CN114437181A - GLP-1R/GCGR/GIPR triple receptor agonist and application thereof - Google Patents

GLP-1R/GCGR/GIPR triple receptor agonist and application thereof Download PDF

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CN114437181A
CN114437181A CN202210085815.9A CN202210085815A CN114437181A CN 114437181 A CN114437181 A CN 114437181A CN 202210085815 A CN202210085815 A CN 202210085815A CN 114437181 A CN114437181 A CN 114437181A
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王宇恩
张凌云
戴政清
马亚平
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Shenzhen Shenchuang Biopharmaceutical Co ltd
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Abstract

The invention discloses a GLP-1R/GCGR/GIPR triple receptor agonist and application thereof. The invention provides a polypeptide compound having a parent peptide represented by the following amino acid sequence: H-Xaa1‑Xaa2‑Glu3‑Gly4‑Xaa5‑Phe6‑Xaa7‑Xaa8‑Asp9‑Val10‑Xaa11‑Ser12‑Xaa13‑Leu14‑Glu15‑Gly16‑Gln17‑Xaa18‑Ala19‑Xaa20‑Glu21‑Phe22‑Ile23‑Ala24‑Trp25‑Leu26‑Val27‑Xaa28‑Gly29‑Arg30‑Gly31‑Lys32‑Arg33‑Asn34‑Arg35‑Asn36‑Asn37‑Ile38‑Ala39‑NH2. The polypeptide compound has the functions of reducing blood sugar, promoting insulin secretion, controlling appetite, regulating metabolic disorder, controlling lipolysis, improving calorie consumption and the like, has the characteristics of good stability, high bioactivity and the like, can be used as a GLP-1R/GCGR/GIPR triple receptor agonist, is expected to be used as a new generation of preventive or therapeutic medicine for diabetes and obesity, and can be used for preventing or treating diseases such as non-alcoholic fatty liver disease, hyperlipidemia, arteriosclerosis and the like.

Description

GLP-1R/GCGR/GIPR triple receptor agonist and application thereof
Technical Field
The invention relates to design and screening of a Glucagon-like Peptide-1 receptor (GLP-1R) and Glucose-dependent Insulinotropic Peptide receptor (GIPR) and Glucagon receptor (GCGR) triple receptor agonist analogue, in particular to a GLP-1R/GCGR/GIPR triple receptor agonist and application thereof.
Background
Diabetes is a metabolic disease characterized by hyperglycemia. Currently, diabetics have more than 4.65 billion worldwide, and more than 5 billion is expected to occur by 2025, and china has become the second largest country of diabetes next to india, with type 2 diabetes accounting for approximately 90% of the total population of diabetics. Type 2 diabetes (T2DM) is a chronic metabolic disorder characterized primarily by increased hepatic glucose output, defective pancreatic beta cell function, insufficient insulin secretion and insulin resistance, which eventually progresses to persistent hyperglycemia (Green et al, Current Pharmaceutical Design,2004, 10). T2DM is also a leading cause of renal failure, blindness and amputation, and is also closely associated with a high risk of death from cardiovascular events. Moreover, with the rapid increase in obesity, T2DM will become more prevalent, a phenomenon that is particularly prominent in developing countries. Although there are many drugs that have been approved by the FDA for the treatment of diabetes, there is still a need for new therapeutic approaches to better achieve blood glucose and weight control. The most effective method for treating type 2 diabetes today is the injection of insulin, but its side effects are the risk of hypoglycemia. Other common small molecule therapeutic drugs for diabetes, such as sulfonylurea and meglitinide, are easy to cause hypoglycemia, metformin affects vitamin absorption, and DPP-4 inhibitors may cause some defects of blood pressure increase, immune response promotion and the like.
La Barre in 1929 proposed the concept of incretins (insulin secretion from the intestine). Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are the two most active incretins found in humans to date.
GLP-1 is an endogenous incretin hormone secreted by L cells of the small intestine. Preproglucagon is a precursor of various gastrointestinal hormones, and active polypeptides such as Glucagon (Glucagon) and GLP-1 can be obtained by cutting after translation. GLP-1 Receptors are members of the B-class family of G protein-coupled Receptors (GPCRs). Class B family GPCRs include GIPR, GCGR, etc. GLP-1 is abundantly expressed in the pancreatic islets, enteric neurons and central nervous system, and moderately expressed in the lung, kidney, heart and peripheral nervous system. GLP-1 plays a role in protecting islet beta cells in the islets of Langerhans, stimulates the islet beta cells to release insulin in a glucose-dependent manner, and effectively controls postprandial blood sugar. Because of its unique mechanism of action, the risk of hypoglycemia is greatly reduced. The expression of GLP-1 in other physiological systems has the advantages of suppressing appetite, delaying gastric emptying and benefiting cardiovascular. However, GLP-1 amide is metabolically inactivated by being cut by enzymes such as dipeptidyl peptidase-4 (DPP-4) and the like in plasma and is rapidly filtered and eliminated by glomeruli, so that the half life in vivo is short and is only 1-2 min.
Oxyntomodulin (OXM) is a 37 amino acid polypeptide whose sequence includes all 29 amino acids of glucagon and an 8 amino acid extension at the C-terminus called IP-1 (interacting peptides,1981,2, Supplement2, 41-44). The sequence of OXM is: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala-NH2。
OXM was shown to activate both the GLP-1 receptor and the glucagon receptor (GCGR). Its effect on inhibiting food intake is most likely achieved by binding to the GLP-1 receptor. OXM did not elicit appetite suppression in GLP-1 receptor knockout mice, but was not affected in glucagon receptor knockout mice. Similar food intake inhibition was produced by the same molar amounts of OXM and GLP-1 (Darkin, et al Endocrinology,2001,142,10, 4244-. Clinical trials in overweight and obese patients have shown that subcutaneous OXM injection 30 minutes before meals can reduce energy intake by 25%. More importantly, all patients administered OXM had a weight loss of around 2.3 kg for 4 consecutive weeks while the control group had a weight loss of only 0.5 kg (Wynne, et al diabetes,54, 82390-.
The main pharmacological activity of glucagon is to promote hepatic glucose breakdown and glycogen neogenesis in hypoglycemic conditions (exton. advanced in Enzyme Regulation,1968, 6, 391) 407, and thus its clinical use is limited to emergency treatment of hypoglycemia caused by insulin injections or as esophageal muscle relaxants. Glucagon also has the effects of increasing lipolysis, increasing satiety, increasing thermogenesis, and energy expenditure (Habegger, t.nature Reviews Endocrinology,2010,6, 689-. Glucagon is a potential obesity treatment target due to the characteristics of increasing satiety, promoting energy consumption and the like, but the application of glucagon is limited due to the effects of increasing blood sugar, accelerating insulin resistance and the like.
Day et al administered a GLP-1/glucagon dual agonist and an equimolar concentration of a single GLP-1 receptor agonist in a DIO animal model and found that the dual agonist and the single selective GLP-1 receptor agonist were able to significantly reduce food intake, body weight and adipose tissue content compared to each other. At the same time, they may also reduce blood glucose and increase glucose tolerance.
The successful development of various GLP-1/GCG and GLP-1/GIP dual receptor agonists led to the study of single molecules that simultaneously agonize 3 target receptors. The appetite suppression effect of GLP-1, the lipolysis effect of GIP and the calorie consumption effect of glucagon are combined to provide a synergistic body mass reduction effect, so that the triple receptor agonist can effectively control blood sugar, improve glucose tolerance and improve insulin sensitivity. GLP-1 and glucagon are both derived from a pro-glucagon precursor, the GIP molecule has extremely high homology with the N-terminal of the GLP-1 and glucagon, and the N-terminal of 3 molecules are sites for affinity binding of a receptor and a ligand, which provides a basis for the guess and design of GLP-1/GCG/GIP triple receptor agonists.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a GLP-1R/GCGR/GIPR triple receptor agonist and application thereof.
GLP-1 and glucagon are both derived from a pro-glucagon precursor, and dual receptor agonists of GLP-1 and glucagon combine the caloric expenditure and lipolysis of glucagon with the delayed gastric emptying and insulinotropic effect of GLP-1, thus producing a significant weight loss effect and buffering the hyperglycemic effect of glucagon, thereby effectively controlling blood glucose. GIP (glucose-dependent insulinotropic peptide) is a polypeptide secreted from neuroendocrine K cells of the small intestine, and its physiological actions are mediated by GIPR, mainly glucose-independent insulinotropic secretion, glucagon secretion enhancement, lipid metabolism enhancement, and the like, and has a very high homology with GLP-1 and the N-terminus of glucagon. And Oxyntomodulin (OXM) has the effects of increasing energy consumption and improving liver fat metabolism by activating GCGR, and in addition to a glucagon sequence, the C-terminal of OXM extends by 8 amino acid residues. The invention mainly utilizes the C-terminal peptide sequence of OXM, GIP and GLP-1 key active sequences and combines long-acting modification to design a series of polypeptide analogs for comparison of hypoglycemic action and insulinotropic action.
The polypeptide compound of the present invention starts from a sequence having GLP-1/glucagon dual receptor agonistic activity, gradually introduces GIP residues to introduce the agonistic activity of GIP, does not destroy the agonistic activity of GLP-1/glucagon, and retains the high potency of 3 natural peptides. The peptide sequence is substituted by different special amino acids at the N-terminal enzymolysis position to prevent DPP-4 enzyme digestion; different fatty acid chains or similar fatty acid chains are connected by Lys or Cys at the 20 th amino acid position of the N terminal to prolong the action time in vivo. The 31 polypeptide compounds analog1-analog31 are designed by adopting unconventional amino acid substitution, phosphorylation amino acid, glycosylation amino acid and other modes for amino acid substitution transformation, adopting different side chain modifications for side chain modification transformation and adopting cyclization closed structure transformation. Finally, the analogs are subjected to sugar-reducing bioactivity screening, HEK293 cells highly expressing glucagon-like peptide-1 receptors are constructed by a transfection technology, GLP-1R/GCGR/GIPR agonists can act on the cells, the cAMP content of the cells stimulated by the compounds is measured, and the GLP-1R/GCGR/GIPR receptor agonistic activity of the compounds is evaluated. The designed analogues analog4, analog13, analog18, analog23 and analog29 have EC50 values below nanomole, show very good blood sugar reducing activity, perform mouse experiments on five analogue polypeptides with better cell experiments and simultaneously reduce blood sugar and promote islet secretion, and show very good blood sugar reducing and islet secretion promoting effects in the mouse experiments, wherein the analog18 has the strongest biological activity and is higher than a control product of a GLP-1 receptor agonist.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a polypeptide compound having a parent peptide represented by the amino acid sequence,
H-Xaa1-Xaa2-Glu3-Gly4-Xaa5-Phe6-Xaa7-Xaa8-Asp9-Val10-Xaa11-Ser12-Xaa13-Leu14-Glu15-Gly16 -Gln17-Xaa18-Ala19-Xaa20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Xaa28-Gly29-Arg30-Gly31-Lys32- Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2(SEQ ID NO.1)；
wherein, Xaa1Selected from His, D-His or Ac-His;
Xaa2selected from Ala, Aib, D-Ala or NmeAla;
Xaa5selected from Thr, D-Thr, Thr (O-Phospho) or Thr (O-beta-D-glucose);
Xaa7selected from Thr, D-Thr, Thr (O-Phospho) or Thr (O-beta-D-glucose);
Xaa8selected from Ser, D-Ser, Ser (O-Phospho) or Ser (O-beta-D-glucose);
Xaa11selected from Ser, D-Ser, Ser (O-Phospho) or Ser (O-beta-D-glucose);
Xaa13selected from Tyr, D-Tyr, Tyr (O-Phospho) or Tyr (O-beta-D-glucose);
Xaa18selected from Ala, Aib, D-Ala or NmeAla;
Xaa20selected from Lys (Oct-gamma Glu-AEEA-AEEA), Lys (Pal-gamma Glu-AEEA-AEEA), Lys (C16-gamma Glu-AEEA-AEEA),
Lys(C20-γGlu-AEEA-AEEA)，Lys(C22-γGlu-AEEA-AEEA)，Lys(PEG-γGlu-AEEA-AEEA)， Orn(Oct-γGlu-AEEA-AEEA)，Dap(Oct-γGlu-AEEA-AEEA)，Dab(Oct-γGlu-AEEA-AEEA)， Lys(Biotin)，
cys (Maleimide), Cys (Maleimide-Oct-gamma Glu-AEEA-AEEA), Cys (Maleimide-Pal-Oct-gamma Glu-AEEA-AEEA) or Cys (Maleimide-C20-Oct-gamma Glu-AEEA-AEEA);
Xaa28selected from Lys, Arg or D-Arg.
Further, the polypeptide compound is Asp9-Lys32Side chain amide bond ringPeptides, Glu3-Lys32Cyclic peptide or Glu with side chain amido bond15-Lys32Side chain amide bond cyclic peptides.
Further, the polypeptide compound screened by the simulation design of the present invention has a parent peptide represented by one of the following amino acid sequences:
(1) analog1-analog13 side chain modified analogue sequence:
analog1(SEQ ID NO.2)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog2(SEQ ID NO.3)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(Pal-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog3(SEQ ID NO.4)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(C16-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly 29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog4(SEQ ID NO.5)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(C20-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly 29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog5(SEQ ID NO.6)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(C22-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly 29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog6(SEQ ID NO.7)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Orn(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly 29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog7(SEQ ID NO.8)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Dap(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly 29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog8(SEQ ID NO.9)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Dab(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly 29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog9(SEQ ID NO.10)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(Biotin)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys 32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog10(SEQ ID NO.11)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Cys(Maleimide)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog11(SEQ ID NO.12)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Cys(Maleimide-Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog12(SEQ ID NO.13)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Cys(Maleimide-Pal-Oct-γGlu-AEEA-AEEA))20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26 -Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog13(SEQ ID NO.14)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-A la18-Ala19-Cys(Maleimide-C20-Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27 -Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
(2) analog14-analog28 amino acid substitution analog sequence:
analog14(SEQ ID NO.15)：
H-His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog15(SEQ ID NO.16)：
H-His1-Aib2-Glu3-Gly4-D-Thr5-Phe6-D-Thr7-D-Ser8-Asp9-Val10-D-Ser11-D-Ser12-Tyr13-Leu14-G lu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val 27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog16(SEQ ID NO.17)：
H-His1-D-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16- Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-G ly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog17(SEQ ID NO.18)：
H-His1-NmeAla2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28- Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog18(SEQ ID NO.19)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Aib18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly 29-D-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog19(SEQ ID NO.20)：
H-His1-D-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16- Gln17-D-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28 -Gly29-D-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog20(SEQ ID NO.21)：
H-His1-Aib2-Glu3-Gly4-D-Thr5-Phe6-Thr7-D-Ser8-Asp9-Val10-Ser11-Ser12-D-Tyr13-Leu14-Glu15- Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-A rg28-Gly29-D-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog21(SEQ ID NO.22)：
H-His1-Aib2-Glu3-Gly4-Thr(O-Phospho)5-Phe6-Thr7-Ser8-Asp9-Val10-Ser(O-Phospho)11-Ser12-T yr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Tr p25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog22(SEQ ID NO.23)：
H-His1-Aib2-Glu3-Gly4-Thr(O-Phospho)5-Phe6-Thr7-Ser(O-Phospho)8-Asp9-Val10-Ser(O-Phosp ho)11-Ser12-Tyr(O-Phospho)13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20- Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-A sn37-Ile38-Ala39-NH2
analog23(SEQ ID NO.24)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser(O-β-D-glucose)8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26- Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog24(SEQ ID NO.25)：
H-His1-Aib2-Glu3-Gly4-Thr(O-β-D-glucose)5-Phe6-Thr7-Ser(O-β-D-glucose)8-Asp9-Val10-Ser11- Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-A la24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH 2
analog25(SEQ ID NO.26)：
H-His1-Aib2-Glu3-Gly4-Thr(O-β-D-glucose)5-Phe6-Thr7-Ser8-Asp9-Val10-Ser(O-β-D-glucose)11- Ser12-Tyr(O-β-D-glucose)13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Gl u21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn 37-Ile38-Ala39-NH2
analog26(SEQ ID NO.27)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser(O-Phospho)8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Gl u15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog27(SEQ ID NO.28)：
Ac-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-G ln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly 29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog28(SEQ ID NO.29)：
D-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-D-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
(3) analog29-analog31 cyclic peptide analogue sequence:
analog29(SEQ ID NO.30)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2(Asp9-Lys32amido bond cyclopeptide)
analog30(SEQ ID NO.31)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2(Glu3-Lys32Amido bond cyclopeptide)
analog31(SEQ ID NO.32)：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gl n17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2(Glu15-Lys32Amido bond cyclopeptide)
In the above technical solution of the present invention, the polypeptide compound is preferably one of the following sequences (shown in the following table):
Xaa1is His, Xaa2Is Aib, Xaa5And Xaa7Is Thr, Xaa8And Xaa11Is Ser, Xaa13Is Tyr, Xaa18Is Ala, Xaa28Is Arg, then Xaa20Lys (C20-gamma Glu-AEEA-AEEA) or Cys (Maleimide-C20-gamma Glu-AEEA-AEEA), namely analog4 and analog 13;
xaa is1Is His, Xaa2Is Aib, Xaa5And Xaa7Is Thr, Xaa8And Xaa11Is Ser, Xaa13Is Tyr, Xaa18Is Aib, Xaa28Is D-Arg, then Xaa20Lys (Oct-gamma Glu-AEEA-AEEA), namely analog 18;
xaa is1Is His, Xaa2Is Aib, Xaa5And Xaa7Is Thr, Xaa8Is Ser (O-beta-D-glucose), Xaa11Is Ser, Xaa13Is Tyr, Xaa18Is Aib, Xaa28Is Arg, then Xaa20Lys (Oct-gamma Glu-AEEA-AEEA), namely analog 23;
asp as described9-Lys32The side chain forms an amide bond ring, thenXaa1Is His, Xaa2Is Aib, Xaa5And Xaa7Is Thr, Xaa8And Xaa11Is Ser, Xaa13Is Tyr, Xaa18Is Aib, Xaa28Is Arg, Xaa20Lys (Oct-gamma Glu-AEEA-AEEA), namely analog 29.
Figure RE-GDA0003585231830000091
The second aspect of the invention provides the application of the polypeptide compound as a GLP-1R/GCGR/GIPR triple receptor agonist.
The third aspect of the present invention provides the use of the polypeptide compound as a medicament for preventing and/or treating metabolic diseases.
In a fourth aspect, the present invention provides a pharmaceutical composition for preventing and/or treating metabolic diseases, which comprises any one of the polypeptide compounds or a composition thereof.
Further, the metabolic disease includes diabetes, hyperglycemia, obesity, hyperlipidemia, arteriosclerosis, fatty liver and/or diabetic complications.
Further, the fatty liver is non-alcoholic fatty liver;
the non-alcoholic fatty liver disease comprises non-alcoholic steatosis, non-alcoholic steatohepatitis, liver fibrosis and/or liver cirrhosis with combined liver fibrosis;
the diabetic complications include diabetic nephropathy, diabetic hypertension, diabetic eye disease and/or diabetic neuropathy.
The invention has the beneficial effects that:
1. the invention utilizes the common means of polypeptide molecule design to carry out reconstruction optimization on a GLP-1R/GCGR/GIPR multi-target agonist polypeptide sequence, designs a series of similar compounds analog1-analog31, and mainly comprises side chain modification, amino acid substitution modification, cyclization and closure structure modification and the like. Finally, the analogs are subjected to sugar-reducing bioactivity screening, HEK293 cells highly expressing glucagon-like peptide-1 receptors are constructed by a transfection technology, polypeptide compounds act on the cells, the cAMP content generated by the cells stimulated by the compounds is measured, and GLP-1R/GCGR/GIPR receptor agonistic activity of the compounds is evaluated. The polypeptide compound has GLP-1R/GCGR/GIPR triple receptor agonistic activity and can be used as a GLP-1R/GCGR/GIPR triple receptor agonist, wherein the EC50 values of analogues analog4, analog13, analog18, analog23 and analog29 are below nanomole, so that the polypeptide compound shows very good blood sugar reducing activity, five analogue polypeptides which are good in cell experiments are subjected to a mouse experiment for reducing blood sugar and promoting islet secretion at the same time, so that the polypeptide compound shows very good blood sugar reducing and promoting islet secretion effects in the mouse experiment, and analog18 has the strongest biological activity and is higher than a control product of the GLP-1 receptor agonist.
2. The designed and synthesized polypeptide compound has a natural polypeptide secondary structure, the alpha helix is stabilized, the alpha helix is close to an endogenous structure and is easy to combine with a target position, the tolerance to the dipeptide acyl peptidase IV is improved, the degradation speed in vivo is reduced, the serum half-life period of the compound is prolonged, and the natural pharmacological activity is kept.
The polypeptide compound of the invention obviously has the functions of reducing blood sugar and promoting insulin secretion, has the functions of suppressing appetite, reducing lipolysis calorie consumption and regulating metabolic disorder, does not generate blood sugar rise generated by glucagon when being continuously used, has the prevention and treatment purposes on obesity, and is expected to be used as an effective candidate medicament for clinically treating metabolic disorder diseases such as hyperglycemia, obesity and the like.
The polypeptide compound can obviously improve fatty liver of mice induced by high fat diet and liver steatosis and nonalcoholic steatoinflammation of db/db diabetic mice, and the GLP-1R/GCGR/GIPR three-target agonist polypeptide is expected to be used as a preventive or therapeutic drug for a new generation of nonalcoholic fatty liver diseases (including nonalcoholic steatodegeneration, nonalcoholic steatohepatitis, hepatic fibrosis and hepatic fibrosis combined cirrhosis). It is also preferable to be a drug for preventing and treating diabetic complications, including but not limited to: diabetic nephropathy, diabetic hypertension, diabetic eye diseases, diabetic neuropathy, etc.
The polypeptide compound provides a new research direction for inhibiting the level of glycometabolism and improving metabolic diseases.
Drawings
FIG. 1 shows the results of HPLC detection of analog 1;
FIG. 2 shows the results of mass spectrometric detection of analog 1.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. Unless otherwise indicated, reagents or equipment used are commercially available.
Abbreviations used in the present invention and their meanings are as follows:
abbreviations and English Means of
Alloc Allyloxycarbonyl radical
DIC Diisopropylcarbodiimide
DIEA Diisopropylethylamine
DMF N, N-dimethylformamide
DCM Methylene dichloride
Fmoc 9-fluorenylmethoxycarbonyl group
Boc Tert-butyloxycarbonyl radical
HBTU O-benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate
HATU 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate
PyBOP (benzotriazole-1-oxo) tripyrrolidinophosphonium hexafluorophosphate
HOBt 1-hydroxybenzotriazoles
HOAt N-hydroxy-7-azobenzotriazol
Pd(PPh3)4 Tetratriphenylphosphine palladium
PhSiH3 Phenyl silane
TFA Trifluoroacetic acid
TIS Trimethylpyridine
PhOH Phenol and its preparation
EDT
1, 2-ethanedithiol
Maleimide Maleimide
GLP-1 control Liraglutide
The invention adopts an N-terminal Fmoc-protection strategy to prepare the polypeptide by a standard solid-phase polypeptide synthesis method. Assembly of the peptide chains was performed manually according to standard Fmoc procedures. Fmoc-Rink Amide resin (1% crosslinking degree, 100-200 mesh, degree of substitution 0.25-0.35mmol/g) purchased from Xian blue-Xiao science and technology Co., Ltd was used as a solid phase carrier.
The following side chain protected amino acids used in the present invention are provided by the Shanghai Jier Biochemical Co., Ltd:
Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Glu (OAll) -OH, Fmoc-Gly (Allyl) -OH, Fmoc-His (Trt) -OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Lys (Alloc) -OH, Fmoc-Met-OH, Fmoc-Nle-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-S (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Trp-OH, Fmoc-Tyr (tBu) -OH, Boc-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Aib-OH, Fmoc-Orn (alloc) -OH, Fmoc-dap (alloc) -OH, Fmoc-dab (alloc) -OH, Fmoc-Glu (Oall) -OH, Fmoc-Asp (Oall) -OH, Fmoc-Thr (O-phoso) -OH, Fmoc-Ser (O-phoso) -OH, Fmoc-T yr (O-phoso) -OH, Fmoc-Thr (O- β -D-glucose) -OH, Fmoc-Ser (O- β -D-glucose) -OH, Fmoc-Tyr (O- β -D-glucose) -OH. All chemicals (from different suppliers including shanghai gill biochemistry, gongkong, etc.) were chemically pure.
The special amino acids such as Fmoc-Cys (Maleimide-Oct-gamma Glu-AEEA-AEEA) -OH, Fmoc-Cys (Maleimide-Pal-gamma Glu-AEEA-AEEA) -OH, Fmoc-Cys (Maleimide-C20-gamma Glu-AEEA-AEEA) -OH and the like are self-made.
Example 1: linear peptide resin preparation of analog1-analog9
62.5 g (20mmol) of Fmoc-Rink-Amide resin (Sub ═ 0.32mmol/g) was weighed into a reaction column, washed 3 times with DMF and swollen with DMF for 30 min. The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF. 18.66 g (60mmol) of Fmoc-Ala-OH, 8.91 g (66mmol) of HOBt were weighed, dissolved in DMF, 11.35 g DIC (90mmol) were added in an ice-water bath at 0 ℃ and activated for 5 minutes, and the mixture was reacted on a reaction column for 2 hours, followed by removal of the Fmoc protecting group with DBLK. Repeating the above procedure by coupling Fmoc-Ile-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (alloc) -OH or Fmoc-dap (alloc) -OH or (Fmoc-Dac-Dalo) -OH, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Thr (tBu) -Phe-OH, Boc-His Trt- (Aib-Glu) (OtBu) -Gly-OH; after the reaction is finished, the reaction product is sequentially washed by DMF, DCM and methanol and dried to obtain the peptide resin.
Example 2: removal of side chain protecting group Alloc from analog1-analog9
The fully protected peptide resin obtained in example 1 was washed 3 times with DCM. 43.2 g of phenylsilane were weighed out, 500ml of DCM were added and reacted for 5 minutes, 11.55 g of Pd were added0(Ph3P)4And the reaction was carried out for 1 hour. The resin was then washed 3 times with DCM, the peptide resin washed with DMF solution for 30 minutes, the resin washed 3 times with DMF and then with DCM to give the selectively Alloc-removed peptide resin for use.
The resulting resin was divided into 10 portions of 2mmol each for the synthesis of analog1 to analog 9.
Example 3: conjugation of analog1 side chain
2.31 g (6mmol) of Fmoc-AEEA-OH, 0.89 g (6.6mmol) of HOBt were weighed, dissolved in DMF, 1.13 g DIC (9mmol) were added in an ice-water bath at 0 ℃ to activate for 5 minutes, the peptide resin of example 2 was added, reacted for 2 hours, and then the Fmoc protecting group was removed with DBLK. Repeating the operation, and coupling Fmoc-AEEA-OH, Fmoc-Glu-OtBu and tert-butyl octadecanedioate according to the sequence; after the reaction was complete, it was washed 6 times with DMF, 3 times again with DCM, then 3X 10 minutes after addition of methanol and dried under vacuum to give 18.8 g of analog1 peptide resin.
Example 4: preparation of analog1
18.8 g of the peptide resin obtained in example 3 was charged into a 500ml single-neck flask, and 190ml of a lysate, TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2O90: 3:3:2:2 (vol.%), the lysate was added to a flask, the reaction was carried out at room temperature for 2.5 hours, the resin was filtered off, the resin was washed with 50ml tfa, the filtrates were combined and added to 1900ml of anhydrous ether to precipitate a white solid, the solid was centrifuged, washed with anhydrous ether, and 9.47 g of crude peptide analog1 was dried in vacuo to a white solid, and the yield was 98.13%. HPLC purity 68.88%. Purifying the crude peptide by HPLC to obtain 3.20 g of analog1 refined peptide with the purity of 98.87%, wherein the total yield of the product is 33.15%; analog1 had an average molecular weight of 5081.03, mass spectrometry showed a 4 charge peak of 1271.28, and was calculated to give an average molecular weight of 5081.12(1271.28 × 4-4 ═ 5081.12) within the target average molecular weight range. The results of mass spectrometry and purity measurements of analog1 are shown in FIGS. 1 and 2.
analog2-analog9 side chain coupling method similar to example 3, selecting amino acids or analogues to be side chain coupled, and coupling according to the side chain number order;
the preparation method of analog2-analog9 is completely identical to that of example 4. analog2-analog9 was calculated to mean molecular weights within the target range of mean molecular weights by HPLC detection and mass spectrometric detection.
Example 5: analog10 peptide resin Synthesis
6.25 g (2mmol) of Fmoc-Rink-Amide resin (Sub ═ 0.31mmol/g) was weighed into the reaction column, washed 3 times with DCM and swollen with DMF for 30 min. The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF. Fmoc-Ala-OH1.87 g (6mmol), HOBt0.89 g (6.6mmol) were weighed, dissolved in DMF, 1.13 g DIC (9mmol) was added in ice water bath at 0 ℃ for 5 min activation, charged to the column, reacted for 2 h and then the Fmoc protecting group was removed with DBLK. Repeating the above procedure by coupling Fmoc-Ile-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Cys (Maleimide) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, and the like, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Thr (tBu) -Phe-OH, Boc-His Trt) -b-Glu (OtBu) -Gly-OH; after the reaction was completed, the reaction mixture was washed with DMF, DCM and methanol in this order, and dried to obtain a peptide resin of analog 10.
The synthetic method of the peptide resin of analog11-analog13 is completely consistent with that of the peptide resin of analog10, different amino acids are selected at the 20 th site according to different peptide sequences, and special amino acids need self-made amino acid monomers.
The preparation method of analog10-analog13 is completely identical to that of example 4. analog10-analog13 was calculated to mean molecular weights within the target range of mean molecular weights by HPLC detection and mass spectrometric detection.
The method for synthesizing linear peptide resin of analog14-analog28 is completely consistent with example 1, and different amino acids are selected according to the peptide sequence design. The deprotection method was completely identical to example 2, the coupling method was completely identical to example 3, and the preparation method was completely identical to example 4. analog14-analog28 was calculated to mean molecular weights within the target range of mean molecular weights by HPLC detection and mass spectrometric detection.
Example 6: peptide resin Synthesis of analog29
62.5 g (2mmol) of Fmoc-Rink-Amide resin (Sub ═ 0.32mmol/g) was weighed into a reaction column, washed 3 times with DMF and swollen with DMF for 30 min. The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF. 1.87 g (6mmol) of Fmoc-Ala-OH and 0.89 g (6.6mmol) of HOBt were weighed, dissolved in DMF, 1.13 g DIC (9mmol) was added in an ice water bath at 0 ℃ to activate for 5 minutes, and the mixture was reacted on a reaction column for 2 hours, followed by removal of the Fmoc protecting group with DBLK. Repeating the above procedure by coupling Fmoc-Ile-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Oct-Glu-AEEA-AEEA) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu-AEEA-OH, Fmoc-Ala-OH, Fmoc-Gly-Arg-Pbf-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-OH, and Fmoc-Arg-OH, and a, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (Oall) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Thr tBu (tBu) -Phe-OH, Boc-His (Trt) -Aib-Glu (OtBu) -Gly-OH; after the reaction is finished, the reaction product is sequentially washed by DMF, DCM and methanol and dried to obtain the peptide resin.
Example 7: analog30 peptide resin Synthesis
62.5 g (2mmol) of Fmoc-Rink-Amide resin (Sub ═ 0.32mmol/g) was weighed into a reaction column, washed 3 times with DMF and swollen with DMF for 30 min. The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF. 1.87 g (6mmol) of Fmoc-Ala-OH and 0.89 g (6.6mmol) of HOBt were weighed, dissolved in DMF, 1.13 g DIC (9mmol) was added in an ice water bath at 0 ℃ to activate for 5 minutes, and the mixture was reacted on a reaction column for 2 hours, followed by removal of the Fmoc protecting group with DBLK. Repeating the above procedure by coupling Fmoc-Ile-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Oct-Glu-AEEA-AEEA) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu-AEEA-OH, Fmoc-Ala-OH, Fmoc-Gly-Arg-Pbf-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-OH, and Fmoc-Arg-OH, and a, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Thr (tBu) -Phe-OH, Fmoc-Gly-OH, Fmoc-Glu (Oall) -OH, Boc-His (Trt) -Aib-OH; after the reaction is finished, the reaction product is sequentially washed by DMF, DCM and methanol and dried to obtain the peptide resin.
Example 8: peptide resin Synthesis of analog31
62.5 g (2mmol) of Fmoc-Rink-Amide resin (Sub ═ 0.32mmol/g) was weighed into a reaction column, washed 3 times with DMF and swollen with DMF for 30 min. The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF. 1.87 g (6mmol) of Fmoc-Ala-OH and 0.89 g (6.6mmol) of HOBt were weighed, dissolved in DMF, 1.13 g DIC (9mmol) was added in an ice water bath at 0 ℃ to activate for 5 minutes, and the mixture was reacted on a reaction column for 2 hours, followed by removal of the Fmoc protecting group with DBLK. Repeating the above procedure by coupling Fmoc-Ile-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Lys (alloc) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Oct-Glu-AEEA-AEEA) -OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu-AEEA-OH, Fmoc-Ala-OH, Fmoc-Gly-Arg-Pbf-OH, Fmoc-Arg-OH, Fmoc-Arg-OH, Fmoc-OH, and Fmoc-Arg-OH, and a, Fmoc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (Oall) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Thr (tBu) -Phe-OH, Boc-His (Trt) -Aib-Glu (OtBu) -Gly-OH; after the reaction is finished, the reaction product is sequentially washed by DMF, DCM and methanol and dried to obtain the peptide resin.
Example 9: removal of Lys and Asp side chain protecting groups of analog29
The fully protected peptide resin obtained in example 6 was washed 3 times with DCM. 8.64 g of phenylsilane was weighed out, 50ml of DCM was added to the reaction column, and after 5 minutes of reaction, 2.31 g of Pd was added0(Ph3P)4Reacting for 1 hour. The resin was then washed 3 times with DCM, the peptide resin washed with DMF solution for 30 minutes, the resin washed 3 times with DMF and then with DCM to give the selectively Alloc/Oall removed peptide resin for use.
Removal of the Lys and Glu side chain protecting groups of analog30 and analog31 is exactly the same as in example 9.
Example 10: side chain looping of analog29
The peptide resin of example 9 was taken, HOAt1.36 g (10mmol) was weighed, dissolved in DMF, 1.89 g DIC (15mmol) was added in ice water bath at 0 ℃, the peptide resin was added and reacted for 2 hours, after the reaction was finished, washed with DMF 6 times, washed again with DCM 3 times, then washed with methanol 3 times for 10 minutes each time, and vacuum-dried to obtain analog29 peptide resin 17.95 g.
Example 11: preparation of analog29
17.95 g of the peptide resin obtained in example 10 was charged into a 500ml single-neck flask, and 180ml of a lysate, TFA: and (3) TIS: EDT (electro-thermal transfer coating): PhOH: h2The lysate was added to a flask and allowed to react at room temperature for 2.5 hours (vol/vol) with filtration of the resin, washing the resin with 50ml tfa, combining the filtrates, adding to 1800ml dry ether to precipitate a white solid, centrifugation, washing the solid with dry ether, and vacuum drying 8.95 g of crude peptide analog29 as a white solid in 97.25% yield. HPLC purity 63.58%. The crude peptide was purified by HPLC to obtain 3.42 g of analog29 refined peptide with 98.33% purity, with a total yield of 33.15%.
The Lys and Glu side chain looping methods of analog30 and analog31 are identical to example 10.
The preparation methods of analog30 and analog31 are completely identical to example 11.
analog29-analog31 was calculated to mean molecular weights within the target range of mean molecular weights by HPLC detection and mass spectrometric detection.
Experimental example 1: test for hypoglycemic Activity
The principle of the method for testing the hypoglycemic activity is based on a time-resolved fluorescence technology, fluorescence resonance energy transfer and a homogeneous competitive analysis mode. cAMP-Gs DYNAMICKIT reagent (Cisbio) was used for the experimentThe cassette detects cAMP and is used to detect the amount of cAMP produced in HEK293/GLP-1R cells by time-resolved homogeneous fluorescence (HTRF). cAMP produced by cells themselves or unlabeled cAMP in standards and test compounds compete with d 2-labeled cAMP for binding Eu3+Cryptate-labeled anti-cAMP mAbs (donors). The specific fluorescence signal generated by fluorescence resonance energy transfer, expressed as a ratio of 665nm/620nm, is inversely proportional to the concentration of cAMP in the standard and control or test sample.
Experimental cells: a HEK293 cell line stably expressing a GLP-1 receptor at a high level (the cell line first confirms the protein level of the GLP-1 receptor in the HEK293 cell).
Sample preparation: analogs (analog1-analog 31);
the preparation method comprises the following steps: accurately weighing about 6mg of analog1-analog31 compound, and adding 1mL of PBS buffer solution; 6mg of the control was weighed out accurately and added with 1mL of PBS buffer to prepare a stock solution of 6 mg/mL.
The specific implementation method comprises the following steps:
(1) and (3) standard curve preparation: different concentrations of solutions were prepared using cAMP Standard stock solution (2768 nM) as the mother liquor and a Standard curve was generated. The concentration of each diluted solution was: 0nM, 0.21nM, 0.76nM, 2.56nM, 10.8nM, 43.8nM, 184nM, 745 nM.
(2) Preparing a reference substance and a test substance: the control (liraglutide) and test article were formulated as 8 concentration samples (745nM to 2.1X 10-1nM)。
(3) The study on the hypoglycemic activity of the reference substance and the sample-adding analogue of the tested substance comprises the following steps: adding 5 mu L of cell suspension into each hole; ② the tested product and the reference product: adding 5 mul of test substance and reference substance solution with each concentration into each hole; cell negative controls: mu.L of each dilution of cAMP-d2 was added to each well. ③ sealing the membrane and incubating for 30 minutes at 25 ℃; adding 5 mu LcAMP-d2 antibody working solution into each hole; sealing the membrane, and incubating for 60 minutes at 25 ℃; sixthly, transferring the sample to an enzyme labeling instrument and detecting the sample by using HTRF665/620 nm.
The EC50 value of the compound was calculated using the software as the ratio of the fluorescence at 665/620nm to the logarithm of the compound concentration.
The results of the above examples for the synthesis of analog1-analog31 compounds and the EC50 of the compounds are shown in the table below.
Results of the Synthesis of analogs and results of EC50
Name (R) Weight (g) Purity of EC50(nM)
analog1 3.20 98.87％ 0.042±0.0044
analog2 3.06 98.33％ 0.059±0.0058
analog3 3.23 98.28％ 0.054±0.0055
analog4 3.40 98.95％ 0.022±0.0024
analog5 2.98 98.11％ 0.062±0.0060
analog6 3.15 98.19％ 0.786±0.0720
analog7 3.20 98.28％ 2.324±0.235
analog8 3.43 99.18％ /
analog9 3.24 98.80％ /
analog10 3.25 98.75％ /
analog11 3.06 98.66％ 0.546±0.0535
analog12 3.14 99.10％ 0.058±0.0055
analog13 2.95 99.22％ 0.030±0.0028
analog14 3.27 98.32％ 0.689±0.0664
analog15 3.41 98.65％ 1.580±0.1664
analog16 3.29 98.48％ 0.988±0.0875
analog17 3.40 98.39％ 0.869±0.0862
analog18 3.38 99.06％ 0.012±0.0013
analog19 3.22 98.33％ 0.436±0.0521
analog20 3.08 98.46％ 0.854±0.0842
analog21 3.19 99.15％ 4.976±0.538
analog22 3.26 98.38％ 3.877±0.487
analog23 3.13 99.08％ 0.032±0.0034
analog24 3.17 98.22％ 0.087±0.0077
analog25 3.33 99.13％ 0.158±0.0189
analog26 3.18 98.46％ 0.218±0.0278
analog27 2.95 98.76％ /
analog28 3.05 98.87％ /
analog29 3.42 98.33％ 0.024±0.0025
analog30 3.28 98.46％ 0.678±0.0588
analog31 3.31 98.89％ 0.216±0.0228
GLP-1 control 99.32％ 0.033±0.0035
All analogues were screened for hypoglycemic bioactivity, and EC50 values of analog4, analog13, analog18, analog23 and analog29 were all below nanomolar, and showed very good hypoglycemic activity compared with the control, so these five compounds were selected for the hypoglycemic and islet-secretory-promoting mouse experiments.
Experimental example 2: blood sugar lowering mouse experiments of analog4, analog13, analog18, analog23 and analog29
Healthy Kunming mice (16-20 g in weight) were fasted overnight and randomized into 8 groups of 5: example analogue group (15mmol glucose solution per kg body weight of mice i.p. and 20nmol analogues analog4, analog13, analog18, analog23, analog 29). Glucose control group (i.p. 15mmol of glucose solution (20%) per kg of mouse body weight); GLP-1 control group (injecting 15mmol glucose solution and 20nmol GLP-1 control substance into abdominal cavity per kilogram mouse body weight); a long-acting drug control group (injected with 15mmol of glucose solution and 20nmol of somaglutide per kilogram of mouse body weight in the abdominal cavity); blood was collected from the tail vein at 0, 30, 60, 90, 120, and 240 minutes, and then a glucose solution was injected, and the blood glucose level was measured with a glucometer.
As shown in the table below, analogue polypeptides analog4, analog13, analog18, analog23, analog29 have hypoglycemic effect compared with the glucose group mice; compared with mice in a GLP-1 control group, the analog polypeptide analog18 can still reduce blood sugar within 240min, the GLP-1 control group can effectively reduce blood sugar within about 90min, the long-acting polypeptide control group can effectively reduce blood sugar within about 120min, and the analog polypeptide analog18 effectively prolongs the drug effect time.
Time of administration 0min 30min 60min 90min 120min 240min
Glucose control 5.12±0.78 12.33±1.69 15.23±2.12 16.67±2.34 17.55±1.98 16.15±1.87
GLP-1 control 4.67±0.97 6.78±0.99 7.23±1.21 8.89±1.37 14.45±1.08 15.36±1.98
Long-acting control 4.88±1.22 7.12±1.28 7.55±1.34 8.12±1.16 8.33±1.36 14.36±1.98
analog4 4.79±1.05 7.89±1.15 9.12±1.19 10.01±2.01 9.89±1.87 13.56±1.85
analog13 5.34±1.18 8.15±1.28 10.23±2.21 10.29±2.05 10.12±1.78 10.26±2.13
analog18 5.01±0.80 6.88±0.97 7.01±1.32 7.78±0.89 7.16±1.12 7.27±0.86
analog23 4.68±0.98 6.64±1.06 8.26±1.86 8.04±1.23 7.98±2.03 10.22±1.79
analog29 4.89±1.16 7.89±1.76 9.03±2.12 8.82±1.67 8.23±1.53 13.17±1.96
Experimental example 3: insulinotropic secretion test of analog4, analog13, analog18, analog23, analog29
NOD mice (non-obese diabetic mice) were divided into 7 groups (5 mice per group), 5 experimental groups, 1 blank group, 1 control group. 80 μ l of blood was collected from the capillary sinus, and 10 μ g of analog4, analog13, analog18, analog23, analog29, 10ug of GLP-1 control, and 200ul of physiological saline were intraperitoneally injected to 7 groups of mice, and blood was collected from the capillary sinus at 0, 10, 20, 30, 40, and 50 minutes. Adding the blood sample into a centrifuge tube containing 80 mul of physiological saline, mixing uniformly, centrifuging to remove red blood cells, measuring the concentration of serum insulin by using a radioimmunoassay kit (taking an average value after removing the maximum value and the minimum value), and detecting the insulin secretion promoting effect of the compound.
As shown in the following table, the results show that both the intraperitoneal injection compound and the GLP-1 control product can remarkably promote the secretion of insulin. The ability of the analog of the invention to stimulate the production of insulin for a long time exceeds the GLP-1 reference substance, which shows that the analog polypeptide of the invention can effectively prolong the in vivo drug effect time of GLP-1.
Insulinotropic action of Compounds
Time of administration 0min 10min 20min 30min 40min 50min
Blank group 2.10×10-6 2.13×10-6 2.08×10-6 2.15×10-6 2.12×10-6 2.01×10-6
GLP-1 control group 2.10×10-6 8.55×10-6 10.31×10-6 10.45×10-6 8.60×10-6 6.24×10-6
analog4 2.18×10-6 7.89×10-6 8.68×10-6 8.45×10-6 7.66×10-6 5.87×10-6
analog13 2.21×10-6 8.23×10-6 10.23×10-6 10.98×10-6 8.75×10-6 7.67×10-6
analog18 2.09×10-6 10.88×10-6 12.15×10-6 11.67×10-6 10.87×10-6 9.89×10-6
analog23 2.15×10-6 6.69×10-6 8.85×10-6 9.02×10-6 8.24×10-6 6.53×10-6
analog29 2.16×10-6 7.87×10-6 9.98×10-6 9.36×10-6 8.24×10-6 5.35×10-6
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (8)

1. A polypeptide compound having a parent peptide represented by the amino acid sequence:
H-Xaa1-Xaa2-Glu3-Gly4-Xaa5-Phe6-Xaa7-Xaa8-Asp9-Val10-Xaa11-Ser12-Xaa13-Leu14-Glu15-Gly16-Gln17-Xaa18-Ala19-Xaa20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Xaa28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2；
wherein, Xaa1His or D-His or Ac-His;
Xaa2ala or Aib or D-Ala or NmeAla;
Xaa5thr or D-Thr or Thr (O-phoso) or Thr (O- β -D-glucose);
Xaa7thr or D-Thr or Thr (O-phoso) or Thr (O- β -D-glucose);
Xaa8ser or D-Ser or Ser (O-phos) or Ser (O- β -D-glucose);
Xaa11ser or D-Ser or Ser(O-Phospho or Ser (O-. beta. -D-glucose);
Xaa13tyr or D-Tyr or Tyr (O-phoso) or Tyr (O- β -D-glucose);
Xaa18ala or Aib or D-Ala or NmeAla;
Xaa20lys (Oct-gamma Glu-AEEA-AEEA) or Lys (Pal-gamma Glu-AEEA-AEEA) or
Lys (C16-gamma Glu-AEEA-AEEA) or Lys (C20-gamma Glu-AEEA-AEEA) or Lys (C22-gamma Glu-AEEA-AEEA) or Lys (PEG-gamma Glu-AEEA-AEEA) or Orn (Oct-gamma Glu-AEEA) or Dap (Oct-gamma Glu-AEEA-AEEA) or Dab (Oct-gamma Glu-AEEA-AEEA) or Lys (Biotin) or Cys (Maleimide) or
Cys (Maleimide-Oct-gamma Glu-AEEA-AEEA) or Cys (Maleimide-Pal-gamma Glu-AEEA-AEEA or Cys (Maleimide-C20-gamma Glu-AEEA-AEEA);
Xaa28-Lys or Arg or D-Arg.
2. The polypeptide compound of claim 1, wherein the polypeptide compound is Asp9-Lys32Cyclic peptide of side chain amide bond, Glu3-Lys32Cyclic peptide or Glu with side chain amido bond15-Lys32Side chain amide bond cyclic peptides.
3. The polypeptide compound of claim 1, wherein the polypeptide compound has a parent peptide represented by one of the following amino acid sequences,
analog1：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog2：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Pal-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog3：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(C16-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog4：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(C20-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog5：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(C22-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog6：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Orn(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog7：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Dap(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog8：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Dab(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog9：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Biotin)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog10：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Cys(Maleimide)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog11：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Cys(Maleimide-Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog12：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Cys(Maleimide-Pal-Oct-γGlu-AEEA-AEEA))20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog13：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Cys(Maleimide-C20-Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog14：
H-His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog15：
H-His1-Aib2-Glu3-Gly4-D-Thr5-Phe6-D-Thr7-D-Ser8-Asp9-Val10-D-Ser11-D-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog16：
H-His1-D-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog17：
H-His1-NmeAla2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog18：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Aib18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-D-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog19：
H-His1-D-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-D-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-D-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog20：
H-His1-Aib2-Glu3-Gly4-D-Thr5-Phe6-Thr7-D-Ser8-Asp9-Val10-Ser11-Ser12-D-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-D-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog21：
H-His1-Aib2-Glu3-Gly4-Thr(O-Phospho)5-Phe6-Thr7-Ser8-Asp9-Val10-Ser(O-Phospho)11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog22：
H-His1-Aib2-Glu3-Gly4-Thr(O-Phospho)5-Phe6-Thr7-Ser(O-Phospho)8-Asp9-Val10-Ser(O-Phospho)11-Ser12-Tyr(O-Phospho)13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog23：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser(O-β-D-glucose)8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog24:
H-His1-Aib2-Glu3-Gly4-Thr(O-β-D-glucose)5-Phe6-Thr7-Ser(O-β-D-glucose)8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog25：
H-His1-Aib2-Glu3-Gly4-Thr(O-β-D-glucose)5-Phe6-Thr7-Ser8-Asp9-Val10-Ser(O-β-D-glucose)11-Ser12-Tyr(O-β-D-glucose)13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog26：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser(O-Phospho)8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog27：
Ac-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog28：
D-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-D-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2
analog29：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2(Asp9-Lys32amido bond cyclopeptide)
analog30：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2(Glu3-Lys32Amido bond cyclopeptide)
analog31：
H-His1-Aib2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18-Ala19-Lys(Oct-γGlu-AEEA-AEEA)20-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-Lys32-Arg33-Asn34-Arg35-Asn36-Asn37-Ile38-Ala39-NH2(Glu15-Lys32Amide bond cyclic peptides).
4. Use of a polypeptide compound according to any one of claims 1-3 as a GLP-1R/GCGR/GIPR triple receptor agonist.
5. Use of the polypeptide compound according to any one of claims 1 to 3 as a medicament for the prophylaxis and/or treatment of metabolic diseases.
6. A pharmaceutical composition for preventing and/or treating metabolic diseases, which comprises the polypeptide compound according to any one of claims 1 to 3 or a composition thereof.
7. The pharmaceutical composition of claim 6, wherein the metabolic disease comprises diabetes, hyperglycemia, obesity, hyperlipidemia, arteriosclerosis, fatty liver, and/or diabetic complications.
8. The use according to claim 7, wherein the fatty liver is non-alcoholic fatty liver;
the non-alcoholic fatty liver disease comprises non-alcoholic steatosis, non-alcoholic steatohepatitis, liver fibrosis and/or liver cirrhosis with combined liver fibrosis;
the diabetic complications include diabetic nephropathy, diabetic hypertension, diabetic eye disease and/or diabetic neuropathy.
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