplary instances that can be used for a7 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, and Gln.
  • exemplary examples that can be used for aa7 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (VII).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • COOHC 18alkyl COOHC 18alkyl -CO- ⁇ Glu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);
  • COOHC 22alkyl COOHC 22alkyl -CO- ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a8 is selected from amino acids with hydrophobic side chains or Pro
  • b8 is an amino acid with hydrophobic side chains
  • c8 is selected from amino acids with hydrophobic side chains or amino acids with positively charged side chains
  • d8 is an amino acid with hydrophobic side chains or Pro
  • e8 is an amino acid with hydrophobic side chains
  • aa8 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8; or
  • a8' is an amino acid with a positively charged side chain, and aa8' is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • exemplary instances that can be used with a8 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.
  • exemplary instances that can be used for b8 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary instances that can be used for c8 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, and Lys.
  • exemplary instances that can be used for d8 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Pro.
  • exemplary instances that can be used with e8 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary examples that can be used for aa8 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • exemplary instances of a8’ that can be used in this disclosure include, but are not limited to, Arg and Lys.
  • exemplary examples that can be used for aa8' of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (VIII-1) or a polypeptide represented by general formula (VIII-2).
  • the Apelin-13 peptide mutants disclosed herein are selected from:
  • COOHC 18Alkyl CO- ⁇ Glu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 12Alkyl CO- ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 14Alkyl CO- ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 18Alkyl CO- ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 22Alkyl CO- ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 18Alkyl CO- ⁇ Glu-4 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 18Alkyl CO-D ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 18alkyl COOHC 18alkyl -CO- ⁇ Glu-8PEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 18alkyl COOHC 18alkyl -CO-2 ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);
  • COOHC 18alkyl COOHC 18alkyl -CO-3 ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);
  • COOHC 18Alkyl CO-Abu- ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 18alkyl COOHC 18alkyl -CO-Abu-2 ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);
  • COOHC 18alkyl COOHC 18alkyl -CO-Abu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);
  • COOHC 18Alkyl CO- ⁇ Glu-5 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • COOHC 18alkyl COOHC 18alkyl -CO- ⁇ Glu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);
  • COOHC 18alkyl COOHC 18alkyl -CO- ⁇ Glu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH 2 (Disulfide bridge: 1-6);
  • COOHC 22alkyl COOHC 22alkyl -CO- ⁇ Glu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH 2 (Disulfide bridge: 1-6);
  • COOHC 16alkyl COOHC 16alkyl -CO- ⁇ Glu-2 ⁇ OEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH 2 (Disulfide bridge:1-6);
  • COOHC 18alkyl COOHC 18alkyl -CO- ⁇ Glu-2 ⁇ OEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH 2 (Disulfide bridge:1-6);
  • COOHC 22alkyl COOHC 22alkyl -CO- ⁇ Glu-2 ⁇ OEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH 2 (Disulfide bridge:1-6);
  • COOHC 22alkyl COOHC 22alkyl -CO- ⁇ Glu-2 ⁇ OEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH 2 (Disulfide bridge:1-6).
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a9 is an amino acid with a polar, uncharged side chain, and aa9 is selected from C11-25 alkylacyl- ⁇ Glu, carboxyl- C10-24 alkylacyl- ⁇ Glu, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 acyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • exemplary instances that can be used with a9 in this disclosure include, but are not limited to, Ser, Thr, Asn, and Gln.
  • exemplary examples that can be used for aa9 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (IX).
  • the Apelin-13 peptide mutant of this disclosure is:
  • COOHC 18alkyl COOHC 18alkyl -CO- ⁇ Glu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe (Disulfide bridge: 1-6);
  • COOHC 22alkyl COOHC 22alkyl -CO- ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6);
  • polypeptide represented by general formula (XI-1) aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe
  • XI-1 aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe
  • a11 is selected from Ala, Phe, Nal, DNal, DMet, Glu or Nle, and aa11 is selected from C 11-25 hydrocarbon acyl- ⁇ Glu, carboxyl-C 10-24 hydrocarbon acyl- ⁇ Glu, C 11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C 11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C 11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl-C 10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5 and n is an integer from 1 to 8;
  • a11' is selected from amino acids with hydrophobic side chains or amino acids with polar, uncharged side chains, His, or Pro; b11' is selected from amino acids with hydrophobic side chains or Gly; aa11' is selected from C11-25 alkylacyl- ⁇ Glu, carboxyl- C10-24 alkylacyl- ⁇ Glu, C11-25 alkylacyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-m ⁇ OEG, C11-25 alkylacyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 alkylacyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • exemplary instances that can be used for a11’ of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, His, and Pro.
  • exemplary instances that can be used for b11’ of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, and Gly.
  • exemplary examples that can be used for aa11 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • exemplary examples that can be used for aa11' of this disclosure include, but are not limited to, C 11-25 hydrocarbon acyl- ⁇ Glu, carboxyl-C 10-24 hydrocarbon acyl- ⁇ Glu, carboxyl-C 10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl-C 10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (XI-1) or a polypeptide represented by general formula (XI-2).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • COOHC 22Alkyl CO- ⁇ Glu-2 ⁇ OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6);
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a12 is selected from amino acids with positively charged side chains, amino acids with hydrophobic side chains, amino acids with polar and uncharged side chains, or amino acids with negatively charged side chains; aa12 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • exemplary instances that can be used for a12 in this disclosure include, but are not limited to, Arg, Lys, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp, and Glu.
  • exemplary examples that can be used for aa12 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (XII).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • COOHC 18alkyl COOHC 18alkyl -CO- ⁇ Glu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe (Disulfide bridge: 1-6);
  • COOHC 18Alkyl COOHC 18Alkyl -CO- ⁇ Glu- ⁇ Glu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH 2 (Disulfide bridge:4-9);
  • COOHC 18Alkyl COOHC 18Alkyl -CO- ⁇ Glu- ⁇ Glu-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH 2 (Disulfide bridge:5-10);
  • COOHC 18Alkyl COOHC 18Alkyl -CO- ⁇ Glu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Arg-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH 2 (Disulfide bridge: 7-12).
  • a13 is selected from amino acids with hydrophobic side chains, amino acids with polar and uncharged side chains, amino acids with negatively charged side chains, or His; aa13 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • exemplary instances that can be used for a13 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp, Glu, and His.
  • exemplary examples that can be used for aa13 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, and PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (XIII).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a14 is selected from amino acids with hydrophobic side chains, amino acids with polar, uncharged side chains, amino acids with negatively charged side chains, His, or Nal; aa14 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, C11-25 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-m ⁇ OEG, C11-25 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-n ⁇ PEG, or PEG, and the cysteine residues at positions 1 and 6 form a disulfide bond, where m is an integer from 1 to 5, and n is an integer from 1 to 8; or
  • exemplary instances that can be used for a14 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Asp, Glu, and His.
  • exemplary examples that can be used for aa14 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (XIV).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a15 and b15 are each independently selected from amino acids with positively charged side chains or Pro
  • c15 is selected from amino acids with hydrophobic side chains or amino acids with positively charged side chains
  • aa15 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG or PEG, and the cysteine residues at positions 1 and 5 form a disulfide bond; or
  • exemplary instances that can be used with the a15 of this disclosure include, but are not limited to, Arg, Lys, and Pro.
  • exemplary instances that can be used for b15 of this disclosure include, but are not limited to, Arg, Lys, and Pro.
  • exemplary instances that can be used for c15 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, and Lys.
  • exemplary examples that can be used for aa15 of this disclosure include, but are not limited to, C11-25 hydrocarbon CO- ⁇ Glu, COOHC10-24 hydrocarbon CO- ⁇ Glu, COOHC10-24 hydrocarbon CO- ⁇ Glu-2 ⁇ OEG, COOHC10-24 hydrocarbon CO- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by the general formula (XV).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a16 is selected from amino acids with positively charged side chains or Pro
  • b16 is selected from amino acids with hydrophobic side chains
  • aa16 is selected from C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG or PEG, and the cysteine residues at positions 1 and 4 form disulfide bonds; or
  • exemplary instances that can be used with the a16 of this disclosure include, but are not limited to, Arg, Lys, and Pro.
  • exemplary instances that can be used for b16 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Arg, Lys, and Pro.
  • exemplary examples that can be used for aa16 of this disclosure include, but are not limited to, C11-25 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-2 ⁇ OEG, carboxyl- C10-24 hydrocarbon acyl- ⁇ Glu-3 ⁇ OEG, or PEG.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by the general formula (XVI).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • 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 positively charged side chains; * is a non-natural amino acid; and * forms a cyclic structure; or
  • exemplary instances that can be used for a17 and b17 of this disclosure include, but are not limited to, Arg, Lys, and Pro.
  • exemplary instances that can be used for b16 in this disclosure include, but are not limited to, Arg and Lys.
  • exemplary examples that can be used in this disclosure include, but are not limited to:
  • exemplary examples of structures that can be used to form a ring structure between the elements of this disclosure include, but are not limited to, staple structures.
  • exemplary examples of structures that can be used to form a ring structure between the elements of this disclosure include, but are not limited to, those that can be used to form a ring structure between the elements of this disclosure.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by general formula (XVII).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a18 is selected from:
  • b18 is selected from amino acids with hydrophobic side chains or amino acids with negatively charged side chains, and the cysteine residues at positions 1 and 6 form a disulfide bond; or
  • exemplary instances that can be used for b18 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Asp, and Glu.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by the general formula (XVIII).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • SEQ ID NO:1 SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8.
  • this disclosure relates to Apelin-13 peptide mutants, which contain
  • a19 is selected from:
  • b19 is selected from amino acids with hydrophobic side chains or amino acids with negatively charged side chains, c19 is selected from amino acids with hydrophobic side chains, and the cysteine residues at positions 1 and 6 form a disulfide bond; or
  • exemplary instances that can be used for b19 in this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, and Trp.
  • exemplary instances that can be used for c19 of this disclosure include, but are not limited to, Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Asp, and Glu.
  • the Apelin-13 peptide mutant of this disclosure is compared with 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 , and P163 4.59.
  • L201 5.38 , V164 4.60 , S205 5.42 , P292 7.36 , M288 7.32 , Q180 ECL2 or Y182 ECL2 combined.
  • the Apelin-13 peptide mutant of this disclosure binds to M183 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 , thereby affecting G protein bias.
  • the Apelin-13 polypeptide mutant of this disclosure consists of a polypeptide represented by the general formula (XIX).
  • the Apelin-13 peptide mutant of this disclosure is selected from:
  • compositions comprising the Apelin-13 polypeptide mutant described herein, and pharmaceutically acceptable carriers, diluents, or excipients.
  • this disclosure relates to methods for treating or preventing diseases or disease states that respond to an agonistic response to the APLNR receptor, comprising administering to an individual in need of the method a therapeutically or preventively effective amount of the Apelin-13 polypeptide mutant or the pharmaceutical composition described in this disclosure.
  • exemplary examples of diseases or disease states that can be used with respect to this disclosure include, but are not limited to, maintaining muscle mass, fat loss, acute decompensated heart failure, acute heart failure, chronic heart failure, pulmonary hypertension, atrial fibrillation, kidney disease, muscle gain, Brugda syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular disease, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes, obesity, peripheral artery disease, cerebrovascular accident, transient ischemic attack, traumatic brain injury, amyotrophic lateral sclerosis, burns, preeclampsia, anti-aging, analgesia, sleep apnea syndrome, pulmonary embolism, asthma, stress-induced gastric ulcer, liver fibrosis, lung injury, brain injury, ischemic stroke, cancer, Alzheimer's disease, Parkinson's disease, stroke, renal hypertension, and neurodegenerative diseases.
  • exemplary examples of individuals that can be used for the purposes of this disclosure include, but are not limited to, mammals.
  • it can be used by individuals of the individuals disclosed herein.
  • this disclosure relates to the Apelin-13 polypeptide mutant described herein for the treatment or prevention of diseases or disease states that respond to an agonistic response to the APLNR receptor.
  • this disclosure relates to the use of the Apelin-13 peptide mutant described herein in the preparation of a medicament for treating or preventing diseases or disease states that respond to an agonistic response to the APLNR receptor.
  • the reagents and equipment used in the embodiments of this disclosure are all conventional and commercially available.
  • Polypeptide sequence If the sequence is ARSS
  • Synthesis sequence synthesized sequentially from the C-terminus to the N-terminus of the peptide chain.
  • Resin swelling Weigh the calculated amount of fmoc-Ser(tbu)-OH resin and pour it into the reaction column. Add DMF and soak for 30 minutes, then dry.
  • the solution in the reaction column was dried, an appropriate amount of DMF was added for washing, nitrogen was agitated for 2 minutes, and then the solution was dried again. This process was repeated 3 times.
  • Cutting Put the dried resin into a suitable round-bottom flask, add an appropriate amount of the prepared cutting solution (1g/10ml), and place it in a constant temperature shaker at 25°C for 2 hours.
  • Filtration Filter out the resin particles using a 50mL sintered glass funnel, then pour the filtrate into a glass container for centrifuge tubes, add 6-8 times the volume of anhydrous diethyl ether while stirring, and the precipitated white solid is the desired crude polypeptide.
  • Washing Dispense the white solid into centrifuge tubes, seal the tubes, and centrifuge at 4000 rpm for 3 minutes. Remove the tubes, discard the supernatant, add ether, stir well with a glass rod, and centrifuge again. Repeat this washing process 5 times.
  • the crude linear peptide was diluted with DMF to 10 ⁇ 3 mol/L, dissolved completely in an ice bath, and then 1.5 times the amount of HOAt, 1.5 times the amount of HATU, and 10 times the amount of DIEA were added. The mixture was stirred in the ice bath for 2 hours. The ice bath was then removed, and the mixture was stirred at room temperature. The solid residue was obtained by vacuum distillation.
  • the polypeptide after being washed five times, was placed in a vacuum desiccator and dried under vacuum for 24 hours.
  • the resulting white powder is the crude polypeptide, which was weighed and prepared for purification.
  • Solution A 80% phenol + 20% anhydrous ethanol
  • Select an ion source Choose an appropriate ion source based on the properties of the sample, such as an electrospray ionization source (ESI) or a chemical ion source (CI) for gas chromatography-mass spectrometry.
  • ESI electrospray ionization source
  • CI chemical ion source
  • Mass spectrometry software is used to analyze the mass spectrum and identify proton peaks or molecular ion peaks. The mass spectrometry data is then used to determine whether the sample contains the target peptide.
  • the purity of the polypeptide sample is determined by the proportion of the main peak to all peaks.
  • the general characterization method for fusion proteins uses SDS-PAGE electrophoresis to detect whether it is the target protein sequence and to determine the protein purity.
  • the target gene was constructed on the TAC expression vector, transformed into E. coli, amplified, and preserved in bacterial culture for subsequent expression and purification in E. coli.
  • Inoculation 1 tube of glycerol culture + 25 ml LB (1:1000A+), incubate overnight at 37°C on a shaker.
  • 5 mL of bacterial culture 500 mL LB (1:1000A+) transferred to a large bottle (the large bottle of LB is sterilized beforehand), incubate at 37°C on a shaker for 2-3 hours, measuring the OD value every hour.
  • the OD value is 0.6-0.8, place the culture in a shaker at 16°C and 220 rpm for pre-cooling.
  • Resuspend Resuspend the bacterial cells in lysis buffer at a ratio of 1:5-10, then pour into a beaker. Add lysozyme (final concentration 0.5 mg/mL), DNase (1:100), and 1 mM PMSF.
  • Lysis Cells are broken up using a high-pressure homogenizer until the solution is clear.
  • Washing Wash with washing buffer for about 20 CV.
  • Elution buffer is used to elute proteins, and the eluent is collected.
  • Concentrated protein eluent Concentrate to 500 ⁇ L using a 10 KD concentrator.
  • Concentration Collect the target protein eluent from the molecular sieve peak position and concentrate it to 250 ⁇ L.
  • SDS-PAGE electrophoresis was used to detect whether the sequence was the target protein sequence and to determine the protein purity.
  • a separation gel solution is prepared by mixing appropriate amounts of acrylamide and bisacrylamide with SDS, TEMED (tetramethylethylenediamine) and APS (ammonium persulfate).
  • a separating gel solution is added between the glass plates, and a comb is inserted to form sample wells.
  • reducing agents such as DTT (dithiothreitol) or ⁇ -mercaptoethanol to break the disulfide bonds in the protein.
  • the initial voltage is set to 80-100 volts, and the voltage can be increased to around 200 volts after the sample enters the separating gel.
  • proteins are separated in a gel according to their molecular weight.
  • a destaining solution such as methanol/acetic acid solution
  • An imaging system can be used to take photos of the gel, or a gel scanner can be used for analysis.
  • the raw materials for synthesis are: lysine, arginine, proline, leucine, glutamic acid, histidine, glycine, D-methionine, D-leucine, alanine, tert-butyl octadecanoate (C18 diacid), and ⁇ -glutamic acid;
  • the reagents used include: condensing agents and organic bases HBTU and NMM, DMF, DCM, methanol, hexahydropyridine, and 98% N2 ;
  • the detection solutions are: solution A: 80% phenol + 20% anhydrous ethanol; solution B: redistilled pyridine; solution C: 5g ninhydrin + 100mL anhydrous ethanol; the deprotection solution is: 20% hexahydropyridine + 80% DMF;
  • the cleavage lysis buffer is: 87.5% TFA + 5% anisole + 2.5% phenol + 2.5% EDT + 2.5% H2O
  • Resin swelling Weigh 1g of fmoc-Phe(tbu)-OH resin and pour it into the reaction column. Add 15mL of DMF and soak for 30 minutes. Then, dry the column (the first amino acid, Phe, is connected to the resin).
  • washing after reaction Drain the solution in the reaction column, add 25 mL of DMF to wash, agitate with nitrogen for 2 minutes, drain, and repeat the operation 3 times.
  • Cutting Put the dried resin into a suitable round-bottom flask, add an appropriate amount of the prepared cutting solution (1g/10mL), and place it in a constant temperature shaker at 25°C for 2 hours.
  • Filtration Filter out the resin particles using a 50mL sintered glass funnel, then pour the filtrate into a glass container for centrifuge tubes, add 6-8 times the volume of anhydrous diethyl ether while stirring, and the precipitated white solid is the desired crude polypeptide.
  • Washing Dispense the white solid into centrifuge tubes, seal the tubes, and centrifuge at 4000 rpm for 3 minutes. Remove the tubes, discard the supernatant, add ether, stir well with a glass rod, and centrifuge again. Repeat this washing process 5 times.
  • the crude linear peptide was diluted with DMF to 10 ⁇ 3 mol/L, dissolved completely in an ice bath, and then 1.5 times the amount of HOAt, 1.5 times the amount of HATU, and 10 times the amount of DIEA were added. The mixture was stirred in the ice bath for 2 hours. The ice bath was then removed, and the mixture was stirred at room temperature. The solid residue was obtained by vacuum distillation.
  • Drying Place the peptides that have been washed 5 times into a vacuum dryer and dry them under vacuum for 24 hours.
  • the final white powder is the crude product of the desired polypeptide, which should be weighed and purified.
  • the preparation process is as described in Preparation Example 1.
  • the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, phenylalanine, tert-butyl octadecanoate, and ⁇ -glutamic acid.
  • the next amino acid, Pro is linked. Steps b-f are then repeated to link Gly, DMet, Pro, Gly, Lys, His, Cys, Leu, Arg, Pro, Arg, and Cys, respectively. This yields the title sequence.
  • the protecting group Fmoc on FmocPhe-OH is removed, exposing the N-terminus.
  • the final condensed amino acids are tert-butyl octadecanoate and ⁇ -glutamic acid. These amino acids do not have Fmoc group protection, therefore, deprotection of Fmoc is not required after condensation.
  • the target peaks were detected at all three positions; therefore, the purified sequence is indeed the title peptide sequence.
  • 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 single letters as QRPRLSHKGPMPF).
  • 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)).
  • the sequence of the target protein is as follows:
  • the target gene was constructed on a TAC expression vector, transformed into E. coli, amplified, and preserved in bacterial culture for subsequent expression and purification in E. coli.
  • Inoculation 1 tube of glycerol culture + 25 ml LB (1:1000A+), incubate overnight at 37°C on a shaker.
  • 5 mL of bacterial culture 500 mL LB (1:1000A+) transferred to a large bottle (the large bottle of LB is sterilized beforehand), incubate at 37°C on a shaker for 2-3 hours, measuring the OD value every hour.
  • the OD value is 0.6-0.8, place the culture in a shaker at 16°C and 220 rpm for pre-cooling.
  • Resuspend Resuspend the bacterial cells in lysis buffer at a ratio of 1:5-10, then pour into a beaker. Add lysozyme (final concentration 0.5 mg/mL), DNase (1:100), and 1 mM PMSF.
  • Lysis Cells are broken up using a high-pressure homogenizer until the solution is clear.
  • Washing Wash with washing buffer for about 20 CV.
  • Elution buffer is used to elute proteins, and the eluent is collected.
  • Concentrated protein eluent Concentrate to 500 ⁇ L using a 10 KD concentrator.
  • Concentration Collect the eluent of the target protein from the molecular sieve peak position and concentrate it to 250 ⁇ L.
  • SDS-page characterization The purified target protein was subjected to protein electrophoresis, and the protein was compared with the protein marker to confirm that the protein was indeed located at the target protein position, indicating that the purified protein sequence was indeed the target protein sequence. In addition, the purity of the target protein was high, reaching over 90%, as confirmed by the bands. The results are shown in Figure 3.
  • the preparation process is as described in Preparation Example 1.
  • the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, phenylalanine, nonadecanoic acid/acyl halide, and ⁇ -glutamic acid.
  • resin with an amide linker was pyrolyzed to automatically form a C-terminal amide.
  • the resin was then loaded into an SPPS reaction column.
  • the resin was fully swollen with DMF (dimethylformamide) for at least 30 minutes.
  • the resin was treated twice with a 20% piperidine/DMF solution for 5-10 minutes each time to remove the initial Fmoc protecting groups on the resin.
  • Fmoc-Phe(tBu)-OH Fmoc-protected phenylalanine, tBu-protected carboxyl group
  • DIC diisopropylcarbodiimide
  • Oxyma Pure (or HOBt) activated, and then added to the resin.
  • the reaction was typically carried out in DMF with stirring for 30-60 minutes. After the first amino acid reaction is complete, the next amino acid, Pro, is ligated.
  • the target peaks were detected at all five positions; therefore, the purified sequence is indeed the title peptide sequence.
  • the preparation process is as described in Preparation Example 1.
  • the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, D-alanine, phenylalanine, nonadecanoic acid/acyl halide, and ⁇ -glutamic acid.
  • resin with an amide linker was pyrolyzed to automatically form a C-terminal amide.
  • the resin was then loaded into an SPPS reaction column.
  • the resin was fully swollen with DMF (dimethylformamide) for at least 30 minutes.
  • the resin was treated twice with a 20% piperidine/DMF solution for 5-10 minutes each time to remove the initial Fmoc protecting groups on the resin.
  • Fmoc-Phe(tBu)-OH Fmoc-protected phenylalanine, tBu-protected carboxyl group
  • DIC diisopropylcarbodiimide
  • Oxyma Pure (or HOBt) activated, and then added to the resin.
  • the reaction was usually carried out in DMF with stirring for 30-60 minutes. After the first amino acid reaction is complete, the next amino acid, Pro, is ligated.
  • the target peaks were detected at all four positions, therefore, the purified sequence is indeed the title peptide sequence.
  • the preparation process is as described in Preparation Example 1.
  • the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, phenylalanine, nonadecanoic acid/acyl halide, ⁇ -glutamic acid, and oligoethylene glycol.
  • resin with an amide linker was pyrolyzed to automatically form a C-terminal amide. The resin was then loaded into an SPPS reaction column. The resin was fully swollen with DMF (dimethylformamide) for at least 30 minutes.
  • the resin was treated twice with a 20% piperidine/DMF solution for 5-10 minutes each time to remove the initial Fmoc protecting groups on the resin.
  • Fmoc-Phe(tBu)-OH Fmoc-protected phenylalanine, tBu-protected carboxyl group
  • DIC diisopropylcarbodiimide
  • Oxyma Pure (or HOBt) activated, and then added to the resin.
  • the reaction was usually carried out in DMF with stirring for 30-60 minutes. After the first amino acid reaction is complete, the next amino acid, Pro, is ligated.
  • the above five steps (bf) are then repeated to ligate DLeu, DMet, Pro, Gly, Arg, His, Cys, Leu, Arg, Pro, Arg, Cys, DArg, Dleu, DArg, and DLys, respectively.
  • the protecting group Fmoc on FmocPhe-OH is then removed, exposing the N-terminus.
  • the final condensed amino acids are C19 alkylcarboxylic acids, oligoethylene glycol, and ⁇ -glutamic acid. These amino acids do not have Fmoc group protection, therefore, deprotection of Fmoc is not required after condensation. Subsequent cleavage and purification steps are as shown in Preparation Example 1.
  • the preparation process is as described in Preparation Example 1.
  • the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, and phenylalanine.
  • resin with an amide linker was pyrolyzed to automatically form a C-terminal amide.
  • the resin was then loaded into an SPPS reaction column.
  • the resin was fully swollen with DMF (dimethylformamide) for at least 30 minutes.
  • the resin was treated twice with a 20% piperidine/DMF solution for 5-10 minutes each time to remove the initial Fmoc protecting groups on the resin.
  • Fmoc-Phe(tBu)-OH Fmoc-protected phenylalanine, tBu-protected carboxyl group
  • DIC diisopropylcarbodiimide
  • Oxyma Pure or HOBt
  • the reaction was usually carried out in DMF with stirring for 30-60 minutes.
  • the next amino acid, Pro is ligated.
  • Steps b-f are then repeated to ligate DLeu, DMet, Pro, Gly, Arg, His, Cys, Leu, Arg, Pro, Arg, Cys, DArg, Dleu, DArg, and DLys, respectively.
  • the protecting group Fmoc on FmocPhe-OH is then removed to expose the N-terminus. The subsequent cleavage and purification steps are as described in Preparation Example 1.
  • the preparation process is as described in Preparation Example 1.
  • the required amino acid raw materials are cysteine, alanine, proline, arginine, leucine, cysteine, histidine, lysine, glycine, D-methionine, D-leucine, D-arginine, phenylalanine, nonadecanoic acid/acyl halide, ⁇ -glutamic acid, and polyethylene glycol.
  • resin with an amide linker was pyrolyzed to automatically form a C-terminal amide. The resin was then loaded into an SPPS reaction column. The resin was fully swollen with DMF (dimethylformamide) for at least 30 minutes.
  • the resin was treated twice with a 20% piperidine/DMF solution for 5-10 minutes each time to remove the initial Fmoc protecting groups on the resin.
  • Fmoc-Phe(tBu)-OH Fmoc-protected phenylalanine, tBu-protected carboxyl group
  • DIC diisopropylcarbodiimide
  • Oxyma Pure (or HOBt) activated, and then added to the resin.
  • the reaction was usually carried out in DMF with stirring for 30-60 minutes. After the first amino acid reaction is complete, the next amino acid, Pro, is ligated.
  • Steps b-f are then repeated to ligate DLeu, DMet, Pro, Gly, Arg, His, Cys, Leu, Arg, Pro, Arg, Cys, DArg, Dleu, DArg, and DLys, respectively.
  • the protecting group Fmoc on FmocPhe-OH is then removed, exposing the N-terminus.
  • the final condensed amino acids are C19 alkylcarboxylic acids, oligoethylene glycol, and ⁇ -glutamic acid. These amino acids do not have Fmoc group protection, therefore, deprotection of Fmoc is not required after condensation. Subsequent cleavage and purification steps are as shown in Preparation Example 1.
  • the target peaks were detected at all three positions, therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all three positions, therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all five positions; therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all four positions; therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all four positions; therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all four positions, therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all four positions; therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all four positions; therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all four positions; therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all four positions, therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all three positions; therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all three positions, therefore, the purified sequence is indeed the title peptide sequence.
  • the target peaks were detected at all three positions, therefore, the purified sequence is indeed the title peptide sequence.
  • Binary Technology is a reporter gene technology for detecting structural complementarity in protein-protein interaction studies.
  • the system consists of two subunits: a large subunit, Large BiT (LgBiT; 18 kDa), and a small subunit, Small BiT (SmBiT; 11 amino acids), which can be fused with target proteins for expression.
  • LgBiT and SmBiT subunits have been optimized to exhibit good stability and minimized self-linking. When the fused target protein carrying these two subunits interacts, the two subunits bind together to form an active enzyme, producing a bright luminescent signal.
  • the above plasmids were co-transfected into 6-well plates containing cultured HEK293T cells for expression. 24 hours after transfection, the culture medium was replaced with complete medium containing 10% FBS and incubated overnight. 24-48 hours after transfection, the transfected 6-well plates were removed, washed once with PBS, digested with 1 ml of trypsin for 1 min, and resuspended in 3 ml of DEMEM complete medium. The resuspended cells were then seeded into 96-well plates and cultured overnight. The next day, the 96-well plates were removed, washed once with D-PBS, and incubated with HBSS-prepared coelenterate 400a substrate for 50 min. Baseline signals were read, and then different concentrations of ligands were added, mixed, and the signals after ligand stimulation were read. The ligand-induced signals were normalized to the baseline signal to show the G protein dissociation response.
  • the BRET assay studies protein-protein interactions by labeling fusion proteins with bioluminescent donors such as Renalis luciferase (Rluc), or fluorescent acceptors such as enhanced green or yellow fluorescent proteins (EGFP or EYFP).
  • Rluc Renalis luciferase
  • EYFP enhanced green or yellow fluorescent proteins
  • the proteins are sufficiently close (in... Within (within), energy will be transferred from Rluc to EYFP.
  • the strict distance between donor and acceptor molecules makes BRET an ideal tool for studying receptor-protein interactions involved in all GPCR functions and regulated by agonists and antagonists.
  • ⁇ -arrestin recruitment was detected using the BRET assay.
  • the APLNR receptor C-terminus was fused with the Rluc8 fragment, and the Venus fragment was inserted into the N-terminus of ⁇ -arrestin2, both cloned into the pBiT1.1 plasmid.
  • These plasmids were co-transfected into 6-well plates containing cultured HEK293T cells for expression. 24 hours after transfection, the culture medium was replaced with complete medium containing 10% FBS and incubated overnight. 24-48 hours after transfection, the 6-well plates were removed, washed once with PBS, digested with 1 ml of trypsin for 1 min, and resuspended in 3 ml of DMEM complete medium.
  • the resuspended cells were then seeded into 96-well plates and cultured overnight. The next day, the 96-well plates were removed, washed once with D-PBS, and 40 ⁇ l of HBSS was added. The transfected cells were then stimulated for 40 min with different concentrations of ligands (5 ⁇ l). Subsequently, the luciferase substrate luciferin was added for 400 hours, and BRET signals were detected: Venus (520-560nm) and RLuc8 (460-485nm).
  • scFv16 antibody 12 mU/ml apyrase (Sigma), and agonists (50 ⁇ M apelin and 50 ⁇ M MM07) were added.
  • the mixture was incubated at room temperature for 1 hour to form the complex.
  • 0.5% (w/v) dodecyl maltose neopentyl glycol and 0.1% (w/v) hemisuccinate cholesterol were added, and the mixture was incubated at 4°C for 2 hours.
  • MBP N-bindse
  • TEV protease was then added for 1 hour to remove MBP protein.
  • the eluent was concentrated in a 100 kDa concentrator, and the sample was injected into a Superose 6increase 10/300GL column (GE Healthcare).
  • the run buffer contained 20 mM HEPES pH 7.5, 100 mM NaCl, 0.00075% dodecyl maltose neopentyl glycol, 0.0002% (w/v) hemisuccinate cholesterol, and 0.00025% (w/v) glycosaminoglycans, along with agonists (50 ⁇ M apelin, 50 ⁇ M MM07).
  • the target peak fraction was collected, concentrated to 10-15 mg/mL, and subjected to electron microscopy.
  • cryo-electron microscopy data acquisition was performed 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 [missing information].
  • Image stacks were acquired at a dose rate of approximately 8.0 electrons/ ⁇ 2/s, with underfocus values ranging from -1.0 to -2.5 ⁇ m.
  • the total exposure time was 8 seconds, with 40 frames recorded per photograph.
  • a total of 2520 apelin-bound complex images were collected.
  • the MM07 complex [the text abruptly ends here, likely due to an incomplete sentence or missing information].
  • a total of 4454 photomicrographs were collected at a pixel size, recorded at a dose rate of 21.3 electrons/ ⁇ 2/sec, with underfocus values ranging from -0.8 to -1.8 ⁇ m.
  • the total exposure time was 2.35 seconds, with 36 frames recorded per photomicrograph.
  • the collected data were processed using RELION 3.1 and CryoSPARC v3.1.
  • the model was docked with cryo-electron microscopy density maps using UCSF Chimera (Pettersen, Goddard et al. 2004).
  • the final model was refined using the "Comprehensive Validation (cryo-EM)" module in Phenix. Structural diagrams were drawn using UCSF Chimera and UCSF Chimera X software.
  • the amino acid sequence of human APLNR is shown in SEQ ID No. 1 (NCBI sequence number: NP_005152.1), and the coding 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), the amino acid sequence of which is shown in SEQ ID No. 3 (NCBI sequence number: NP_001027619.1), and the coding gene sequence is shown in SEQ ID No. 4 (NCBI sequence number: NM_001032447.1).
  • the experimental results are shown in Figure 1.
  • the results demonstrate that we have resolved the high-resolution cryo-electron microscopy structure of the apelin-APLNR-Gi1 complex (Figure 1, A and B), allowing for effective analysis of the interaction between the ligand and receptor.
  • the results are shown in Figure 1, C.
  • the EC50 for apelin activating G protein signaling was 9.793 ⁇ 10 ⁇ 10 M
  • the EC50 for recruiting ⁇ -arrestin was 8.688 ⁇ 10 ⁇ 9 .
  • the EC50 for MM07 activating G protein signaling was 1.377 ⁇ 10 ⁇ 8 M, and the EC50 for recruiting ⁇ -arrestin was 5.495 ⁇ 10 ⁇ 6 M.
  • MM07's ability to activate arrestin was nearly 1000-fold weaker, while its G protein activity was about 10-fold weaker.
  • M11 and F13 of apelin and MM07 are inserted into the two pockets, respectively, interacting with the key amino acid residues of the "twin hotspots.” Therefore, altering the 11-Met and 13-Phe positions that interact with the receptor can help design G protein-biased peptide agonists.
  • the detection method was as described in Example 1.
  • M288 7.32 , Q180 ECL2 , and Y182 ECL2 are key amino acid residues that affect receptor-ligand interaction.
  • ⁇ pEC50 and Emax show that alanine mutations at these amino acid sites significantly weaken APLNR-mediated G protein signaling, and EC50 is reduced relative to wild type (WT). Therefore, these amino acids are key amino acids for receptor-ligand interaction.
  • Subcutaneous administration Rats were fasted overnight. The test compound was dissolved in PBS (concentration 0.2 mg/mL), and the dose was 1 mg/kg. At 0.5 h, 2 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h after administration, blood was continuously collected from the jugular vein (0.2 mL each time) and placed in EP tubes containing EDTA-K2. After centrifugation at 4000 g for 5 min at 4°C, the supernatant plasma was collected and stored at -80°C. The plasma was analyzed by LC-MS/MS. Based on the obtained blood drug concentration-time data, the pharmacokinetic parameters were calculated using WinNonlin software.
  • Subcutaneous administration Cynomolgus monkeys were fasted overnight. The test compound was dissolved in PBS (concentration 0.5 mg/mL), and the dosage was 0.5 mg/kg. Blood samples (0.3 mL each time) were continuously collected from peripheral veins at 0.5 h, 2 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 240 h, 336 h, and 504 h after administration. The samples were placed in EP tubes containing EDTA-K2, centrifuged at 2000 x g for 10 min at 2-8 °C, and the supernatant plasma was collected and stored at -80 °C. The plasma was analyzed by LC-MS/MS. Based on the obtained blood drug concentration-time data, pharmacokinetic parameters were calculated using WinNonlin software.
  • Group 1 DIO mice treated with a combination of tirzepatide (0.05 mpk, subcutaneous injection) and sequence 216 (5 mpk, subcutaneous injection).
  • Group 2 DIO mice treated with a combination of telpolide (0.05 mpk, subcutaneous injection) and BGE-105 (0.01 mpk, gavage).
  • Group 3 Blank control for DIO mice (administered the same volume of PBS solution subcutaneously).
  • Group 4 GLP-1 receptor modulator, telpokinesin (0.05 mpk, subcutaneous injection) in DIO mice
  • Key evaluation indicators include:
  • Body composition was measured weekly, including muscle mass, muscle mass, fat mass, and fat mass; body composition was compared at the endpoint using the muscle weight/fat tissue weight ratio at baseline (week 2, week 3, and week 4) or after completion of administration.
  • the body composition was determined by tissue dissection and collection of muscle and fat mass.
  • Tissues Half of the tissue samples were frozen for molecular biological analysis, and the other half were fixed in 10% neutral buffered formaldehyde (or embedded in OTC) for histological analysis to measure and quantify fats: groin fat, perirenal fat, epididymal fat, brown fat; muscles: soleus, extensor digitorum longus, tibialis anterior, gastrocnemius; heart.
  • Figures 4A and 4B show the changes in body weight of DIO mice in each group during the administration period (28 days). All telpotetide treatment groups showed a significant decrease in body weight. Group 1: DIO mice treated with a combination of telpotetide (0.05 mpk, subcutaneous injection) and sequence 216 (5 mpk, subcutaneous injection) showed a more pronounced trend of weight loss.
  • Figures 4C and 4D show the changes in Q2D and cumulative food intake of DIO mice in each group during the administration period (28 days). All telpotetide treatment groups showed a significant reduction in food intake.
  • Figures 5A to 5C show the changes in muscle mass in DIO mice in each group before administration, at 1, 2, 3, and 4 weeks after administration. Compared with treatment with telpolide alone, treatment with the combined dose of sequence 216 and telpolide significantly increased muscle mass during days 21 to 28.
  • Figures 6A to 6C show the changes in fat content in DIO mice in each group before administration, at 1, 2, 3, and 4 weeks after administration. Compared with treatment with telpolide alone, treatment with the combined dose of sequence 216 and telpolide significantly reduced fat content during days 21 to 28.
  • Figures 7A to 7C show the changes in fat mass in DIO mice in each group before administration, at 1, 2, 3, and 4 weeks after administration. Compared with treatment with telpolide alone, treatment with the combined dose of sequence 216 and telpolide significantly reduced fat content during days 21 to 28.
  • Figures 8A and 8B show the changes in muscle mass in DIO mice in each group before administration, at 1, 2, 3, and 4 weeks after administration. Compared with treatment with telpolide alone, muscle mass recovered in the group treated with the combined dose of sequence 216 and telpolide on day 21.
  • Figures 9A and 9B show the changes in muscle/fat mass in DIO mice at 1, 2, 3, and 4 weeks prior to drug administration. Mice receiving the combined dose of sequence 216 and telpolide showed an increased muscle/fat ratio compared to telpolide alone.
  • Figures 10A to 10C show the total muscle mass of anatomical tissues at the end of drug administration. Compared with telpolide alone, the group receiving the combined dose of sequence 216 and telpolide showed a significant increase in the ratio of muscle mass to adipose tissue.
  • sequence 216 can significantly enhance the weight loss effect of telpolide and overcome the muscle loss side effect that telpolide may cause.
  • Figures 11A to 11B show that mice treated with sequence 216 alone had an 11% decrease in body weight compared to the control group, but their appetite was not affected.
  • Figures 12A to 12D show that mice treated with sequence 216 alone had significantly less epididymal fat and significantly more brown fat compared to the control group. This indicates that sequence 216 treatment can remodel the adipose tissue composition of mice, exerting its potential weight control or metabolic improvement effects by reducing energy-storing white fat and increasing energy-consuming brown fat.
  • mice treated with a combination of telpolide and sequence 216 experienced weight loss while maintaining muscle function and reducing muscle loss.
  • Immunofluorescence of cardiomyocytes To observe the effects of apelin and its analogues (sequences 221, 250, and 253) on cardiomyocyte morphology, immunofluorescence experiments were performed using the rat cardiomyocyte cell line H9C2. Cells were treated with 1 ⁇ M PE (Phorbol 12-myristate 13-acetate) for 36 hours, either directly or without treatment. After treatment, the cell nuclei were stained with blue fluorescence using DAPI, and the actin cytoskeleton was stained with red fluorescence using phalloidin. Cell size was photographed under a fluorescence microscope.
  • PE Phorbol 12-myristate 13-acetate
  • TAC Modeling and Drug Administration A pressure overload-induced heart failure model was established via transverse aortic constriction (TAC) surgery. Mice were anesthetized with 3% isoflurane inhalation and underwent open-chest surgery. A ligation was performed at the aortic arch (specifically, between the bifurcation of the brachiocephalic artery and the left common carotid artery) using 7-0 sutures, and a needle was used to assist in creating a local stenosis before the needle was withdrawn. One week post-surgery, mice were intraperitoneally injected with 1 mpk sequence 221, while the control group received an equal volume of PBS buffer. Echocardiography was performed on mice at weeks 3 and 5 post-surgery to assess changes in cardiac structure and function.
  • FIG 14 shows that Apelin, sequence 221, sequence 250, and sequence 253 all have inhibitory effects on PE-induced cardiomyocyte hypertrophy. However, Apelin can induce hypertrophy in normal cardiomyocytes, while sequence 221, sequence 250, and sequence 253 have no hypertrophic effect on normal cardiomyocytes.
  • FIG. 15 shows that during the five-week intervention period, the TAC+ sequence 221 group significantly improved cardiac function indicators in heart failure mice compared with the TAC group: it inhibited the increase of left ventricular end-systolic diameter (LVIDs) and left ventricular mass (LV Mass), and prevented the decrease of ejection fraction (EF%) and fractional shortening (FS%).
  • LVIDs left ventricular end-systolic diameter
  • LV Mass left ventricular mass
  • Figure 16 shows that the drug administration groups of sequence 221, sequence 250, and sequence 253 significantly inhibited cardiomyocyte hypertrophy.
  • Figure 17 shows that these drugs also significantly reversed TAC-induced overall cardiac hypertrophy, with sequence 221 showing the best results when administered daily or every three days.
  • Figure 18 shows that, at the molecular level, the sequence 221 administration group effectively reduced the expression of atrial natriuretic peptide (ANP) and inhibited the increase in the Myh7/Myh6 ratio induced by TAC.
  • ABP atrial natriuretic peptide
  • the Sirius red staining results in Figure 19 show the degree of myocardial fibrosis.
  • the level of myocardial fibrosis was quantified by calculating the ratio of fibrotic area to cell area.
  • the results showed that the myocardial fibrosis area in the daily and once-every-three-day administration groups of sequence 221 and the administration group of sequence 253 was significantly smaller than that in the TAC group, indicating that sequence 221 and sequence 253 treatment significantly inhibited the occurrence of cardiac fibrosis.
  • relational terms such as first and second are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

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Abstract

公开了Apelin-13多肽突变体、包含该Apelin-13多肽突变体的药物组合物及其用途。

Description

Apelin-13多肽突变体
相关申请的引用
本公开要求于2024年7月5日向中华人民共和国国家知识产权局提交的申请号为202410906618.8、发明名称为“Apelin-13多肽突变体”的中国发明专利申请的全部权益，并通过引用的方式将其全部内容并入本公开。
领域
本公开大体上涉及生物医药领域，更具体地，涉及多肽领域。
背景
心血管疾病是威胁人类身心健康的主要疾病之一，患病率逐年增加，各国在心血管疾病预防和治疗方面面临重大挑战，造成了主要的社会和经济负担。Apelin受体(APLNR)是一种典型的A类GPCR，在心血管系统中具有重要功能，包括促进心脏收缩、增强左心室搏量、血管舒张、利尿、降低全身血压等。
概述
一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(I)所示的多肽：
aa1-Arg-Pro-Arg-Leu-b1-His-cc1-Gly-Pro-DMet-Gly-Pro-Phe
通式(I)
其中，b1为侧链带负电荷的氨基酸，aa1选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，cc1选自C11-25烃酰基-γGlu-Lys、羧基-C10-24烃酰基-γGlu-Lys、C11-25烃酰基-m×OEG-γGlu-Lys、羧基-C10-24烃酰基-m×OEG-γGlu-Lys或PEG-Lys，并且1、6位的氨基酸残基形成酰胺键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(I)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(II)所示的多肽：
aa2-Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe
通式(II)
其中，a2选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，aa2选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(II)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(III-1)所示的多肽：
aa3-Cys-Arg-a3-Arg-b3-Cys-His-c3-Gly-Pro-d3-e3-f3-g3
通式(III-1)
其中，a3选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，b3为具有疏水侧链的氨基酸，c3为侧链带正电荷的氨基酸，d3和e3各自独立地选自具有疏水侧链的氨基酸或Gly，f3为具有疏水侧链的氨基酸或Pro，g3为具有疏水侧链的氨基酸，aa3选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)通式(III-2)所示的多肽：
aa3’-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3’
通式(III-2)
其中，a3’为具有疏水侧链的氨基酸，aa3’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(3)与通式(III-1)所示的多肽或者通式(III-2)具有至少70％同源性并且具有相同功能的多肽。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(IV)所示的多肽：
aa4-Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe
通式(IV)
其中，a4为具有疏水侧链的氨基酸，aa4选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(IV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(V-1)所示的多肽：
aa5-Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5
通式(V-1)
其中，a5选自具有疏水侧链的氨基酸或者Pro，b5为具有疏水侧链的氨基酸，c5为侧链带正电荷的氨基酸，d5和e5各自独立地选自具有疏水侧链的氨基酸或Gly，f5为具有疏水侧链的氨基酸或Pro，g5为具有疏水侧链的氨基酸，aa5选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)通式(V-2)所示的多肽：
aa5’-Cys-Arg-a5’-Arg-b5’-Cys-His-c5’-Gly-Pro-Nal-Pro-d5’
通式(V-2)
其中，a5’选自具有疏水侧链的氨基酸或者Pro，b5’为具有疏水侧链的氨基酸，c5’为侧链带正电荷的氨基酸，d5’为具有疏水侧链的氨基酸，aa5’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(3)与通式(V-1)所示的多肽或者通式(V-2)具有至少70％同源性并且具有相同功能的多肽。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(VII)所示的多肽：
aa7-Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe
通式(VII)
其中，a7选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，aa7选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(VII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(VIII-1)所示的多肽：
aa8-Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8
通式(VIII-1)
其中，a8选自具有疏水侧链的氨基酸或者Pro，b8为具有疏水侧链的氨基酸，c8选自具有疏水侧链的氨基酸或者侧链带正电荷的氨基酸，d8为具有疏水侧链的氨基酸或Pro，e8为具有疏水侧链的氨基酸，aa8选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-3×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数，或者
(2)通式(VIII-2)所示的多肽：
aa8’-Cys-Arg-Pro-Arg-Trp-Cys-His-a8’-Gly-Pro-Nal-Pro-Phe
通式(VIII-2)
其中，a8’为侧链带正电荷的氨基酸，aa8’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(3)与通式(VIII-1)所示的多肽或者通式(VIII-2)具有至少70％同源性并且具有相同功能的多肽。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(IX)所示的多肽：
aa9-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe
通式(IX)
其中，a9为侧链为极性不带电荷的氨基酸，aa9选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(IX)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XI-1)所示的多肽：
aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe
通式(XI-1)
其中，a11选自选自Ala、Phe、Nal、DNal、DMet、Glu或者Nle，aa11选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；
(2)通式(XI-2)所示的多肽：
aa11’-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11’-b11’-Pro-Phe
通式(XI-2)
其中，a11’选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸、His或者Pro，b11’选自具有疏水侧链的氨基酸或者Gly，aa11’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(3)与通式(XI-1)所示的多肽或者通式(XI-2)具有至少70％同源性并且具有相同功能的多肽。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XII)所示的多肽：
aa12-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe
通式(XII)
其中，a12选自侧链带正电荷的氨基酸、具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸或者侧链带负电荷的氨基酸，aa12选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(XII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XIII)所示的多肽：
aa13-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe
通式(XIII)
其中，a12选自具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸、侧链带负电荷的氨基酸或者His，aa13选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(XIII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XIV)所示的多肽：
aa14-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14
通式(XIV)
其中，a14选自具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸、侧链带负电荷的氨基酸、His或者Nal，aa14选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(XIV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XV)所示的多肽：
aa15-Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe
通式(XV)
其中，a15和b15各自独立地选自侧链带正电荷的氨基酸或者Pro，c15选自具有疏水侧链的氨基酸或者侧链带正电荷的氨基酸，aa15选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、5位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(XV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XVI)所示的多肽：
aa16-Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe
通式(XVI)
其中，a16选自侧链带正电荷的氨基酸或者Pro，b16选自具有疏水侧链的氨基酸、侧链带正电荷的氨基酸或者Pro，aa16选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、4位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(XVI)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XVII)所示的多肽：
*-Arg-a17-b17-Leu-*-His-C17-Gly-Pro-DMet-Gly-Pro-Phe
通式(XVII)
其中，a17和b17各自独立地选自脯氨酸或精氨酸，或者a17和/或b17不存在，c17为侧链带正电荷的氨基酸，*为非天然氨基酸，并且*之间形成环状结构；或者
(2)与通式(XVII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XVIII)所示的多肽：
a18-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b18-Pro-Phe
通式(XVIII)
其中，a18选自：

b18选自具有疏水侧链的氨基酸或者侧链带负电荷的氨基酸，并且1、6位的半胱氨酸残基形成二硫键；或者
(2)与通式(XVIII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XIV)所示的多肽：
a19-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b19-c19-Pro-Phe
通式(XIV)
其中，a19选自：

b19选自具有疏水侧链的氨基酸或者侧链带负电荷的氨基酸，c19选为具有疏水侧链的氨基酸，并且1、6位的半胱氨酸残基形成二硫键；或者
(2)与通式(XIV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
又一方面，本公开涉及药物组合物，其包含本公开所述的Apelin-13多肽突变体，以及药物可接受的的载体、稀释剂或赋形剂。
在某些实施方案中，本公开所述的药物组合物还包含至少另一种活性成分。
在某些实施方案中，能够用于本公开的药物组合物中的至少另一种活性成分的示例性实例包括但不限于与GLP1R、GLP2R、GIPR、GCGR、FGF21R、NPY2R或GDF15作用的活性成分。
在某些实施方案中，能够用于本公开的药物组合物中的至少另一种活性成分的示例性实例包括但不限于Vurolenatide、exenatide、ROSE-010、GX-G6、orforglipron、FNP-120、UTMD-GLP-1、GLP-1mimetics、VTC-G15、curaglutide、Exendin-4、P-11、CTRS-101、HD-7671、HTL-097、PGN-OB2、GL-0034、度拉糖肽、司美格鲁肽、ecnoglutide、supalutai、rE-4、HS-20004、BPI-3016、ZT002、ECC-5004、TJ-103、SHR-2042、MDR-001、LPXT-007、Tirzepatide、CT-868、NNC-0090-2746、AMG133、CT-388、LY3493269、VK-2735、DA4-JC、HISHS-3001、HISHS-2001、LBT-6030、SCO-094、HS-20094、GMA106、HZ010、BGM0504、HZ012、XW003+XW017、THDBH120、BEBT-808、RAY-1225、Cotadutide、Pegapamodutide、BI-456906、Mazdutide/IBI362、efinopegdutide、Pemvidutide、NN-6177、ZP-2929、oxyntomodulin、G-49、AGM-217、JNJ-54728518、DA-1726、HM-14220、DD01、PB-718、TB001、SHR-1816、TB-001、YH-25724、HEC88473、AP-026、GLP-1-Fc-PYY、ZP-7570(dapiglutide)、HTL-30023、QL-1005、LY3437943(retatrutide)、HM15211、SAR441255、HISHS-3001、MWN-101和DR10624。
另一方面，本公开涉及治疗或预防对APLNR受体的激动响应的疾病或疾病状态的方法，其包括向需要所述方法的个体给予治疗或预防有效量的本公开所述的Apelin-13多肽突变体或者本公开所述的药物组合物。
再一方面，本公开涉及用于治疗或预防对APLNR受体的激动响应的疾病或疾病状态的本公开所述的Apelin-13多肽突变体。
又一方面，本公开涉及本公开所述的Apelin-13多肽突变体在制备用于治疗或预防对APLNR受体的激动响应的疾病或疾病状态的药物中的用途。
附图简要说明
图1为apelin-APLNR-Gi1复合物的结构图、配体与受体之间的相互作用分析结果图，其中，A：apelin-APLNR-Gi1复合物的冷冻电镜密度图和分子模型，B：多肽配体apelin和MM07的冷冻电镜密度图和分子模型，C：apelin和MM07诱导Gi蛋白解离(左)和β-arrestin招募(右)的剂量依赖曲线。
图2为APLNR偏向性信号转导机制分析结果图，其中，A：apelin与APLNR中影响偏向性的关键氨基酸残基相互作用的密度图，B：MM07与APLNR中影响偏向性的关键氨基酸残基相互作用的密度图，C-F：为与M11和F13相互作用的关键氨基酸突变的G蛋白激活和β-arrestin招募检测，G：受体上“twin hotspots”与肽激动剂的M11和F13相互作用模式图。
图3示出了本公开的融合蛋白的SDS-page表征结果。
图4示出了不同给药组对DIO小鼠体重和摄食量的影响，其中，A：给药期间体重下降质量变化，B：下降体重占原体重比例，C：给药期间Q2D摄食量变化，D：累计摄食量的统计。
图5示出了不同给药组对DIO小鼠肌肉含量变化的影响，其中，A：不同给药组给药期间肌肉含量变化，B：肌肉含量变化占初始肌肉含量比例变化，C：肌肉含量变化显著性差异统计学分析。
图6示出了不同给药组对DIO小鼠脂肪含量变化的影响，其中，A：不同给药组给药期间脂肪含量变化，B：脂肪含量变化占初始脂肪含量比例的变化，C：脂肪含量变化显著性差异统计学分析。
图7示出了不同给药组对DIO小鼠脂肪质量变化的影响，其中，A：不同给药组给药期间脂肪质量变化，B：脂肪质量变化占初始脂肪质量比例的变化，C：脂肪质量变化显著性差异统计学分析。
图8示出了不同给药组对DIO小鼠肌肉质量变化的影响，其中，A：不同给药组给药期间肌肉质量变化，B：肌肉质量变化占初始脂肪质量比例的变化。
图9示出了不同给药组对DIO小鼠肌肉质量/脂肪质量变化的影响，其中，A：不同给药组给药期间肌肉质量/脂肪质量变化，B：肌肉质量/脂肪质量变化显著性统计学分析。
图10示出了序列216对DIO小鼠解剖后肌肉和脂肪组织重量变化的影响，其中，A：解剖后肌肉组织称重(g)显著性分析，B：解剖后脂肪组织称重(g)显著性分析，C：解剖后肌肉组织质量/脂肪组织质量变化显著性分析。
图11示出了序列216对DIO小鼠体重和摄食量的影响，其中，A：给药期间体重下降质量(g)和体重下降百分比(％)，B：给药期间累计摄食量统计。
图12示出了序列216对DIO小鼠不同脂肪组织重量的影响，其中，A：给药28天后解剖肾周白色脂肪rpWAT，B：附睾白色脂肪eWAT，C：腹股沟白色脂肪iWAT，D：棕色脂肪BAT的质量统计。
图13示出了TAC小鼠模型及给药方案。
图14示出了心肌细胞免疫荧光。
图15示出了TAC模型心超检测结果。
图16示出了心肌细胞肥大检测结果。
图17示出了心脏肥大检测结果。
图18示出了心脏肥大相关因子水平检测结果。
图19示出了心肌纤维化检测结果。
详述
在以下的说明中，包括某些具体的细节以对各个公开的实施方案提供全面的理解。然而，相关领域的技术人员会认识到，不采用一个或多个这些具体的细节，而采用其他方法、部件、材料等的情况下可实现实施方案。
除非本公开中另外要求，在整个说明书和其后的权利要求书中，词语“包括”和“包含”应解释为开放式的、含括式的意义，即“包括但不限于”。
在本公开和所附权利要求书中使用时，除非上下文另有明确规定，否则不带数量指示的单数指称物包括复数指称物。
在整个本说明书中提及的“一实施方案”或“另一实施方案”或“实施方案”或“某些实施方案”意指在至少一实施方案中包括与该实施方案所述的相关的具体参考要素、结构或特征。因此，在整个说明书中不同位置出现的短语“一实施方案”或“实施方案”或“另一实施方案”不必全部指同一实施方案。此外，具体要素、结构或特征可以任何适当的方式在一个或多个实施方案中结合。
应当理解，在本公开的说明书和所附的权利要求书中用到的单数形式的冠词“一”(对应于英文“a”、“an”和“the”)包括复数的对象，除非文中另外明确地规定。
定义
因此，除非另有相反的说明，否则说明书及所附权利要求中所用的下列术语具有以下的意思：
本公开中命名的某些化学基团前面所置的简写符号表示在所指示的化学基团存在的碳原子总数。例如，C1-4烷基描述如下文所定义的具有总共1至4个碳原子的烷基，而C3-C10环烷基描述如下文所定义的具有总共3至10个碳原子的环烷基。简写符号中的碳总数不包含可能存在于所述基团的取代基中的碳。
在本公开中，术语“烃基”系指脂肪族烃基团。烃基部分可以是“饱和的烃基”基团，意为其不包含任何烯或炔部分，也即烷基。烃基部分还可以是“不饱和的烃基”部分，意为其包含至少一个烯或炔部分。“烯”部分，也即烯基，是指由12至24个碳原子和至少一个碳-碳双键组成的基团，并且由单键与分子的其余部分连接的直链或支链烃链基团，例如乙烯基、丙-1-烯基、丁-1-烯基、戊-1-烯基、戊-1,4-二烯基等，并且“炔”部分是指由12至24个碳原子和至少一个碳-碳三键组成的基团，并且由单键与分子的其余部分连接的直链或支链烃链基团。烃基部分，无论饱和的或不饱和的，可以是支链的或直链的。
烃基基团可具有12至24个碳原子(在本公开中每次出现时，诸如“12至14”的数值范围是指给定范围中的每一整数；如“12至24”意为所述烃基基团可由12个碳原子、13个碳原子、14个碳原子、15个碳原子等直至并包括24个碳原子，尽管本定义还涵盖未指定数值范围的术语“烃基”的出现)。
在本公开中，术语“C11-25烃酰基-γGlu”系指具有如下结构的基团：
在本公开中，术语“羧基-C10-24烃酰基-γGlu”系指具有如下结构的基团：
在本公开中，术语“C11-25烃酰基-γGlu-m×OEG”系指具有如下结构的基团：
在本公开中，术语“羧基-C10-24烃酰基-γGlu-m×OEG”系指具有如下结构的基团：
在本公开中，术语“C11-25烃酰基-γGlu-n×PEG”系指具有如下结构的基团：
在本公开中，术语“羧基-C10-24烃酰基-γGlu-n×PEG”系指具有如下结构的基团：
在本公开中，术语“C11-25烃酰基-γGlu-Lys”系指具有如下结构的基团：
在本公开中，术语“羧基-C10-24烃酰基-γGlu-Lys”系指具有如下结构的基团：
在本公开中，术语“PEG”系指具有如下结构的基团：
n＝2-10
在本公开中，野生型Apelin-13多肽系指具有如下氨基酸序列的多肽：Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe，其用单字母表示为QRPRLSHKGPMPF。
在本公开中，术语“1、6位的半胱氨酸残基形成二硫键”系指本公开的Apelin-13多肽突变体在对应的野生型Apelin-13多肽的1、6位上的半胱氨酸残基形成二硫键。
在本公开中，术语“Disulfide bridge:a-b(a、b位二硫键)”系指本公开的Apelin-13多肽突变体在对应的野生型Apelin-13多肽的a、b位形成二硫键。
在本公开中，术语“Disulfide bridge:1-6(1、6位二硫键)”系指本公开的Apelin-13多肽突变体在对应的野生型Apelin-13多肽的1、6位形成二硫键。
在本公开中，术语“药物可接受的载体、稀释剂或赋形剂”包括但不限于已经被美国食品与药品管理局认可的而可用于人类或动物的任何佐剂、载体、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、香味增强剂、表面活性剂、润湿剂、分散剂、助悬剂、稳定剂、等渗压剂、溶剂或乳化剂等对组成药物组合物无副作用的各种形式的载体。
在本公开中，术语“治疗有效量”系指改善、减弱或消除特定疾病或病况和特定疾病或病况的症状、或者避免或延迟特定疾病或病况或者特定疾病或病况的症状的发病的化合物或化合物组合的量。根据化合物、疾病状态及其严重性、以及待治疗哺乳动物的年龄、体重等，构成“治疗有效量”的本公开中所述的化合物的量将会不同，但是本领域的技术人员根据其自身的知识以及本公开可以依惯例确定本公开中所述的化合物的量。
本公开所用的“进行治疗”或“治疗”涵盖患有相关疾病或病症的哺乳动物例如人类中治疗相关的疾病或疾病状态，并且包括：
(i)预防疾病或疾病状态在哺乳动物中发生，尤其是当该哺乳动物易感于所述疾病状态，但尚未被诊断出患有这种疾病状态时；
(ii)抑制疾病或疾病状态，即阻止其发生；或者
(iii)缓解疾病或疾病状态，即使疾病或疾病状态消退或不进展。
正如本公开所用的那样，术语“疾病”和“疾病状态”可以相互交换使用，或者可以是不同的，因为特殊的疾病或疾病状态可能并没有已知的致病因子(因此不能用病因学解释)，因此其不被公认为是疾病，而是被认为是不期望的疾病状态或病症，其中临床医生已经鉴定出或多或少的特定系列的症状。
具体实施方式
一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(I)所示的多肽：
aa1-Arg-Pro-Arg-Leu-b1-His-cc1-Gly-Pro-DMet-Gly-Pro-Phe
通式(I)
其中，b1为侧链带负电荷的氨基酸，aa1选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，cc1选自C11-25烃酰基-γGlu-Lys、C11-25烃酰基-γGlu-Lys、C11-25烃酰基-m×OEG-γGlu-Lys、C11-25烃酰基-m×OEG-γGlu-Lys或PEG-Lys，并且1、6位的氨基酸残基形成酰胺键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(I)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a1的示例性实例包括但不限于自Arg、His、Lys、Asp、Glu、Ser、Thr、Asn、Gln、Cys、Sec、Gly、Pro、Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的b1的示例性实例包括但不限于Glu和Asp。
在某些实施方案中，能够用于本公开的aa1的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，能够用于本公开的cc1的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(I)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体为：
C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Ph e(1，6位酰胺键)。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(II)所示的多肽：
aa2-Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe
通式(II)
其中，a2选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，aa2选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(II)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a2的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn和Gln。
在某些实施方案中，能够用于本公开的aa2的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(II)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C19烷基-CO-γGlu-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-2×OEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG2-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG3-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG5-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
PEG8-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(III-1)所示的多肽：
aa3-Cys-Arg-a3-Arg-b3-Cys-His-c3-Gly-Pro-d3-e3-f3-g3
通式(III-1)
其中，a3选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，b3为具有疏水侧链的氨基酸，c3为侧链带正电荷的氨基酸，d3和e3各自独立地选自具有疏水侧链的氨基酸或Gly，f3为具有疏水侧链的氨基酸或Pro，g3为具有疏水侧链的氨基酸，aa3选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)通式(III-2)所示的多肽：
aa3’-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3’
通式(III-2)
其中，a3’为具有疏水侧链的氨基酸，aa3’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(3)与通式(III-1)所示的多肽或者通式(III-2)具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a3的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn和Gln。
在某些实施方案中，能够用于本公开的b3的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的c3的示例性实例包括但不限于Lys和Arg。
在某些实施方案中，能够用于本公开的d3和e3的示例性实例包括但不限于Ala、Val、Ile、Leu、DMet、Phe、Tyr、Trp和Gly。
在某些实施方案中，能够用于本公开的f3的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
在某些实施方案中，能够用于本公开的g3的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的aa3的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，能够用于本公开的a3’的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的aa3’的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(III-1)所示的多肽或通式(III-2)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6)；
C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfide bridge:1-6)；
C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Trp(Disulfide bridge:1-6)。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(IV)所示的多肽：
aa4-Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe
通式(IV)
其中，a4为具有疏水侧链的氨基酸，aa3选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(IV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a4的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的aa4的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、180ECL2或182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(IV)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C19烷基-CO-γGlu-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG2-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG3-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG5-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
PEG8-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(V-1)所示的多肽：
aa5-Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5
通式(V-1)
其中，a5选自具有疏水侧链的氨基酸或者Pro，b5为具有疏水侧链的氨基酸，c5为侧链带正电荷的氨基酸，d5和e5各自独立地选自具有疏水侧链的氨基酸或Gly，f5为具有疏水侧链的氨基酸或Pro，g5为具有疏水侧链的氨基酸，aa1选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)通式(V-2)所示的多肽：
aa5’-Cys-Arg-a5’-Arg-b5’-Cys-His-c5’-Gly-Pro-Nal-Pro-d5’
通式(V-2)
其中，a5’选自具有疏水侧链的氨基酸或者Pro，b5’为具有疏水侧链的氨基酸，c5’为侧链带正电荷的氨基酸，d5’为具有疏水侧链的氨基酸，aa5’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(3)与通式(V-1)所示的多肽或者通式(V-2)具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a5的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
在某些实施方案中，能够用于本公开的b5的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的c5的示例性实例包括但不限于Lys和Arg。
在某些实施方案中，能够用于本公开的d5和e5的示例性实例包括但不限于Ala、Val、Ile、Leu、DMet、Phe、Tyr、Trp和Gly。
在某些实施方案中，能够用于本公开的f5的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
在某些实施方案中，能够用于本公开的g5的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的aa5的示例性实例包括但不限于C11-25烃基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，能够用于本公开的a5’的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
在某些实施方案中，能够用于本公开的b5’的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的c5’的示例性实例包括但不限于Lys和Arg。
在某些实施方案中，能够用于本公开的d5’的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的aa5’的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(V-1)所示的多肽或通式(V-2)所示的多肽组成。
在某些实施方案中，本公开的的Apelin-13多肽突变体选自：
C17烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C21-烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC16烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C11烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C13烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C15烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C19烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C21烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC16烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-4×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-5×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-PheC17烷CO(Disulfide bridge:1-6)；
C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)；以及
PEG8-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(VII)所示的多肽：
aa7-Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe
通式(VII)
其中，a7选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，aa7选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(VII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a7的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn和Gln。
在某些实施方案中，能够用于本公开的aa7的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(VII)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG2-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG3-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG5-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
PEG8-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(VIII-1)所示的多肽：
aa8-Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8
通式(VIII-1)
其中，a8选自具有疏水侧链的氨基酸或者Pro，b8为具有疏水侧链的氨基酸，c8选自具有疏水侧链的氨基酸或者侧链带正电荷的氨基酸，d8为具有疏水侧链的氨基酸或Pro，e8为具有疏水侧链的氨基酸，aa8选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)通式(VIII-2)所示的多肽：
aa8’-Cys-Arg-Pro-Arg-Trp-Cys-His-a8’-Gly-Pro-Nal-Pro-Phe
通式(VIII-2)
其中，a8’为侧链带正电荷的氨基酸，aa8’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(3)与通式(VIII-1)所示的多肽或者通式(VIII-2)具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a8的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
在某些实施方案中，能够用于本公开的b8的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的c8的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Arg和Lys。
在某些实施方案中，能够用于本公开的d8的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
在某些实施方案中，能够用于本公开的e8的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的aa8的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，能够用于本公开的a8’的示例性实例包括但不限于Arg和Lys。
在某些实施方案中，能够用于本公开的aa8’的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(VIII-1)所示的多肽或通式(VIII-2)所示的多肽组成。
在某些实施方案中，本公开的的Apelin-13多肽突变体选自：
C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C21烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC16烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C11烷基CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C13烷基CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C15烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C19烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C21烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC16烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOH-C18烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-4×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-5×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-3×OEG-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-DγGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-8PEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-2×γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-3×γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
Phenyl-C17烷基-CO-PAMBA-βAla-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-Abu-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-Abu-2×γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-Abu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC12烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC14烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC16烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC20烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-4×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-2×OEG-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-DγGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-8PEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-2×γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-3×γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-PAMBA-βAla-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-Abu-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-Abu-2×γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-Abu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-5×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC16烷基-CO-γGlu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
COOHC16烷基-CO-γGlu-2×OEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-2×OEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-2×OEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；以及
COOHC22烷基-CO-γGlu-2×OEG-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(IX)所示的多肽：
aa9-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe
通式(IX)
其中，a9为侧链为极性不带电荷的氨基酸，aa9选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(IX)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a9的示例性实例包括但不限于Ser、Thr、Asn和Gln。
在某些实施方案中，能够用于本公开的aa9的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(IX)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体为：
C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG2-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG3-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
PEG8-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XI-1)所示的多肽：
aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe
通式(XI-1)
其中，a11选自选自Ala、Phe、Nal、DNal、DMet、Glu或者Nle，aa11选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；
(2)通式(XI-2)所示的多肽：
aa11’-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11’-b11’-Pro-Phe
通式(XI-2)
其中，a11’选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸、His或者Pro，b11’选自具有疏水侧链的氨基酸或者Gly，aa11’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(3)与通式(XI-1)所示的多肽或者通式(XI-2)具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a11’的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn、Gln、His和Pro。
在某些实施方案中，能够用于本公开的b11’的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Gly。
在某些实施方案中，能够用于本公开的aa11的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，能够用于本公开的aa11’的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XI-1)所示的多肽或通式(XI-2)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)；
C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
PEG2-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
PEG3-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；以及
PEG8-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XII)所示的多肽：
aa12-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe
通式(XII)
其中，a12选自侧链带正电荷的氨基酸、具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸或者侧链带负电荷的氨基酸，aa12选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(XII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a12的示例性实例包括但不限于Arg、Lys、Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn、Gln、Asp和Glu。
在某些实施方案中，能够用于本公开的aa12的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XII)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe(Disulfide bridge:1-6)；
C19烷基-CO-2×γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
C19烷基-CO-2×γGlu-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
Ac-DLys(2×OEG-γGlu-C19烷基-CO)-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
C19烷基-CO-γGlu-2×OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
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:3-8)；
Ac-Lys-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:5-10)；
Ac-Leu-Arg-Gly-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:5-10)；
Ac-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC16烷基-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC18烷基-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC16烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Leu-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC16烷基-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC18烷基-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC18烷基-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC16烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)；
COOHC18烷基-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:5-10)；
COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:5-10)；
COOHC18烷基-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:6-11)；
COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:6-11)；以及
COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Arg-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:7-12)。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XIII)所示的多肽：
aa13-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe
通式(XIII)
其中，a13选自具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸、侧链带负电荷的氨基酸或者His，aa13选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(XIII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a13的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn、Gln、Asp、Glu和His。
在某些实施方案中，能够用于本公开的aa13的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG和PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XIII)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe(Disulfide bridge:1-6)；
COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe(Disulfide bridge:1-6)；以及
COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe(Disulfide bridge:1-6)。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XIV)所示的多肽：
aa14-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14
通式(XIV)
其中，a14选自具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸、侧链带负电荷的氨基酸、His或者Nal，aa14选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
(2)与通式(XIV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a14的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn、Gln、Asp、Glu和His。
在某些实施方案中，能够用于本公开的aa14的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XIV)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr(Disulfide bridge:1-6)；以及
PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr(Disulfide bridge:1-6)。
又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XV)所示的多肽：
aa15-Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe
通式(XV)
其中，a15和b15各自独立地选自侧链带正电荷的氨基酸或者Pro，c15选自具有疏水侧链的氨基酸或者侧链带正电荷的氨基酸，aa15选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG，并且1、5位的半胱氨酸残基形成二硫键；或者
(2)与通式(XV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a15的示例性实例包括但不限于Arg、Lys和Pro。
在某些实施方案中，能够用于本公开的b15的示例性实例包括但不限于Arg、Lys和Pro。
在某些实施方案中，能够用于本公开的c15的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Arg和Lys。
在某些实施方案中，能够用于本公开的aa15的示例性实例包括但不限于C11-25烃CO-γGlu、COOHC10-24烃CO-γGlu、COOHC10-24烃CO-γGlu-2×OEG、COOHC10-24烃CO-γGlu-3×OEG或PEG。
在某些实施方案中，Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XV)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C19烷基-CO-γGlu-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
C17烷基-CO-γGlu-2×OEG-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XVI)所示的多肽：
aa16-Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe
通式(XVI)
其中，a16选自侧链带正电荷的氨基酸或者Pro，b16选自具有疏水侧链的氨基酸、侧链带正电荷的氨基酸或者Pro，aa16选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG，并且1、4位的半胱氨酸残基形成二硫键；或者
(2)与通式(XVI)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a16的示例性实例包括但不限于Arg、Lys和Pro。
在某些实施方案中，能够用于本公开的b16的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Arg、Lys和Pro。
在某些实施方案中，能够用于本公开的aa16的示例性实例包括但不限于C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XVI)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
C19烷基-CO-γGlu-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
PEG5-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
再一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XVII)所示的多肽：
*-Arg-a17-b17-Leu-*-His-c17-Gly-Pro-DMet-Gly-Pro-Phe
通式(XVII)
其中，a17和b17各自独立地选自侧链带正电荷的氨基酸，或者a17和/或b17不存在，c17为侧链带正电荷的氨基酸，*为非天然氨基酸，并且*之间形成环状结构；或者
(2)与通式(XVII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的a17和b17的示例性实例包括但不限于Arg、Lys和Pro。
在某些实施方案中，能够用于本公开的a17和b17均不存在。
在某些实施方案中，能够用于本公开的b16的示例性实例包括但不限于Arg和Lys。
在某些实施方案中，能够用于本公开的*的示例性实例包括但不限于：
在某些实施方案中，能够用于本公开的*之间形成环状结构的示例性实例包括但不限于订书钉结构。
在某些实施方案中，能够用于本公开的*之间形成环状结构的示例性实例包括但不限于
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XVII)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：

又一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XVIII)所示的多肽：
a18-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b18-Pro-Phe
通式(XVIII)
其中，a18选自：

b18选自具有疏水侧链的氨基酸或者侧链带负电荷的氨基酸，并且1、6位的半胱氨酸残基形成二硫键；或者
(2)与通式(XVIII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的b18的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Asp和Glu。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XVIII)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:6和SEQ ID NO:8。
另一方面，本公开涉及Apelin-13多肽突变体，其包含
(1)通式(XIX)所示的多肽：
a19-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b19-c19-Pro-Phe
通式(XIX)
其中，a19选自：

b19选自具有疏水侧链的氨基酸或者侧链带负电荷的氨基酸，c19选为具有疏水侧链的氨基酸，并且1、6位的半胱氨酸残基形成二硫键；或者
(2)与通式(XIX)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
在某些实施方案中，能够用于本公开的b19的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
在某些实施方案中，能够用于本公开的c19的示例性实例包括但不限于Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Asp和Glu。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
在某些实施方案中，本公开的Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39或Y2997.49结合，从而影响G蛋白偏向性。
在某些实施方案中，本公开的Apelin-13多肽突变体由通式(XIX)所示的多肽组成。
在某些实施方案中，本公开的Apelin-13多肽突变体选自：
SEQ ID NO:2和SEQ ID NO:5。
又一方面，本公开涉及药物组合物，其包含本公开所述的Apelin-13多肽突变体，以及药物可接受的的载体、稀释剂或赋形剂。
另一方面，本公开涉及治疗或预防对APLNR受体的激动响应的疾病或疾病状态的方法，其包括向需要所述方法的个体给予治疗或预防有效量的本公开所述的Apelin-13多肽突变体或者本公开所述的药物组合物。
在某些实施方案中，能够用于本公开的疾病或疾病状态的示例性实例包括但不限于维持肌肉质量、减脂、急性代偿失调性心力衰竭、急性心力衰竭、慢性心力衰竭、肺性高血压、心房颤动、肾病、增肌、布鲁格达氏综合征、室性心动过速、动脉粥样硬化、高血压、再狭窄、局部缺血性心血管疾病、心肌病、心脏纤维化、心律不齐、水潴留、糖尿病、肥胖症、外周动脉病、脑血管意外、暂时性缺血发作、创伤性脑损伤、肌萎缩侧索硬化、灼伤、先兆子痫、抗衰老、镇痛，睡眠呼吸暂停综合征、肺栓塞、哮喘、应激性胃溃疡、肝脏纤维化、肺损伤、脑损伤、缺血性中风、癌症、阿尔茨海默病、帕金森病、脑卒中、肾性高血压和神经退行性疾病。
在某些实施方案中，能够用于本公开的个体的示例性实例包括但不限于哺乳动物。
在某些实施方案中，能够用于本公开的个体的人类。
再一方面，本公开涉及用于治疗或预防对APLNR受体的激动响应的疾病或疾病状态的本公开所述的Apelin-13多肽突变体。
又一方面，本公开涉及本公开所述的Apelin-13多肽突变体在制备用于治疗或预防对APLNR受体的激动响应的疾病或疾病状态的药物中的用途。
下文中，本公开将通过如下实施例进行详细解释以便更好地理解本申请的各个方面及其优点。然而，应当理解，以下的实施例是非限制性的而且仅用于说明本公开的某些实施方案。
本公开的实施例中使用的试剂和设备均为常规的并且可商购的。
多肽的通用制备工艺
多肽序列：假如序列是ARSS
一、多肽的合成
合成顺序：从肽链C端向N端依次合成
a.树脂溶胀：称取算好量的fmoc-Ser(tbu)-OH树脂倒入反应柱中，加入DMF浸泡30分钟，抽干。
b.脱保护：
向反应柱中加入适量脱保护溶液(哌啶＝20％六氢吡啶+80％DMF)，通氮气搅拌鼓动30分钟，抽干，向反应柱中加入适量DMF，氮气鼓动2分钟，抽干，重复操作6次。
c.称料：
称取3倍摩尔量的保护氨基酸，再称取2.85倍摩尔量的HBTU(缩剂)备用
d.投料：
将已备好的保护氨基酸和HBTU加入反应柱中，再加入树脂6倍摩尔量的NMM(有机碱)，氮气搅拌鼓动30分钟。
e.反应后洗涤：
将反应柱中的溶液抽干，加入适量DMF洗涤，氮气鼓动2分钟，抽干，重复操作3次。
f.检测：
取适量(10-20颗)树脂于小试管中，加入A，B，C液各两滴。放入干式加热器中加热3分钟(110摄氏度)。取出后若溶液显蓝色，树脂有杂色不透明，则反应没有完全，需要重新再反应一次；若溶液颜色微黄，树脂无色透明则为反应完全，可以连接下一个氨基酸，具体步骤重复以上b-f这五个步骤，直到连完最后一个氨基酸。即得到ADSS这个序列。最后脱除FmocAla-OH上面的保护基Fmoc，使N端裸露出来。最后缩合的氨基酸是十八烷二酸单叔丁酯，此氨基酸没有Fmoc基团保护，因此缩合后不需要脱Fmoc保护。
g.合成完毕后的洗涤和干燥：
在连完最后一个氨基酸并已脱保护洗涤之后，抽干，向反应柱中加入适量甲醇，氮气鼓动2分钟，抽干，再加入适量DCM，氮气鼓动2分钟，抽干重复操作3次。最后反应釜中加入适量甲醇，氮气鼓动2分钟，抽干，重复操作2次，将树脂装入合适器皿中置于真空干燥器内真空干燥12小时待切割。
二、多肽的切割(裂解)
切割：将干燥后的树脂装入合适的圆底烧瓶中，加入适量配好的切液(1g/10ml)，放置于恒温摇床中25℃恒温振荡2小时。
过滤：用50mL的砂芯漏斗过滤掉树脂颗粒，然后将滤液倒入离心管的玻璃容器内，加入6-8倍体积量的无水乙醚，边加边搅拌，析出的白色固体为即所需多肽粗品。
洗涤：将白色固体分装进离心管中，离心管密封后放入离心机中以4000r/min的转速离心3分钟，取出，将上层清液倒掉，再加入乙醚，用玻璃棒搅拌均匀，再次离心；如此重复操作洗涤5次。
液相合环合成：
将直链肽粗品用DMF稀释至10-3mol/L，置于冰浴中充分溶解后加入1.5倍量HOAt、1.5倍量HATU、10倍量DIEA，继续冰浴搅拌2小时，撤去冰浴，继续常温下搅拌，减压蒸馏后得固体残余物。
干燥：将已洗涤5次的后的多肽放入真空干燥器中真空干燥24小时。最后的所得白色粉末即为所需多肽的粗品，称量，待纯化。
注：用到的氨基酸及试剂
A.氨基酸
Fmoc-Arg(pbf)-OH
Fmoc-Ala-OH
Fmoc-Ser(tbu)-OH
B.缩合剂及有机碱
HBTU
NMM
C.溶剂和氮气：
DMF
DCM
甲醇
六氢吡啶
98％N2
D.检测液
A液：80％的苯酚+20％的无水乙醇
B液：重蒸吡啶
C液：5g茚三酮+100mL无水乙醇
E.脱保护溶液的配制
20％六氢吡啶+80％DMF
G.切割的裂解液
87.5％TFA+5％苯甲硫醚+2.5％苯酚+2.5％EDT+2.5％H2O
三、纯化方法步骤
1.先进行粗品的10-100％分析，确定主峰的保留时间之后，再进行线性梯度分析。
2.试样品的溶解性水和乙腈(4:1)可以充分的溶解多肽。
3.取200mg多肽溶解于10mL的溶解瓶中，超声溶解后，用0.45μm的有机相滤头过滤得澄清液。
4.线性梯度制备收峰，并确定MS的对错。
5.对所收的液体进行纯度的跟踪反馈，得符合纯度要求的液体量。
6.对所收集的合格液体进行低温浓缩，浓缩温度：38℃，浓缩至50-100mL的液体量(特殊情况一般不浓缩直接冷冻)。
7.把浓缩好的液体量放在100mL的烧杯中，液氮或干冰冷冻成固体状态。
8.干燥
9.称量
多肽的通用表征方法
一、能够在纯化过程中通过MS确定目的峰是否为制备的多肽样品：
1.准备待测样品，确保样品适用于质谱分析，通常需要进行溶解和提取。
2.调整仪器参数
选择离子源：根据样品性质选择适当的离子源，如电喷雾离子源(ESI)或气相色谱-质谱联用的化学离子源(CI)。
3.设置质谱分析模式：选择质谱分析模式为离子监测模式。
4.进行质谱分析
启动离子源并等待其稳定，将多肽样品注入质谱仪。
5.数据采集
启动质谱分析，实时采集质谱数据，包括质谱图谱和相对丰度。
6.分析质谱数据
利用质谱软件对质谱图进行解析，识别质子峰或分子离子峰。根据质谱数据确定样品中存在的是否为目标多肽。
二、随后通过HPLC检测多肽的纯度：
1.打开HPLC工作站，来凝结好流动相管道，连接检测系统。
2.调节流量，初次使用新的流动相，可以先试一下压力，流速越大，压力越大，一般不要超过2000。点击injure，选用合适的流速，点击on，走基线，观察基线的情况。
3.设置走样方法，稳流2-5min，基线归零，进样阀loading-inject转换，设置走样时间。
将得到的多肽样品过滤上样。方法走完后，点击postrun，可记录数据和做标记等。全部样品走完后，再用上面的方法走一段基线，洗掉剩余物。
4.统计主峰占所有峰的比例，即为多肽样品的纯度。
融合蛋白通用表征方法时使用SDS-PAGE电泳检测是否为目的蛋白序列，并确定蛋白纯度。
融合蛋白的通用制备方法
将目的基因构建在TAC表达载体上，转化到大肠杆菌中扩增保存菌液，用于后续在大肠杆菌中表达纯化。
1.接菌：1管甘油菌+25ml LB(1:1000A+)，37℃摇床过夜，5mL菌液：500mL LB(1:1000A+)转大瓶(大瓶LB提前灭菌)，37℃摇床2-3h，每小时测OD值，OD值0.6-0.8时，将菌放置16℃，220rpm摇床预冷。
2.诱导：0.5M IPTG终浓度0.3mM加入菌液中，16℃，220rpm摇床20h。
3.收菌：6000g，10min。
4.重悬：1:5-10加入裂解缓冲液重悬菌体，后倒入烧杯。加入溶菌酶(终浓度0.5mg/mL)，DNase(1:100)和1mM PMSF。
5.搅拌：4℃搅拌0.5h。
6.裂解：利用高压均质机破碎细胞至溶液澄清。
7.离心：30000g，4℃，30min。
8.收上清：将离心上清液收到烧杯中，并于上清中加入30mM咪唑。
9.平衡Ni柱：取3mL beads于柱中，先水后平衡缓冲液分别洗5CV。
10.挂柱：将上清缓慢加入Ni柱。重复1至2遍。
11.洗涤:洗涤缓冲液洗约20CV。
12.洗脱:洗脱缓冲液洗脱蛋白，收集洗脱液。
13.测浓度：检测蛋白含量。
14.浓缩蛋白洗脱液：用10KD浓缩管浓缩至500μL。
15.离心：浓缩后转移至1.5mL EP管中12000rpm离心10min。
16.过分子筛。
17.浓缩：收集分子筛出峰位置目的蛋白洗脱液，浓缩至250μL。
18.离心，转移到新的EP管中，测浓度，分装至0.2mg每管，液氮速冻，存于-80℃保存。
融合蛋白的通用表征方法
使用SDS-PAGE电泳检测是否为目的蛋白序列，并确定蛋白纯度。
步骤如下：
1.制备凝胶：
准备分离胶和浓缩胶。
将适量的丙烯酰胺和双丙烯酰胺与SDS、TEMED(四甲基乙二胺)和APS(过硫酸铵)混合，制备成分离胶溶液。
在玻璃板之间加入分离胶溶液，插入梳子形成样品孔。
等待分离胶凝固后，在上方加入浓缩胶溶液，插入梳子形成样品孔。
2.样品制备：
将蛋白质样品与SDS样品缓冲液混合，加热至95℃使蛋白质变性。
加入还原剂如DTT(二硫苏糖醇)或β-巯基乙醇，以断裂蛋白质中的二硫键。
上样：
将凝固的凝胶和玻璃板放入电泳槽中，加入电泳缓冲液。
小心移除梳子，露出样品孔。
将变性的蛋白质样品加入样品孔中。
3.电泳：
接通电源，开始电泳。通常，起始电压设置为80-100伏，待样品进入分离胶后，可以增加电压至200伏左右。
电泳过程中，蛋白质根据分子量大小在凝胶中被分离。
4.染色和脱色：
电泳完成后，取出凝胶，通常使用考马斯亮蓝R-250或银染法对蛋白质进行染色。
染色后，使用脱色液(如甲醇/醋酸溶液)去除未结合的染料，使蛋白质条带清晰可见。
5.分析：
可以使用成像系统拍摄凝胶照片，或使用凝胶扫描仪进行分析。
使用适当的软件对蛋白质条带进行量化分析，如分子量估算、蛋白质含量测定等。
制备实施例1
COOHC16烷基
-CO-γGlu-Arg-Lys-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Dleu-Pro-Phe(Disulfide bridge:5-10)的制备
合成原料为：赖氨酸、精氨酸、脯氨酸、亮氨酸、谷氨酸、组氨酸、甘氨酸、D型甲硫氨酸、D型亮氨酸、丙氨酸、十八烷二酸单叔丁酯(C18 diacid)、γ谷氨酸；所用试剂包含：缩合剂及有机碱HBTU和NMM、DMF、DCM、甲醇、六氢吡啶、98％N2；检测液(A液：80％苯酚+20％无水乙醇；B液：重蒸吡啶；C液：5g茚三酮+100mL无水乙醇)；脱保护溶液：20％六氢吡啶+80％DMF；切割的裂解液：87.5％TFA+5％苯甲硫醚+2.5％苯酚+2.5％EDT+2.5％H2O。
合成步骤如下：
a.树脂溶胀：称取1g的fmoc-Phe(tbu)-OH树脂倒入反应柱中，加入15mL DMF浸泡30分钟，抽干(第一个氨基酸Phe与树脂连接)。
b.脱保护：向反应柱中加入25ml脱保护溶液(哌啶＝20％六氢吡啶+80％DMF)，通氮气搅拌鼓动30分钟，抽干，向反应柱中加入25mL DMF，氮气鼓动2分钟，抽干，重复操作6次。
c.称料：称取4.8g的保护氨基酸，再称取0.562g的HBTU(缩剂)备用。
d.投料：将已备好的保护氨基酸和HBTU加入反应柱中，再加入树脂0.608mL的NMM(有机碱)，氮气搅拌鼓动30分钟。
e.反应后洗涤：将反应柱中的溶液抽干，加入25mL DMF洗涤，氮气鼓动2分钟，抽干，重复操作3次。
f.检测：取20颗树脂于小试管中，加入A，B，C液各两滴。放入干式加热器中加热3分钟(110摄氏度)。
取出后若溶液显蓝色，树脂有杂色不透明，则反应没有完全，需要重新再反应一次；若溶液颜色微黄，树脂无色透明则为反应完全，开始连接下一个氨基酸Pro，随后重复以上b-f这五个步骤，分别将Phe、Pro、Dleu、DMet、Pro、Gly、Lys、His、Cys、Glu、Leu、Arg、Pro、Arg、Cys、Arg、Lys、Arg连接。
即得到标题序列。最后脱除FmocAla-OH上面的保护基Fmoc，使N端裸露出来。最后缩合的氨基酸是十八烷二酸单叔丁酯和γ谷氨酸，这些氨基酸没有Fmoc基团保护，因此缩合后不需要脱Fmoc保护。
g.合成完毕后的洗涤和干燥：
在连完最后一个氨基酸并已脱保护洗涤之后，抽干，向反应柱中加入适量甲醇，氮气鼓动2分钟，抽干，再加入适量DCM，氮气鼓动2分钟，抽干重复操作3次。最后反应釜中加入适量甲醇，氮气鼓动2分钟，抽干，重复操作2次，将树脂装入合适器皿中置于真空干燥器内真空干燥12小时待切割
二、多肽的切割(裂解)
切割：将干燥后的树脂装入合适的圆底烧瓶中，加入适量配好的切液(1g/10mL)，放置于恒温摇床中25℃恒温振荡2小时。
过滤：用50mL的砂芯漏斗过滤掉树脂颗粒，然后将滤液倒入离心管的玻璃容器内，加入6-8倍体积量的无水乙醚，边加边搅拌，析出的白色固体为即所需多肽粗品。
洗涤：将白色固体分装进离心管中，离心管密封后放入离心机中以4000r/min的转速离心3分钟，取出，将上层清液倒掉，再加入乙醚，用玻璃棒搅拌均匀，再次离心；如此重复操作洗涤5次。
液相合环合成：
将直链肽粗品用DMF稀释至10-3mol/L，置于冰浴中充分溶解后加入1.5倍量HOAt、1.5倍量HATU、10倍量DIEA，继续冰浴搅拌2小时，撤去冰浴，继续常温下搅拌，减压蒸馏后得固体残余物。
干燥：将已洗涤5次的后的多肽放入真空干燥器中真空干燥24小时。
最后的所得白色粉末即为所需多肽的粗品，称量，待纯化。
三、纯化方法步骤
1.先进行粗品的10-100％分析，确定主峰的保留时间之后，再进行线性梯度分析。
2.试样品的溶解性水和乙腈(4:1)可以充分的溶解多肽。
3.取200mg多肽溶解于10ml的溶解瓶中，超声溶解后，用0.45um的有机相滤头过滤得澄清液。
4.线性梯度制备收峰，并确定MS的对错。
5.对所收的液体进行纯度的跟踪反馈，得符合纯度要求的液体量。
6.对所收集的合格液体进行低温浓缩，浓缩温度：38℃，浓缩至50-100mL的液体量(特殊情况一般不浓缩直接冷冻)
7.把浓缩好的液体量放在100mL的烧杯中，液氮或干冰冷冻成固体状态。
8.干燥
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝507.1，[M+4H]4+＝633.6，[M+3H]3+＝844.4。在这几个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.61％。
制备实施例2
C19烷基
-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:2-7)的制备
制备工艺如制备实施例1所述，所需氨基酸原料为半胱氨酸、丙氨酸、脯氨酸、精氨酸、亮氨酸、半胱氨酸、组氨酸、赖氨酸、甘氨酸、D型甲硫氨酸、苯丙胺酸、十八烷二酸单叔丁酯、γ谷氨酸。其他方法制备实施例1，在第一个氨基酸反应完全后，开始连接下一个氨基酸Pro，随后重复以上b-f这五个步骤，分别将Gly、DMet、Pro、Gly、Lys、His、Cys、Leu、Arg、Pro、Arg、Cys连接。即得到标题序列。最后脱除FmocPhe-OH上面的保护基Fmoc，使N端裸露出来。最后缩合的氨基酸是十八烷二酸单叔丁酯和γ谷氨酸，这些氨基酸没有Fmoc基团保护，因此缩合后不需要脱Fmoc保护。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+4H]4+＝513.0，[M+3H]3+＝683.6，[M+2H]2+＝1025.6。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.17％。
野生型apelin-13多肽的氨基酸序列为Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe(用单字母表示为QRPRLSHKGPMPF)。
MM07氨基酸序列为Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Met-Pro-Phe(disulfide bridge 1-6，即1、6位的Cys形成二硫键)(用单字母表示为CRPRLCHKGPMPF(disulfide bridge1-6))。
制备实施例3
FC融合蛋白的制备：
合成目的蛋白序列如下：
将目的基因构建在TAC表达载体上，转化到大肠杆菌中扩增保存菌液，用于后续在大肠杆菌中表达纯化。
1.接菌：1管甘油菌+25ml LB(1:1000A+)，37℃摇床过夜，5mL菌液：500mL LB(1:1000A+)转大瓶(大瓶LB提前灭菌)，37℃摇床2-3h，每小时测OD值，OD值0.6-0.8时，将菌放置16℃，220rpm摇床预冷。
2.诱导：0.5M IPTG终浓度0.3mM加入菌液中，16℃，220rpm摇床20h。
3.收菌：6000g，10min
4.重悬：1:5-10加入裂解缓冲液重悬菌体，后倒入烧杯。加入溶菌酶(终浓度0.5mg/mL)，DNase(1:100)和1mM PMSF。
5.搅拌：4℃搅拌0.5h。
6.裂解：利用高压均质机破碎细胞至溶液澄清。
7.离心：30000g，4℃，30min。
8.收上清：将离心上清液收到烧杯中，并于上清中加入30mM咪唑。
9.平衡Ni柱：取3ml beads于柱中，先水后平衡缓冲液分别洗5CV。
10.挂柱：将上清缓慢加入Ni柱。重复1至2遍。
11.洗涤:洗涤缓冲液洗约20CV。
12.洗脱:洗脱缓冲液洗脱蛋白，收集洗脱液。
13.测浓度：检测蛋白含量。
14.浓缩蛋白洗脱液：用10KD浓缩管浓缩至500μL。
15.离心：浓缩后转移至1.5mL EP管中12000rpm离心10min。
16.过分子筛。
17.浓缩：收集分子筛出峰位置目的蛋白洗脱液，浓缩至250μL。
18.离心，转移到新的EP管中，测浓度，分装至0.2mg每管，液氮速冻，存于-80℃保存。
19.SDS-page表征：将纯化得到的目的蛋白进行蛋白质电泳，与蛋白maker比较蛋白确实在目的蛋白的位置，表明纯化得到的确实是目标蛋白序列，此外，通过条带确定目的蛋白纯度较高，可达到90％以上。结果如图3所示。
本申请中使用到的其余突变体多肽序列如表1所示。
表1

制备实施例4
C19烷基
-CO-2×γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)的制备
制备工艺如制备实施例1所述，所需氨基酸原料为半胱氨酸、丙氨酸、脯氨酸、精氨酸、亮氨酸、半胱氨酸、组氨酸、赖氨酸、甘氨酸、D型甲硫氨酸、D型亮氨酸、D型精氨酸、苯丙胺酸、十九烷基羧酸/酰卤、γ谷氨酸。其他方法制备实施例1，取带有酰胺接头的树脂，裂解后自动形成C端酰胺，将树脂装入SPPS反应柱中。用DMF(二甲基甲酰胺)充分溶胀树脂至少30分钟。用20％哌啶/DMF溶液处理树脂2次，每次5-10分钟，以去除树脂上的初始Fmoc保护基团。将Fmoc-Phe(tBu)-OH(Fmoc保护的苯丙氨酸，tBu保护羧基)与DIC(二异丙基碳二亚胺)和Oxyma Pure(或HOBt)混合，活化后加到树脂上。反应通常在DMF中进行，搅拌30-60分钟。在第一个氨基酸反应完全后，开始连接下一个氨基酸Pro，随后重复以上b-f这五个步骤，分别将DLeu、DMet、Pro、Gly、Arg、His、Cys、Leu、Arg、Pro、Arg、Cys、DArg、Dleu、DArg连接。随后脱除FmocPhe-OH上面的保护基Fmoc，使N端裸露出来。最后缩合的氨基酸是C19烷基羧酸和γ谷氨酸，这些氨基酸没有Fmoc基团保护，因此缩合后不需要脱Fmoc保护。随后裂解纯化步骤如制备实施例1所示。
MS表征：质谱显示的是多肽的质荷比，5个目标峰：[M+6H]6+＝449.1，[M+5H]5+＝538.7，[M+4H]4+＝673.0，[M+3H]3+＝897.0，[M+2H]2+＝1345.0。在这5个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.20％。
制备实施例5
C19烷基
-CO-2×γGlu-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)的制备
制备工艺如制备实施例1所述，所需氨基酸原料为半胱氨酸、丙氨酸、脯氨酸、精氨酸、亮氨酸、半胱氨酸、组氨酸、赖氨酸、甘氨酸、D型甲硫氨酸、D型亮氨酸、D型精氨酸、D型丙氨酸、苯丙胺酸、十九烷基羧酸/酰卤、γ谷氨酸。其他方法制备实施例1，取带有酰胺接头的树脂，裂解后自动形成C端酰胺，将树脂装入SPPS反应柱中。用DMF(二甲基甲酰胺)充分溶胀树脂至少30分钟。用20％哌啶/DMF溶液处理树脂2次，每次5-10分钟，以去除树脂上的初始Fmoc保护基团。将Fmoc-Phe(tBu)-OH(Fmoc保护的苯丙氨酸，tBu保护羧基)与DIC(二异丙基碳二亚胺)和Oxyma Pure(或HOBt)混合，活化后加到树脂上。反应通常在DMF中进行，搅拌30-60分钟。在第一个氨基酸反应完全后，开始连接下一个氨基酸Pro，随后重复以上b-f这五个步骤，分别将DLeu、DMet、Pro、Gly、Arg、His、Cys、Leu、Arg、Pro、Arg、Cys、DArg、DAla、DArg、DArg、Dleu、DArg连接。随后脱除FmocPhe-OH上面的保护基Fmoc，使N端裸露出来。最后缩合的氨基酸是C19烷基羧酸和γ谷氨酸，这些氨基酸没有Fmoc基团保护，因此缩合后不需要脱Fmoc保护。随后裂解纯化步骤如制备实施例1所示。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+6H]6+＝498.7，[M+5H]5+＝598.3，[M+4H]4+＝747.6，[M+3H]3+＝996.4。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.30％。
制备实施例6
Ac-DLys(2×OEG-γGlu-C19烷基
-CO)-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)的制备
制备工艺如制备实施例1所述，所需氨基酸原料为半胱氨酸、丙氨酸、脯氨酸、精氨酸、亮氨酸、半胱氨酸、组氨酸、赖氨酸、甘氨酸、D型甲硫氨酸、D型亮氨酸、D型精氨酸、苯丙胺酸、十九烷基羧酸/酰卤、γ谷氨酸、寡聚乙二醇。其他方法制备实施例1，取带有酰胺接头的树脂，裂解后自动形成C端酰胺，将树脂装入SPPS反应柱中。用DMF(二甲基甲酰胺)充分溶胀树脂至少30分钟。用20％哌啶/DMF溶液处理树脂2次，每次5-10分钟，以去除树脂上的初始Fmoc保护基团。将Fmoc-Phe(tBu)-OH(Fmoc保护的苯丙氨酸，tBu保护羧基)与DIC(二异丙基碳二亚胺)和Oxyma Pure(或HOBt)混合，活化后加到树脂上。反应通常在DMF中进行，搅拌30-60分钟。在第一个氨基酸反应完全后，开始连接下一个氨基酸Pro，随后重复以上b-f这五个步骤，分别将DLeu、DMet、Pro、Gly、Arg、His、Cys、Leu、Arg、Pro、Arg、Cys、DArg、Dleu、DArg、DLys连接。随后脱除FmocPhe-OH上面的保护基Fmoc，使N端裸露出来。最后缩合的氨基酸是C19烷基羧酸、寡聚乙二醇和γ谷氨酸，这些氨基酸没有Fmoc基团保护，因此缩合后不需要脱Fmoc保护。随后裂解纯化步骤如制备实施例1所示。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝604.8，[M+4H]4+＝755.8，[M+3H]3+＝1007.5。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.52％。
制备实施例7
DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)的制备
制备工艺如制备实施例1所述，所需氨基酸原料为半胱氨酸、丙氨酸、脯氨酸、精氨酸、亮氨酸、半胱氨酸、组氨酸、赖氨酸、甘氨酸、D型甲硫氨酸、D型亮氨酸、D型精氨酸、苯丙胺酸。其他方法制备实施例1，取带有酰胺接头的树脂，裂解后自动形成C端酰胺，将树脂装入SPPS反应柱中。用DMF(二甲基甲酰胺)充分溶胀树脂至少30分钟。用20％哌啶/DMF溶液处理树脂2次，每次5-10分钟，以去除树脂上的初始Fmoc保护基团。将Fmoc-Phe(tBu)-OH(Fmoc保护的苯丙氨酸，tBu保护羧基)与DIC(二异丙基碳二亚胺)和Oxyma Pure(或HOBt)混合，活化后加到树脂上。反应通常在DMF中进行，搅拌30-60分钟。在第一个氨基酸反应完全后，开始连接下一个氨基酸Pro，随后重复以上b-f这五个步骤，分别将DLeu、DMet、Pro、Gly、Arg、His、Cys、Leu、Arg、Pro、Arg、Cys、DArg、Dleu、DArg、DLys连接。随后脱除FmocPhe-OH上面的保护基Fmoc，使N端裸露出来，随后裂解纯化步骤如制备实施例1所示。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝422.1，[M+4H]4+＝527.4，[M+3H]3+＝702.9。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.70％。
制备实施例8
C19烷基
-CO-γGlu-2×OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)的制备
制备工艺如制备实施例1所述，所需氨基酸原料为半胱氨酸、丙氨酸、脯氨酸、精氨酸、亮氨酸、半胱氨酸、组氨酸、赖氨酸、甘氨酸、D型甲硫氨酸、D型亮氨酸、D型精氨酸、苯丙胺酸、十九烷基羧酸/酰卤、γ谷氨酸、寡聚乙二醇。其他方法制备实施例1，取带有酰胺接头的树脂，裂解后自动形成C端酰胺，将树脂装入SPPS反应柱中。用DMF(二甲基甲酰胺)充分溶胀树脂至少30分钟。用20％哌啶/DMF溶液处理树脂2次，每次5-10分钟，以去除树脂上的初始Fmoc保护基团。将Fmoc-Phe(tBu)-OH(Fmoc保护的苯丙氨酸，tBu保护羧基)与DIC(二异丙基碳二亚胺)和Oxyma Pure(或HOBt)混合，活化后加到树脂上。反应通常在DMF中进行，搅拌30-60分钟。在第一个氨基酸反应完全后，开始连接下一个氨基酸Pro，随后重复以上b-f这五个步骤，分别将DLeu、DMet、Pro、Gly、Arg、His、Cys、Leu、Arg、Pro、Arg、Cys、DArg、Dleu、DArg、DLys连接。随后脱除FmocPhe-OH上面的保护基Fmoc，使N端裸露出来。最后缩合的氨基酸是C19烷基羧酸、寡聚乙二醇和γ谷氨酸，这些氨基酸没有Fmoc基团保护，因此缩合后不需要脱Fmoc保护。随后裂解纯化步骤如制备实施例1所示。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝570.9，[M+4H]4+＝713.3，[M+3H]3+＝950.6。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.20％。
制备实施例9
Ac-Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝427.0，[M+4H]4+＝570.1，[M+3H]3+＝855.0。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.10％。
制备实施例10
Ac-Lys-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝424.4，[M+4H]4+＝565.5，[M+3H]3+＝848.0。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.22％。
制备实施例11
Ac-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:3-8)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝424.4，[M+4H]4+＝565.5，[M+3H]3+＝847.4。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为97.20％。
制备实施例12
Ac-Lys-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:3-8)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝374.2，[M+4H]4+＝467.4，[M+4H]3+＝622.8。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为98.67％。
制备实施例13
Ac-Arg-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:3-8)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝428.1，[M+4H]4+＝534.7。在这2个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为6.26％。
制备实施例14
Ac-Lys-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:5-10)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝428.1，[M+4H]4+＝534.7。在这2个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.61％。
制备实施例15
Ac-Leu-Arg-Gly-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:5-10)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝413.8，[M+4H]4+＝516.9。在这2个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.18％。
制备实施例16
Arg-Leu-Arg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝422.1，[M+4H]4+＝527.2，[M+3H]3+＝702.7。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.27％。
制备实施例17
Ac-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]5+＝430.4，[M+4H]4+＝537.8，[M+3H]3+＝716.6。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.75％。
制备实施例18
Ac-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝424.2，[M+4H]4+＝566.0，[M+3H]3+＝847.4。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.40％。
制备实施例19
COOHC16烷基
-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝496.0，[M+4H]4+＝619.7，[M+3H]3+＝826。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.28％。
制备实施例20
COOHC18烷基
-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]6+＝418.1，[M+5H]5+＝501.5，[M+4H]4+＝626.8，[M+3H]3+＝835.2，[M+4H]2+＝1252.3。在这5个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.48％。
制备实施例21
COOHC16烷基
-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]5+＝554.0，[M+4H]4+＝692.3，[M+3H]3+＝922.7，[M+5H]6+＝1383.8。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.51％。
制备实施例22
COOHC18烷基
-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Leu-His-Arg-Gly-Pro-DMet-GPF-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]6+＝559.7，[M+5H]5+＝699.33，[M+4H]4+＝932.1，[M+3H]3+＝1397.6。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.84％。
制备实施例23
COOHC16烷基
-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]5+＝521.8，[M+5H]4+＝652.0，[M+4H]3+＝868.8，。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为96.37％。
制备实施例24
COOHC18烷基
-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-GPF-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]5+＝527.4，[M+4H]4+＝659.0，[M+3H]3+＝878.3，[M+3H]2+＝1316.8。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.76％。
制备实施例25
COOHC18烷基
-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]5+＝495.9，[M+5H]4+＝619.6，[M+4H]3+＝826.0，[M+3H]2+＝1283.3。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.49％。
制备实施例26
COOHC16烷基
-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]5+＝551.6，[M+4H]4+＝689.2，[M+3H]3+＝918.6，[M+3H]2+＝1377.3。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.85％。
制备实施例26
COOHC18烷基
-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝554.1，[M+4H]4+＝692.3，[M+3H]3+＝922.7。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为97.32％。
制备实施例27
COOHC18烷基
-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-PF-NH2(Disulfide bridge:5-10)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，4个目标峰：[M+5H]5+＝552.8，[M+4H]4+＝690.9，[M+3H]3+＝920.7，[M+3H]2+＝1380.4。在这4个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.23％。
制备实施例28
COOHC18烷基
-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-PF-NH2(Disulfide bridge:5-10)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]6+＝487.7，[M+5H]5+＝585.1，[M+4H]4+＝731.0，[M+3H]3+＝974.4。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.72％。
制备实施例29
COOHC18烷基
-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-PF-NH2(Disulfide bridge:6-11)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝604.2，[M+4H]4+＝760.3，[M+3H]3+＝1012.2。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为97.32％。
制备实施例30
COOHC18烷基
-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-PF-NH2(Disulfide bridge:6-11)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，3个目标峰：[M+5H]5+＝604.5，[M+4H]4+＝756.2，[M+3H]3+＝1002.0。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为97.32％。
制备实施例31
COOHC18烷基
-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Ar g-Leu-Arg-Arg-Gly-Pro-DMet-DLeu-PF-NH2(Disulfide bridge:7-12)的制备
制备步骤同制备实施例4。
MS表征：质谱显示的是多肽的质荷比，5个目标峰：[M+5H]6+＝525.6，[M+5H]5+＝630.6，[M+4H]4+＝788.1，[M+3H]3+＝1050.2，[M+5H]2+＝1575.1，。在这3个位置均检测到目的峰，因此，纯化得到的确实是标题多肽序列。
HPLC结果显示，标题多肽纯度为95.32％。
生物学实施例
1.Nanobit实验：
实验原理：Binary Technology(NanoBiT)是一种用于检测蛋白相互作用研究的结构互补的报告基因技术。system由2个亚基组成，大亚基Large BiT(LgBiT；18kDa)和小亚基Small BiT(SmBiT；11氨基酸)，可与靶标蛋白融合表达。LgBiT和SmBiT亚基已经过优化，拥有良好的稳定性和最小化的自联。当带有这2个亚基的融合靶标蛋白相互作用后，2个亚基会相互结合形成有活性的酶，而产生明亮的发光信号。我们将Gα蛋白与LgBiT相连，Gγ与SmBiT相连，并将受体基因克隆到pcDNA3.1载体上，通过检测Gα和Gγ的解离来反映配体激活受体偶联G蛋白的能力。
实验方法：将上述质粒共同转染到培养好HEK293T细胞的6孔板中进行表达，细胞转染24小时后将培养基换成含10％FBS的完全培养基过夜。待细胞转染24h-48h后取出转染的6孔板，用PBS清洗一遍，加1ml胰酶消化1min后用3mlDEMEM完全培养基重悬细胞，将重悬后的细胞接种到96孔板中过夜培养。次日，取出96孔板，用D-PBS清洗一遍，先加入HBSS配制的腔肠素400a底物孵育50min，读取基底信号，再加入不同浓度的配体混匀后读取配体刺激之后的信号。配体诱导的信号比上基线信号进行归一化处理来显示G蛋白的解离反应。
2.BRET实验：
实验原理：BRET实验是通过标记生物发光供体如海肾的荧光素酶(Rluc)或者荧光受体如增强型绿色或黄色荧光蛋白(EGFP或EYFP)的融合蛋白来研究蛋白之间的相互作用。当相互作用的部分在细胞内共表达，在底物腔肠素存在的情况下，蛋白距离足够近(在以内)，将发生能量从Rluc到EYFP的转移。依靠供体和受体分子的严格距离使BRET成为研究涉及到所有GPCR功能和通过激动剂和拮抗剂的调节的受体蛋白相互作用的理想工具。
实验方法：
采用BRET法检测β-arrestin招募情况。APLNR受体C端融合Rluc8片段，并将Venus片段插入β-arrestin2的N端，分别克隆到pBiT1.1质粒中。将上述质粒共同转染到培养好HEK293T细胞的6孔板中进行表达，细胞转染24小时后将培养基换成含10％FBS的完全培养基过夜。待细胞转染24h-48h后取出转染的6孔板，用PBS清洗一遍，加1ml胰酶消化1min后用3ml DMEM完全培养基重悬细胞，将重悬后的细胞接种到96孔板中过夜培养。次日，取出96孔板，用D-PBS清洗一遍，并加入40μl HBSS，然后用不同浓度的配体(5μl)刺激转染细胞40min。随后加入荧光素酶底物腔长素400h，检测BRET信号：Venus(520-560nm)，RLuc8(460-485nm)。
3.蛋白纯化
制备APLNR-Gi1结合apelin-、MM07-复合物的冷冻电镜样品。我们将人野生型(WT)APLNR、Gi1-Gβγ、scfv16基因克隆到pFastBac1载体(Invitrogen)中，并在Sf9昆虫细胞中以1：1：1的表达。为了纯化APLNR-Gi1复合物，将细胞颗粒悬浮在含有20mM HEPES pH 7.5、2mM MgCl2、100mM NaCl和蛋白酶抑制剂混合物(Bimake)的缓冲液中，然后加入1.2mg scFv16抗体、12mU/ml apyrase(Sigma)和激动剂(50μM apelin、50μM MM07)。将混合物在室温下孵育1小时以形成复合物。然后加入0.5％(w/v)十二烷基麦芽糖新戊二醇和0.1％(w/v)半琥珀酸胆固醇，4℃孵育2h。用MBP(NEB)孵育后，用20mM麦芽糖洗脱蛋白质。然后加入TEV蛋白酶1小时，去除MBP蛋白。洗脱液用100kDa的浓缩管浓缩，样品注入Superose 6increase 10/300GL柱中(GE Healthcare)，运行缓冲液含有20mM HEPES pH 7.5,100mM NaCl,0.00075％十二烷基麦麸糖新戊二醇，0.0002％(w/v)半乙酰胆固醇和0.00025％(w/v)糖皂苷元，激动剂(50μM apelin,50μM MM07)。收集目的峰组分，浓缩至10-15mg/ml，进行电镜实验。
4.数据收集及结构解析
对于APLNR-Gi1复合物，在300kV加速电压下，在Titan Krios上进行冷冻电镜数据采集。使用SerialEM软件(Schorb,Haberbosch et al.2019)，使用Gatan K2探测器在超分辨率模式下记录显微照片，apelin复合物像素大小为在约8.0电子/埃2/秒的剂量速率下获得图像堆栈，欠焦值范围为-1.0～-2.5微米。总曝光时间为8秒，每张显微照片记录40帧。共收集到2520张apelin结合的复合物照片。对于MM07复合物，以像素大小收集了共4454张显微照片，以21.3电子/埃2/秒的剂量速率记录显微照片，欠焦值范围为-0.8～-1.8微米。总曝光时间为2.35秒，每张显微照片记录36帧。使用RELION 3.1和CryoSPARC v3.1对收集的数据进行处理。使用UCSF Chimera(Pettersen,Goddard et al.2004)将模型与冷冻电镜密度图对接。使用Phenix中的“综合验证(cryo-EM)”模块精细化最终的模型。使用UCSF Chimera和UCSF Chimera X软件绘制结构图。
实施例1
APLNR-G蛋白复合物结构解析
人源APLNR的氨基酸序列如SEQ ID No.1所示(NCBI序列号：NP_005152.1)，编码基因序列如SEQ ID No.2所示(NCBI序列号：NM_005161.6)。G蛋白为人源Gi1(Gi1为Gi蛋白的一种亚型)，氨基酸序列如SEQ ID No.3所示(NCBI序列号：NP_001027619.1)，编码基因序列如SEQ ID No.4所示(NCBI序列号：NM_001032447.1)。
为了研究APLNR的配体识别机制，我们解析了平衡激动剂apelin和G蛋白偏向性激动剂MM07(CAS号：1876450-21-3)激活APLNR-Gi复合物的冷冻电镜结构，分辨率分别在和
首先，通过杆状病毒在昆虫表达系统表达，并借助scFv16抗体(实验室表达纯化，氨基酸序列SEQ ID No.5所示)稳定G蛋白三聚体的方法，纯化得到稳定的apelin-13-APLNR-Gi1(apelin-13与APLNR、Gi1复合物)和MM07-APLNR-Gi1(MM07与APLNR、Gi1复合物)蛋白复合物。随后，利用300KV冷冻电镜进行数据收集。使用RELION软件进行数据处理与三维重构，使用WIN烷COOT和PHENIX软件进行建模与结构精修，以获得高分辨的三元复合物结构。
实验结果如图1所示。实验结果表明我们解析了高分辨率的apelin-APLNR—Gi1复合物冷冻电镜结构(图1中A和B)，我们可以很好的分析配体与受体之间的相互作用。我们在HEK293细胞中过表达APLNR受体，通过nanobit实验检测apelin和MM07激活APLNR招募G蛋白能力，bret实验检测配体激活arrestin的能力。实验结果如图1中C所示，apelin激活G蛋白信号的EC50为9.793×10-10M，招募β-arrestin的EC50为8.688×10-9。MM07激活G蛋白信号的EC50为1.377×10-8M，招募β-arrestin的EC50为5.495×10-6M。与apelin相比，MM07激活arrestin的能力减弱了近1000倍，而G蛋白活性比apelin弱10倍左右。这些结果表明MM07是偏向G蛋白的激动剂，但仍然保留了相当的arrestin活性。
实施例2
APLNR偏向性信号转导机制分析
通过比较两个配体激活APLNR结合口袋的差异，明确不同激动剂与APLNR结合的特异性氨基酸位点，阐述内源性多肽apelin和G蛋白偏向性激动剂MM07的识别和激活机制。对APLNR中参与配体识别的关键氨基酸位点进行突变，并通过nanobit实验和BRET实验分别检测配体激活下游G蛋白和β-arrestin活性，以确定不同配体作用下，点突变对APLNR结合下游G蛋白和β-arrestin能力的影响，阐释偏向性信号转导机制。
结果如图2所示。apelin(A)和MM07(B)结合的APLNR结构的配体结合口袋的残基展示，多肽配体的M11和F13以及受体上与这两个氨基酸相互作用的残基的密度用表面图展示。功能实验结果表明位于“twin hotspots”的关键氨基酸突变会影响G蛋白或β-arrestin活性(图2中C、E展示的氨基酸突变后β-arrestin活性降低比G蛋白更多，突变后受体偏向结合G蛋白；图2中D、F展示的氨基酸突变后G蛋白活性降低比β-arrestin更多，突变后受体偏向结合β-arrestin)，表明受体上两个口袋“twin hotspots”是影响APLNR对下游信号分子选择性的关键。apelin和MM07的M11和F13分别插入两个口袋，与“twin hotspots”的关键氨基酸残基相互作用。因此改变与受体的相互作用的11位Met和13位Phe有助于设计G蛋白偏向的多肽激动剂。
实施例3
检测方法如实施例1所述，我们解析了激动剂激活受体结合G蛋白复合物结构，对配体结合口袋进行分析，我们确定了如图27所示的所有影响配体与受体结合的关键氨基酸残基包括：Y351.39、F782.53、W852.60、Y882.63、Y93ECL1、I1093.32、F1103.33、M1133.36、R1684.64、M183ECL2、Y185ECL2、E1985.35、Y2646.51、K2686.55、Y2716.58、S2756.62、M2887.32、F2917.35、T2957.39、Y2997.43、M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Q180ECL2和P2927.36。
我们使用与实例2相同的信号检测方法，通过nanobit检测了Gα和Gβγ解离，确定所有与apelin和MM07相互作用的氨基酸位点突变后对受体活性的影响，发现M183ECL2、F1103.33、I1093.32、K2656.55、Y351.39、Y2997.49、W852.60、Y882.63、Y93ECL1、R1684.64、Y185ECL2、E1985.35、Y2716.58、M2887.32、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2、Y182ECL2是影响受体相互作用的关键氨基酸残基，如下表△pEC50和Emax显示，这些氨基酸位点进行丙氨酸突变后，显著减弱APLNR介导的G蛋白信号，EC50相对野生型(WT)降低，因此这些氨基酸是受体和配体相互作用的关键氨基酸。
数据以平均值±SEM表示。经单因素方差分析，*P<0.05，**P<0.01，***P<0.001，ns：不显著，再经Dunnett多重比较检验，与对照组比较。ND，活性完全丧失，无法确定EC50。
表2.配体诱导野生型(WT)和APLNR突变体的Gα-Gγ解离

我们还鉴定了影响受体偏向性的关键氨基酸残基，如图2及表3所示，M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63是影响偏向性的关键氨基酸，和这些氨基酸相互作用差异，会直接影响受体对G蛋白(表2)和β-arrestin(表3)的偏向性，这也是我们多肽设计的依据。
数据以平均值±SEM表示。经单因素方差分析，*P<0.05，**P<0.01，***P<0.001，ns：不显著，再经Dunnett多重比较检验，与对照组比较。
表3.配体诱导野生型(WT)和APLNR突变体的β-arrestin招募

实施例4
为了获得活性稳定的多肽序列，我们采用多种策略进行优化，如N端加脂肪酸修饰、PEG、融合蛋白等，使用制备实施例1至3所述的方法制备本公开的多肽突变体，功能实验结果如表4所示。β-arrestin活性丧失，G蛋白活性保留，完全偏向G蛋白。
表4.多肽序列的G蛋白和β-arrestin活性及偏向性统计









实施例5
大鼠模型药代动力学性质测试
皮下给药：大鼠禁食过夜，将待测化合物用PBS溶解(浓度0.2mg/mL)，给药剂量1mg/kg。于给药后0.5h，2h，8h，24h，48h，72h，96h，120h，144h，分别从颈静脉连续取血(每次0.2mL)置于含有EDTA-K2的EP管中，4°下4000g离心5min后取上层血浆，-80℃冻存，用LC-MS/MS分析，根据测试所得的血药浓度-时间数据，采用WinNonlin软件求算药代动力学参数。
食蟹猴模型药代动力学性质测试
皮下给药：食蟹猴禁食过夜，将待测化合物用PBS溶解(浓度0.5mg/mL)，给药剂量0.5mg/kg。于给药后0.5h，2h，8h，24h，48h，72h，96h，120h，144h，168h，192h，240h，336h，504h分别从外周静脉连续取血(每次0.3mL)置于含有EDTA-K2的EP管中，2-8℃下2000xg离心10min后取上层血浆，-80℃冻存，用LC-MS/MS分析，根据测试所得的血药浓度-时间数据，采用WinNonlin软件求算药代动力学参数。
生物学实施例5
DIO小鼠造模和药效检测
饮食诱导的肥胖(DIO)小鼠通过将5周的C57BL/6小鼠置于高脂肪饮食(60kcal％脂肪)喂养约19周而生成。在高脂肪饮食喂养前记录初始体重，然后每周监测一次体重(典型的DIO小鼠体重在40至50克之间)。为随机化测量体重、体成分和摄食量。随机分组后，小鼠按以下组别进行治疗(n＝8)：共计给药34天，每日给药。
组1：DIO小鼠中替尔泊肽(tirzepatide)(0.05mpk，皮下注射)+序列216(5mpk，皮下注射)联合用药
组2：DIO小鼠中替尔泊肽(0.05mpk，皮下注射)+BGE-105(0.01mpk，灌胃给药)联合用药
组3：DIO小鼠的空白对照(给药同等体积PBS溶液，皮下注射)
组4：DIO小鼠中GLP-1受体调节剂，替尔泊肽(0.05mpk，皮下注射)
主要评估指标包括：
每日笼边观察；
两日一次体重，摄食量统计；
每周一次身体成分测量，包括肌肉质量，肌肉含量，脂肪质量，脂肪含量等；体成分通过(基线、第2周，第3周及第4周)或在给药完成之后利用肌肉重量/脂肪组织重量比在终点进行比较。
实验结束后，通过组织解剖收集和肌肉，脂肪重含量的终点身体组成。
解剖当天，收集血浆和组织/器官。称重组织并进行进一步测试。
组织：一半的组织样本冷冻用于分子生物学分析，另一半固定于10％中性缓冲甲醛(或包埋于OTC)中进行组织学分析，以测量和量化脂肪：腹股沟脂肪、肾周脂肪，附睾脂肪，棕色脂肪；肌肉：比目鱼肌，趾长伸肌，胫骨前肌、腓肠肌；心脏。
图4A和图4B显示在给药期间(28天)各组DIO小鼠的体重变化状况。所有替尔泊肽治疗组都显示出体重显著下降。而组1：DIO小鼠中替尔泊肽(0.05mpk，皮下注射)+序列216(5mpk，皮下注射)联合用药表现出更为明显的体重下降趋势。图4C和图4D显示在给药期间(28天)各组DIO小鼠的Q2D摄食量和累积摄食量变化情况。所有替尔泊肽治疗组都显示出食物摄入量显著减少。
图5A至5C显示在给药前，给药1周、2周、3周以及4周各组DIO小鼠的肌肉含量变化情况。与单独使用替尔泊肽给药治疗相比，在第21天到第28天期间，使用序列216和替尔泊肽联合剂量治疗显著增加了肌肉含量。
图6A至6C显示在给药前，给药1周、2周、3周以及4周各组DIO小鼠的脂肪含量变化情况。与单独使用替尔泊肽给药治疗相比，在第21天到第28天期间，使用序列216和替尔泊肽联合剂量治疗显著降低了脂肪含量。
图7A至7C显示在给药前，给药1周、2周、3周以及4周各组DIO小鼠的脂肪质量变化情况。与单独使用替尔泊肽给药治疗相比，在第21天到第28天期间，使用序列216和替尔泊肽联合剂量治疗显著降低了脂肪含量。
图8A至8B显示在给药前，给药1周、2周、3周以及4周各组DIO小鼠的肌肉质量变化情况。与单独使用替尔泊肽给药治疗相比，在第21天时，使用序列216和替尔泊肽联合剂量治疗组的肌肉质量回升。
图9A至9B显示在给药前，给药1周、2周、3周以及4周各组DIO小鼠的肌肉质量/脂肪质量变化情况。与单独使用替尔泊肽相比，接受序列216和替尔泊肽联合剂量治疗组的小鼠肌肉/脂肪比增加。
图10A至10C显示给药终末时，各组解剖组织总肌肉含量，与单独使用替尔泊肽相比，接受序列216和替尔泊肽联合剂量治疗组的小鼠肌肉组织质量/脂肪组织质量比的显著性增加。
以上结果表明序列216能够显著增强替尔泊肽的减肥效果，并能够克服替尔泊肽可能导致的肌肉流失副作用。
图11A至11B显示单独接受序列216治疗组的小鼠体重与对照组相比体重下降11％，却不影响食欲。
图12A至12D显示单独接受序列216治疗组的小鼠体重与对照组相比附睾脂肪显著减少，棕色脂肪显著增加。表明序列216治疗能够重塑小鼠的脂肪组织构成，通过减少储存能量的白色脂肪、增加消耗能量的棕色脂肪，来发挥其潜在的体重控制或代谢改善作用。
结果：使用替尔泊肽和序列216联合给药处理的小鼠(治疗组1)导致体重减轻，同时保持肌肉功能并减少肌肉损失。
生物学实施例6
心肌细胞免疫荧光：为观察Apelin及其类似物序列221、序列250、序列253对心肌细胞形态的影响，使用大鼠心肌细胞系H9C2进行了免疫荧光实验。细胞在给予1μM PE(Phorbol 12-myristate 13-acetate)处理或不处理的基础上，分别加10μM处理细胞36小时。处理结束后，使用DAPI对细胞核进行蓝色荧光染色，鬼笔环肽(Phalloidin)对肌动蛋白细胞骨架进行红色荧光染色，在荧光显微镜下拍摄细胞大小。
TAC造模和给药：通过主动脉缩窄(Transverse Aortic Constriction,TAC)手术建立压力超负荷诱导的心力衰竭模型。小鼠经3％异氟醚吸入麻醉后，行开胸手术，于主动脉弓处(具体位置为无名动脉与左颈总动脉分叉之间)利用7-0缝合线进行结扎，并用针头辅助形成局部狭窄，随后撤出针头。术后一周，开始对小鼠进行腹腔注射给药1mpk序列221，对照组则注射等体积的PBS缓冲液。在给药后的第三周和第五周，对小鼠进行心脏超声检测，以评估心脏结构和功能的变化。
心脏超声检测：使用Vevo2100超声系统，麻醉小鼠(1％异氟醚)后获取胸骨旁短轴视图。检测左心室舒张末期内径(LVIDd)和收缩末期内径(LVIDs)。射血分数(EF％)＝[(LVIDd3-LVIDs3)/LVIDd3]×100；缩短分数(FS％)＝[(LVIDd-LVIDs)/LVIDd]×100。数据取5个连续心动周期的平均值。
QPCR：将提取的心脏样品进行RNA提取，反转录为cDNA，对ANP，Myh7，Myh6表达水平进行QPCR检测。扩增条件如下95℃预变性30秒→35个循环(95℃变性15秒→60℃退火/延伸30秒)。处理数据采用ΔΔCt法，以至少3次独立实验的mRNA表达量平均值作为结果。
心脏切片免疫组化：对TAC造模实验小鼠心肌组织用4％多聚甲醛固定，用天狼星红标记胶原纤维，Alexa Fluor 488偶联WGA染色细胞边界。Leica TCS-SP8共聚焦显微镜(20倍物镜)拍摄图像，NIH Image J软件量化细胞表面积。
图14结果表明，对于PE诱导的心肌细胞肥大，Apelin、序列221、序列250和序列253均具有抑制作用。但Apelin可引起正常心肌细胞肥大，相比之下，序列221、序列250和序列253对正常心肌细胞则无促肥大作用。
图15结果表明，在五周的干预期内，TAC+序列221组相较于TAC组，显著改善了心衰小鼠的心脏功能指标：抑制了左心室收缩末期内径(LVIDs)与左心室质量(LV Mass)的增大，并阻止了射血分数(EF％)和缩短分数(FS％)的降低。
图16表明，序列221、序列250、序列253给药组显著抑制了心肌细胞肥大。
图17表明，这些药物同样显著逆转了TAC诱导的整体心脏肥大，序列221每天给药和三天一次给药效果最佳。
图18表明，在分子水平上，序列221给药组有效降低了心房钠尿肽(ANP)的表达，并抑制了TAC引起的Myh7/Myh6比值升高。
图19中的天狼星红染色结果显示了心肌纤维化的程度。通过计算纤维化面积与细胞面积的比值，量化了心肌纤维化的水平。结果显示，序列221每天给药和三天一次给药组以及序列253给药组的心肌纤维化面积显著小于TAC组，表明序列221和序列253处理显著抑制了心脏纤维化的发生。
在本公开中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。
从前述中可以理解，尽管为了示例性说明的目的描述了本公开的具体实施方案，但是在不偏离本公开的精神和范围的条件下，本领域所述技术人员可以作出各种变形或改进。这些变形或修改都应落入本公开所附权利要求的范围。

Claims (142)

  1. Apelin-13多肽突变体，其包含
    (1)通式(I)所示的多肽：
    aa1-Arg-Pro-Arg-Leu-b1-His-cc1-Gly-Pro-DMet-Gly-Pro-Phe
    通式(I)
    其中，b1为侧链带负电荷的氨基酸，aa1选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，cc1选自C11-25烃酰基-γGlu-Lys、羧基-C10-24烃酰基-γGlu-Lys、C11-25烃酰基-m×OEG-γGlu-Lys、羧基-C10-24烃酰基-m×OEG-γGlu-Lys或PEG-Lys，并且1、6位的氨基酸残基形成酰胺键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(I)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  2. 如权利要求1所述的Apelin-13多肽突变体，其中所述aa1选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  3. 如权利要求1或2所述的Apelin-13多肽突变体，其中所述b1选自Glu和Asp。
  4. 如权利要求1至3中任一权利要求所述的Apelin-13多肽突变体，其中所述cc1选自C11-25烃酰基-γGlu-Lys、羧基-C10-24烃酰基-γGlu-Lys、羧基-C10-24烃酰基-2×OEG-γGlu-Lys、羧基-C10-24烃酰基-3×OEG-γGlu-Lys或PEG-Lys。
  5. 如权利要求1至4中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  6. 如权利要求1至5中任一权利要求所述的Apelin-13多肽突变体，其由通式(I)所示的多肽组成。
  7. 如权利要求1至6中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(I)所示的多肽为：
    C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(1，6位酰胺键)。
  8. Apelin-13多肽突变体，其包含
    (1)通式(II)所示的多肽：
    aa2-Cys-a2-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe
    通式(II)
    其中，a2选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，aa2选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(II)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  9. 如权利要求8所述的Apelin-13多肽突变体，其中所述a2选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn和Gln。
  10. 如权利要求8或9所述的Apelin-13多肽突变体，其中所述aa2选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  11. 如权利要求8至10中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  12. 如权利要求8至11中任一权利要求所述的Apelin-13多肽突变体，其由通式(II)所示的多肽组成。
  13. 如权利要求8至12中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(II)所示的多肽选自：
    C19烷基-CO-γGlu-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOH-C18烷基-CO-γGlu-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOH-C22烷基-CO-γGlu-2×OEG-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG2-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG3-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG5-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    PEG8-Cys-Ser-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
  14. Apelin-13多肽突变体，其包含
    (1)通式(III-1)所示的多肽：
    aa3-Cys-Arg-a3-Arg-b3-Cys-His-c3-Gly-Pro-d3-e3-f3-g3
    通式(III-1)
    其中，a3选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，b3为具有疏水侧链的氨基酸，c3为侧链带正电荷的氨基酸，d3和e3各自独立地选自具有疏水侧链的氨基酸或Gly，f3为具有疏水侧链的氨基酸或Pro，g3为具有疏水侧链的氨基酸，aa3选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)通式(III-2)所示的多肽：
    aa3’-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-a3’
    通式(III-2)
    其中，a3’为具有疏水侧链的氨基酸，aa3’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基二酸-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (3)与通式(III-1)所示的多肽或者通式(III-2)具有至少70％同源性并且具有相同功能的多肽。
  15. 如权利要求14所述的Apelin-13多肽突变体，其中所述a3选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn和Gln。
  16. 如权利要求14或15所述的Apelin-13多肽突变体，其中所述b3选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  17. 如权利要求14至16中任一权利要求所述的Apelin-13多肽突变体，其中所述c3选自Lys和Arg。
  18. 如权利要求14至17中任一权利要求所述的Apelin-13多肽突变体，其中所述d3和e3各自独立地选自Ala、Val、Ile、Leu、DMet、Phe、Tyr、Trp和Gly。
  19. 如权利要求14至18中任一权利要求所述的Apelin-13多肽突变体，其中所述f3选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
  20. 如权利要求14至19中任一权利要求所述的Apelin-13多肽突变体，其中所述g3选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  21. 如权利要求14至20中任一权利要求所述的Apelin-13多肽突变体，其中所述a3’选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  22. 如权利要求14至21中任一权利要求所述的Apelin-13多肽突变体，其中所述aa3和aa3’各自独立地选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  23. 如权利要求14至22中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、180ECL2或182ECL2结合。
  24. 如权利要求14至23中任一权利要求所述的Apelin-13多肽突变体，其由通式(III-1)所示的多肽或通式(III-2)所示的多肽组成。
  25. 如权利要求14至24中任一权利要求所述的Apelin-13多肽突变体，其中通式(III-1)所示的多肽选自：
    C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Leu(Disulfide bridge:1-6)；
    C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfide bridge:1-6)；
    C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
  26. 如权利要求14至24中任一权利要求所述的Apelin-13多肽突变体，其中通式(III-2)所示的多肽选自：
    C15烷基-CO-γGlu-Cys-Arg-Trp-Arg-Phe-Cys-His-Lys-Gly-Pro-Nal-Pro-Trp(Disulfide bridge:1-6)。
  27. Apelin-13多肽突变体，其包含
    (1)通式(IV)所示的多肽：
    aa4-Cys-Arg-Pro-a4-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe
    通式(IV)
    其中，a4为具有疏水侧链的氨基酸，aa3选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(IV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  28. 如权利要求27所述的Apelin-13多肽突变体，其中所述a4选自Ala、Val、Ile、Leu、Met、Phe、Tyr或Trp。
  29. 如权利要求27或28所述的Apelin-13多肽突变体，其中所述aa4选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  30. 如权利要求27至29中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、180ECL2或182ECL2结合。
  31. 如权利要求27至30中任一权利要求所述的Apelin-13多肽突变体，其由通式(IV)所示的多肽组成。
  32. 如权利要求27至31中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(IV)所示的多肽选自：
    C19烷基-CO-γGlu-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG2-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG3-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG5-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    PEG8-Cys-Arg-Pro-Ala-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
  33. Apelin-13多肽突变体，其包含
    (1)通式(V-1)所示的多肽：
    aa5-Cys-Arg-a5-Arg-b5-Cys-His-c5-Gly-Pro-d5-e5-f5-g5
    通式(V-1)
    其中，a5选自具有疏水侧链的氨基酸或者Pro，b5为具有疏水侧链的氨基酸，c5为侧链带正电荷的氨基酸，d5和e5各自独立地选自具有疏水侧链的氨基酸或Gly，f5为具有疏水侧链的氨基酸或Pro，g5为具有疏水侧链的氨基酸，aa1选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)通式(V-2)所示的多肽：
    aa5’-Cys-Arg-a5’-Arg-b5’-Cys-His-c5’-Gly-Pro-Nal-Pro-d5’
    通式(V-2)
    其中，a5’选自具有疏水侧链的氨基酸或者Pro，b5’为具有疏水侧链的氨基酸，c5’为侧链带正电荷的氨基酸，d5’为具有疏水侧链的氨基酸，aa5’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (3)与通式(V-1)所示的多肽或者通式(V-2)具有至少70％同源性并且具有相同功能的多肽。
  34. 如权利要求33所述的Apelin-13多肽突变体，其中所述a5选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
  35. 如权利要求33或34所述的Apelin-13多肽突变体，其中所述b5选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  36. 如权利要求33至35中任一权利要求所述的Apelin-13多肽突变体，其中所述c5选自Lys和Arg。
  37. 如权利要求33至36中任一权利要求所述的Apelin-13多肽突变体，其中所述d5和e5各自独立地选自Ala、Val、Ile、Leu、DMet、Phe、Tyr、Trp和Gly。
  38. 如权利要求33至37中任一权利要求所述的Apelin-13多肽突变体，其中所述f5选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
  39. 如权利要求33至38中任一权利要求所述的Apelin-13多肽突变体，其中所述g5选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  40. 如权利要求33至39中任一权利要求所述的Apelin-13多肽突变体，其中所述aa5选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  41. 如权利要求33至40中任一权利要求所述的Apelin-13多肽突变体，其中所述a5’选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
  42. 如权利要求33至41中任一权利要求所述的Apelin-13多肽突变体，其中所述b5’选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  43. 如权利要求33至42中任一权利要求所述的Apelin-13多肽突变体，其中所述c5’选自Lys和Arg。
  44. 如权利要求33至43中任一权利要求所述的Apelin-13多肽突变体，其中所述d5’选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  45. 如权利要求33至33中任一权利要求所述的Apelin-13多肽突变体，其中所述aa5’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  46. 如权利要求33至45中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  47. 如权利要求33至46中任一权利要求所述的Apelin-13多肽突变体，其由通式(V-1)所示的多肽或通式(V-2)所示的多肽组成。
  48. 如权利要求33至47中任一权利要求所述的Apelin-13多肽突变体，通式(V-1)所示的多肽选自：
    C17烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C21烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC16烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC16烷基-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C11烷基CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C13烷基CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C15烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C19烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C21烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC16烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-4×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    C17烷基-CO-γGlu-5×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
  49. 如权利要求33至47中任一权利要求所述的Apelin-13多肽突变体，通式(V-2)所示的多肽选自：
    C19烷CO-γGlu-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)；
    C17烷CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)；以及
    PEG8-Cys-Arg-Pro-Arg-Trp-Cys-His-Arg-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)。
  50. Apelin-13多肽突变体，其包含
    (1)通式(VII)所示的多肽：
    aa7-Cys-Arg-Pro-Arg-Leu-Cys-a7-Lys-Gly-Pro-DMet-Gly-Pro-Phe
    通式(VII)
    其中，a7选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸，aa7选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(VII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  51. 如权利要求50所述的Apelin-13多肽突变体，其中所述a7选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn和Gln。
  52. 如权利要求50或51所述的Apelin-13多肽突变体，其中所述aa7选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  53. 如权利要求50至52中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  54. 如权利要求50至53中任一权利要求所述的Apelin-13多肽突变体，其由通式(VII)所示的多肽组成。
  55. 如权利要求50至54中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(VII)所示的多肽选自：
    C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG2-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG3-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG5-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    PEG8-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe。
  56. Apelin-13多肽突变体，其包含
    (1)通式(VIII-1)所示的多肽：
    aa8-Cys-Arg-a8-Arg-b8-Cys-His-c8-Gly-Pro-DMet-Gly-d8-e8
    通式(VIII-1)
    其中，a8选自具有疏水侧链的氨基酸或者Pro，b8为具有疏水侧链的氨基酸，c8选自具有疏水侧链的氨基酸或者侧链带正电荷的氨基酸，d8为具有疏水侧链的氨基酸或Pro，e8为具有疏水侧链的氨基酸，aa8选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)通式(VIII-2)所示的多肽：
    aa8’-Cys-Arg-Pro-Arg-Trp-Cys-His-a8’-Gly-Pro-Nal-Pro-Phe
    通式(VIII-2)
    其中，a8’为侧链带正电荷的氨基酸，aa8’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (3)与通式(VIII-1)所示的多肽或者通式(VIII-2)具有至少70％同源性并且具有相同功能的多肽。
  57. 如权利要求56所述的Apelin-13多肽突变体，其中所述a8选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
  58. 如权利要求56或57所述的Apelin-13多肽突变体，其中所述b8选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  59. 如权利要求56至58中任一权利要求所述的Apelin-13多肽突变体，其中所述c8选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Arg和Lys。
  60. 如权利要求56至59中任一权利要求所述的Apelin-13多肽突变体，其中所述d8选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Pro。
  61. 如权利要求56至60中任一权利要求所述的Apelin-13多肽突变体，其中所述e8选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  62. 如权利要求56至61中任一权利要求所述的Apelin-13多肽突变体，其中所述aa8选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  63. 如权利要求56至62中任一权利要求所述的Apelin-13多肽突变体，其中所述a8’选自Arg和Lys。
  64. 如权利要求56至63中任一权利要求所述的Apelin-13多肽突变体，其中所述aa8’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  65. 如权利要求56至64中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  66. 如权利要求56至65中任一权利要求所述的Apelin-13多肽突变体，其由通式(VIII-1)所示的多肽或通式(VIII-2)所示的多肽组成。
  67. 如权利要求56至66中任一权利要求所述的Apelin-13多肽突变体，通式(VIII-1)所示的多肽选自：
    C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C21烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC16烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C11烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C13烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C15烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C19烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C21烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC16烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-4×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-5×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-3×OEG-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷CO-DγGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-8PEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-2×γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-3×γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    Phenyl-C17烷基-CO-PBMBA-βAla-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-Abu-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-Abu-2×γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-Abu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC12烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC14烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC16烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC20烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-4×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-2×OEG-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-DγGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-8PEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-2×γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-3×γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-PBMBA-βAla-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-Abu-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-Abu-2×γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-Abu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-5×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-3×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    COOHC18烷基-CO-γGlu-OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe。
  68. 如权利要求56至66中任一权利要求所述的Apelin-13多肽突变体，通式(VIII-2)所示的多肽为：
    C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG3-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG5-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    PEG8-Cys-Arg-Pro-Arg-Leu-Cys-Ser-Lys-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
  69. Apelin-13多肽突变体，其包含
    (1)通式(IX)所示的多肽：
    aa9-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-a9-Pro-DMet-Gly-Pro-Phe
    通式(IX)
    其中，a9为侧链为极性不带电荷的氨基酸，aa9选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(IX)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  70. 如权利要求69所述的Apelin-13多肽突变体，其中所述a9选自Ser、Thr、Asn和Gln。
  71. 如权利要求69或70所述的Apelin-13多肽突变体，其中aa9选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  72. 如权利要求69至71中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  73. 如权利要求69至72中任一权利要求所述的Apelin-13多肽突变体，其由通式(IX)所示的多肽组成。
  74. 如权利要求69至73中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(IX)所示的多肽为：
    C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC22烷基
    -CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG2-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG3-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    PEG8-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Ser-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
  75. Apelin-13多肽突变体，其包含
    (1)通式(XI-1)所示的多肽：
    aa11-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11-Pro-Phe
    通式(XI-1)
    其中，a11选自选自Ala、Phe、Nal、DNal、DMet、Glu或者Nle，aa11选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；
    (2)通式(XI-2)所示的多肽：
    aa11’-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-a11’-b11’-Pro-Phe
    通式(XI-2)
    其中，a11’选自具有疏水侧链的氨基酸或者侧链为极性不带电荷的氨基酸、His或者Pro，b11’选自具有疏水侧链的氨基酸或者Gly，aa11’选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (3)与通式(XI-1)所示的多肽或者通式(XI-2)具有至少70％同源性并且具有相同功能的多肽。
  76. 如权利要求75所述的Apelin-13多肽突变体，其中所述a11’选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn、Gln、His和Pro。
  77. 如权利要求75或76所述的Apelin-13多肽突变体，其中所述b11’选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp和Gly。
  78. 如权利要求75至77中任一权利要求所述的Apelin-13多肽突变体，其中所述aa11和aa11’各自独立地选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  79. 如权利要求75至78中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  80. 如权利要求75至79中任一权利要求所述的Apelin-13多肽突变体，其由通式(XI-1)所示的多肽或通式(XI-2)所示的多肽组成。
  81. 如权利要求75至80中任一权利要求所述的Apelin-13多肽突变体，通式(XI-1)所示的多肽选自：
    C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)。
  82. 如权利要求75至80中任一权利要求所述的Apelin-13多肽突变体，通式(XI-2)所示的多肽选自：
    C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Nal-Pro-Phe(Disulfide bridge:1-6)；
    C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
    COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
    PEG2-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
    PEG3-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；
    PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)；以及
    PEG8-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-DMet-Pro-Phe(Disulfide bridge:1-6)。
  83. Apelin-13多肽突变体，其包含
    (1)通式(XII)所示的多肽：
    aa12-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-a12-Pro-Phe
    通式(XII)
    其中，a12选自侧链带正电荷的氨基酸、具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸或者侧链带负电荷的氨基酸，aa12选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(XII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  84. 如权利要求83所述的Apelin-13多肽突变体，其中所述a12选自Arg、Lys、Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn、Gln、Asp和Glu。
  85. 如权利要求83或84所述的Apelin-13多肽突变体，其中所述aa12选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  86. 如权利要求83至85中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  87. 如权利要求83至86中任一权利要求所述的Apelin-13多肽突变体，其由通式(XII)所示的多肽组成。
  88. 如权利要求83至87中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(XII)所示的多肽选自：
    C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe(Disulfide bridge:1-6)；
    C19烷基-CO-2×γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
    C19烷基-CO-2×γGlu-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
    Ac-DLys(2×OEG-γGlu-C19烷基-CO)-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
    DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
    C19烷基-CO-γGlu-2×OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:1-6)；
    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:3-8)；
    Ac-Lys-Leu-Arg-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:5-10)；
    Ac-Leu-Arg-Gly-Ala-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:5-10)；
    Ac-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
    DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC16烷基-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC18烷基-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC16烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Leu-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC16烷基-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC18烷基-CO-γGlu-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC18烷基-CO-γGlu-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC16烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-Gly-Met-Pro-Phe-NH2(Disulfide bridge:4-9)；
    COOHC18烷基-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:5-10)；
    COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:5-10)；
    COOHC18烷基-CO-γGlu-γGlu-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:6-11)；
    COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:6-11)；以及
    COOHC18烷基-CO-γGlu-OEG-OEG-DArg-DLeu-DArg-DAla-DArg-DAla-Cys-Arg-Pro-Arg-Leu-Arg-Arg-Gly-Pro-DMet-DLeu-Pro-Phe-NH2(Disulfide bridge:7-12)。
  89. Apelin-13多肽突变体，其包含
    (1)通式(XIII)所示的多肽：
    aa13-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-a13-Phe
    通式(XIII)
    其中，a13选自具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸、侧链带负电荷的氨基酸或者His，aa13选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(XIII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  90. 如权利要求89所述的Apelin-13多肽突变体，其中所述a13选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn、Gln、Asp、Glu和His。
  91. 如权利要求89或90所述的Apelin-13多肽突变体，其中所述aa13选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  92. 如权利要求89至91中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  93. 如权利要求89至92中任一权利要求所述的Apelin-13多肽突变体，其由通式(XIII)所示的多肽组成。
  94. 如权利要求89至93中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(XIII)所示的多肽选自：
    C15烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Tyr-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe(Disulfide bridge:1-6)；
    COOHC18烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe(Disulfide bridge:1-6)；以及
    COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-DLeu-Phe(Disulfide bridge:1-6)。
  95. Apelin-13多肽突变体，其包含
    (1)通式(XIV)所示的多肽：
    aa14-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-a14
    通式(XIV)
    其中，a14选自具有疏水侧链的氨基酸、侧链为极性不带电荷的氨基酸、侧链带负电荷的氨基酸、His或者Nal，aa14选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、6位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(XIV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  96. 如权利要求95所述的Apelin-13多肽突变体，其中所述a14选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Ser、Thr、Asn、Gln、Asp、Glu、His和Nal。
  97. 如权利要求95或96所述的Apelin-13多肽突变体，其中所述aa14选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  98. 如权利要求95至97中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  99. 如权利要求95至98中任一权利要求所述的Apelin-13多肽突变体，其由通式(XIV)所示的多肽组成。
  100. 如权利要求95至99中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(XIV)所示的多肽选自：
    C19烷基-CO-γGlu-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr(Disulfide bridge:1-6)；以及
    PEG5-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-DMet-Gly-Pro-DTyr(Disulfide bridge:1-6)。
  101. Apelin-13多肽突变体，其包含
    (1)通式(XV)所示的多肽：
    aa15-Cys-a15-b15-c15-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe
    通式(XV)
    其中，a15和b15各自独立地选自侧链带正电荷的氨基酸或者Pro，c15选自具有疏水侧链的氨基酸或者侧链带正电荷的氨基酸，aa15选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、5位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(XV)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  102. 如权利要求101所述的Apelin-13多肽突变体，其中所述a15和b15各自独立地选自Arg、Lys和Pro。
  103. 如权利要求101或102所述的Apelin-13多肽突变体，其中c15选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Arg和Lys。
  104. 如权利要求101至103中任一权利要求所述的Apelin-13多肽突变体，其中所述aa15选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  105. 如权利要求101至104中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  106. 如权利要求101至105中任一权利要求所述的Apelin-13多肽突变体，其由通式(XV)所示的多肽组成。
  107. 如权利要求101至106中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(XV)所示的多肽选自：
    C19烷基-CO-γGlu-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    C17烷基-CO-γGlu-2×OEG-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
  108. Apelin-13多肽突变体，其包含
    (1)通式(XVI)所示的多肽：
    aa16-Cys-a16-b16-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe
    通式(XVI)
    其中，a16选自侧链带正电荷的氨基酸或者Pro，b16选自具有疏水侧链的氨基酸、侧链带正电荷的氨基酸或者Pro，aa16选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、C11-25烃酰基-γGlu-m×OEG、羧基-C10-24烃酰基-γGlu-m×OEG、C11-25烃酰基-γGlu-n×PEG、羧基-C10-24烃酰基-γGlu-n×PEG或PEG，并且1、4位的半胱氨酸残基形成二硫键，其中m为1至5的整数，n为1至8的整数；或者
    (2)与通式(XVI)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  109. 如权利要求108所述的Apelin-13多肽突变体，其中所述a16选自Arg、Lys和Pro。
  110. 如权利要求108或109所述的Apelin-13多肽突变体，其中b16选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Arg、Lys和Pro。
  111. 如权利要求108至110中任一权利要求所述的Apelin-13多肽突变体，其中所述aa16选自C11-25烃酰基-γGlu、羧基-C10-24烃酰基-γGlu、羧基-C10-24烃酰基-γGlu-2×OEG、羧基-C10-24烃酰基-γGlu-3×OEG或PEG。
  112. 如权利要求108至111中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  113. 如权利要求108至112中任一权利要求所述的Apelin-13多肽突变体，其由通式(XVI)所示的多肽组成。
  114. 如权利要求108至113中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(XVI)所示的多肽选自：
    C19烷基-CO-γGlu-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；
    COOHC22烷基-CO-γGlu-2×OEG-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)；以及
    PEG5-Cys-Arg-Leu-Cys-His-Arg-Gly-Pro-DMet-Gly-Pro-Phe(Disulfide bridge:1-6)。
  115. Apelin-13多肽突变体，其包含
    (1)通式(XVII)所示的多肽：
    *-Arg-a17-b17-Leu-*-His-C17-Gly-Pro-DMet-Gly-Pro-Phe
    通式(XVII)
    其中，a17和b17各自独立地选自侧链带正电荷的氨基酸，或者a17和/或b17不存在，c17为侧链带正电荷的氨基酸，*为非天然氨基酸，并且*形成环状结构；或者
    (2)与通式(XVII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  116. 如权利要求115所述的Apelin-13多肽突变体，其中所述a17和b17各自独立地选自Arg、Lys和Pro。
  117. 如权利要求115所述的Apelin-13多肽突变体，其中所述a17和b17均不存在。
  118. 如权利要求115至117中任一权利要求所述的Apelin-13多肽突变体，其中b16选自Arg和Lys。
  119. 如权利要求115至118中任一权利要求所述的Apelin-13多肽突变体，其中*选自
  120. 如权利要求115至119中任一权利要求所述的Apelin-13多肽突变体，其中*之间形成环状结构为订书肽结构。
  121. 如权利要求如权利要求115至120中任一权利要求所述的Apelin-13多肽突变体，其中*之间形成环状结构为
  122. 如权利要求115至121中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  123. 如权利要求115至122中任一权利要求所述的Apelin-13多肽突变体，其由通式(XVII)所示的多肽组成。
  124. 如权利要求115至123中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(XVII)所示的多肽选自：

  125. Apelin-13多肽突变体，其包含
    (1)通式(XVIII)所示的多肽：
    a18-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b18-Pro-Phe通式(XVIII)
    其中，a18选自：
    MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGA
    MKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGA
    MKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGGAGGAGGAGGAGGA
    MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGARLR
    MKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGARLR
    MKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGGAGGAGGAGGAGGARLR
    b18选自具有疏水侧链的氨基酸或者侧链带负电荷的氨基酸，并且1、6位的半胱氨酸残基形成二硫键；或者
    (2)与通式(XVIII)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  126. 如权利要求125所述的Apelin-13多肽突变体，其中所述b18选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Asp和Glu。
  127. 如权利要求125或126所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  128. 如权利要求125至127中任一权利要求所述的Apelin-13多肽突变体，其由通式(XVIII)所示的多肽组成。
  129. 如权利要求125至128中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(XVIII)所示的多肽选自：
    SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:6和SEQ ID NO:8。
  130. Apelin-13多肽突变体，其包含
    (1)通式(XIX)所示的多肽：
    a19-Cys-Arg-Pro-Arg-Leu-Cys-His-Lys-Gly-Pro-b19-c19-Pro-Phe
    通式(XIX)
    其中，a19选自：
    MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGA
    MKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGA
    MKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGGAGGAGGAGGAGGA
    MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGARLR
    MKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGAGGAGGAGGAGGARLR
    MKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLGGAGGAGGAGGAGGARLR
    b19选自具有疏水侧链的氨基酸或者侧链带负电荷的氨基酸，c19选为具有疏水侧链的氨基酸，并且1、6位的半胱氨酸残基形成二硫键；或者
    (2)与通式(XIX)所示的多肽具有至少70％同源性并且具有相同功能的多肽。
  131. 如权利要求130所述Apelin-13多肽突变体，其中所述b19选自Ala、Val、Ile、Leu、Met、Phe、Tyr和Trp。
  132. 如权利要求130或131所述Apelin-13多肽突变体，其中所述c19选自Ala、Val、Ile、Leu、Met、Phe、Tyr、Trp、Asp和Glu。
  133. 如权利要求130至132中任一权利要求所述的Apelin-13多肽突变体，其中所述Apelin-13多肽突变体与M183ECL2、F1103.33、I1093.32、K2686.55、Y2646.51、K2656.55、Y351.39、T2957.39、Y2997.49、F782.53、W852.60、Y882.63、Y93ECL1、M1133.36、R1684.64、Y185ECL2、E1985.35、Y2716.58、S2756.62、M2887.32、F2917.35、P1634.59、L2015.38、V1644.60、S2055.42、P2927.36、M2887.32、Q180ECL2或Y182ECL2结合。
  134. 如权利要求130至133中任一权利要求所述的Apelin-13多肽突变体，其由通式(XIX)所示的多肽组成。
  135. 如权利要求130至134中任一权利要求所述的Apelin-13多肽突变体，其中所述通式(XIX)所示的多肽选自：
    SEQ ID NO:2和SEQ ID NO:5。
  136. 药物组合物，其包含权利要求1至135中任一权利要求所述的Apelin-13多肽突变体，以及药物可接受的的载体、稀释剂或赋形剂。
  137. 如权利要求136所述的药物组合物，其还包含至少另一种活性成分。
  138. 如权利要求137所述的药物组合物，其中所述至少另一种活性成分能够与GLP1R、GLP2R、GIPR、GCGR、FGF21R、NPY2R或GDF15作用。
  139. 如权利要求137或138所述的药物组合物，其中所述至少另一种活性成分选自Vurolenatide、exenatide、ROSE-010、GX-G6、orforglipron、FNP-120、UTMD-GLP-1、GLP-1 mimetics、VTC-G15、curaglutide、Exendin-4、P-11、CTRS-101、HD-7671、HTL-097、PGN-OB2、GL-0034、度拉糖肽、司美格鲁肽、ecnoglutide、supalutai、rE-4、HS-20004、BPI-3016、ZT002、ECC-5004、TJ-103、SHR-2042、MDR-001、LPXT-007、Tirzepatide、CT-868、NNC-0090-2746、AMG133、CT-388、LY3493269、VK-2735、DA4-JC、HISHS-3001、HISHS-2001、LBT-6030、SCO-094、HS-20094、GMA106、HZ010、BGM0504、HZ012、XW003+XW017、THDBH120、BEBT-808、RAY-1225、Cotadutide、Pegapamodutide、BI-456906、Mazdutide/IBI362、efinopegdutide、Pemvidutide、NN-6177、ZP-2929、oxyntomodulin、G-49、AGM-217、JNJ-54728518、DA-1726、HM-14220、DD01、PB-718、TB001、SHR-1816、TB-001、YH-25724、HEC88473、AP-026、GLP-1-Fc-PYY、ZP-7570(dapiglutide)、HTL-30023、QL-1005、LY3437943(retatrutide)、HM15211、SAR441255、HISHS-3001、MWN-101和DR10624。
  140. 治疗或预防对APLNR受体的激动响应的疾病或疾病状态的方法，其包括向需要所述方法的个体给予治疗或预防有效量的权利要求1至135中任一权利要求所述的Apelin-13多肽突变体或者权利要求136至139中任一权利要求所述的药物组合物。
  141. 如权利要求140所述的方法，其中所述疾病或疾病状态选自急性代偿失调性心力衰竭、急性心力衰竭、慢性心力衰竭、肺性高血压、心房颤动、肾病、增肌、肌肉萎缩，布鲁格达氏综合征、室性心动过速、动脉粥样硬化、高血压、再狭窄、局部缺血性心血管疾病、心肌病、心脏纤维化、心律不齐、水潴留、糖尿病、肥胖症、外周动脉病、脑血管意外、暂时性缺血发作、创伤性脑损伤、肌萎缩侧索硬化、灼伤、先兆子痫、抗衰老、镇痛，睡眠呼吸暂停综合征、肺栓塞、哮喘、应激性胃溃疡、肝脏纤维化、肺损伤、脑损伤、缺血性中风、癌症、阿尔茨海默病、帕金森病、脑卒中、肾性高血压和神经退行性疾病。
  142. 如权利要求140或141所述的方法，其中所述个体为哺乳动物，优选人类。
PCT/CN2025/101655 2024-07-05 2025-06-18 Apelin-13多肽突变体 Pending WO2026007693A1 (zh)

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