阅读说明：本技术 GIP-Exendin-4嵌合肽的分子改构及其二聚体在治疗糖尿病中的应用 (Molecular modification of GIP-Exendin-4 chimeric peptide and application of dimer thereof in treating diabetes ) 是由 唐松山 张旭东 罗群 杨莉 谭宏梅 唐婧晅 于 2020-03-18 设计创作，主要内容包括：本发明提供了基于胰高血糖素样肽-1受体激活剂(GLP-1R)中葡萄糖依赖性胰岛素营养多肽(GIP)-Exendin 4嵌合多肽分子变构体和其二聚体在降糖和治疗代谢综合征中的应用。本发明二聚体是两个相同的含有单个半胱氨酸变构的GIP-Exendin-4嵌合肽单体通过半胱氨酸形成的二硫键连接而成。本发明的H型二聚体(分子内单Ser→Cys替换)在不降低活性的情况下,显著增加了该二聚体的降糖持续时间,本发明的二聚体在体内持续活性长达22天,较阳性对照药Lixisenatide(2天)有明显延长。与Lixisenatide比较,相同摩尔浓度的二聚体2G21产生相似降糖活性和体重降低、诱导产生成倍胰岛素分泌并改善器官毒性的效果,拓展了二聚体的医药用途,如在代谢综合征的治疗,包括糖尿病和肥胖症治疗中的用途,以及用于减少过量食物摄取等。(The invention provides a glucagon-like peptide-1 receptor activator (GLP-1R) -based glucose-dependent insulin nutrition polypeptide (GIP) -Exendin 4 chimeric polypeptide molecular variant and application of a dimer thereof in reducing blood sugar and treating metabolic syndrome. The dimer is formed by connecting two identical GIP-Exendin-4 chimeric peptide monomers containing single cysteine allosteric through a disulfide bond formed by cysteine. The H-type dimer (single Ser → Cys replacement in a molecule) obviously prolongs the blood sugar-reducing duration time of the dimer under the condition of not reducing the activity, and the dimer has the sustained activity in vivo for 22 days, and is obviously prolonged compared with a positive control medicament Lixisenatide (2 days). Compared with Lixisenatide, the dimer 2G21 with the same molar concentration produces similar hypoglycemic activity and weight reduction, induces the production of doubled insulin secretion and improves the effect of organ toxicity, and expands the medical application of the dimer, such as the application in the treatment of metabolic syndrome, including the treatment of diabetes and obesity, and the application in reducing excessive food intake and the like.)

1. A glucagon-like peptide-1 receptor (GLP-1R) activator analog peptide is characterized in that the GLP-1R activator analog peptide is formed by the modification of a fatty acid chain on a lysine epsilon-amino group and the sequence of a GIP-Exendin-4 chimeric peptide is allosterically changed.

2. The GLP-1R activator analog peptide of claim 1, wherein the analog peptide has a specific sequence of any one of:

(1)(HN₂)Y-αAib-EGTFTCDYSI-X₁₃-LD-X₁₆-IAQ-X₂₀-AFVQWLIAGGPSSGAPP-X₃₈or;

(2)(HN₂)Y-αAib-EGTFTSDYCI-X₁₃-LD-X₁₆-IAQ-X₂₀-AFVQWLIAGGPSSGAPP-X₃₈or;

(3)(HN₂)Y-αAib-EGTFTSDYSI-X₁₃-LD-X₁₆-IAQ-X₂₀-AFVQWLIAGGPCSGAPP-X₃₈or;

(4)(HN₂)Y-αAib-EGTFTSDYSI-X₁₃-LD-X₁₆-IAQ-X₂₀-AFVQWLIAGGPSCGAPP-X₃₈or;

wherein, X₁₃Is Ala or alpha-aminoisobutyric acid (alpha Aib); x₁₆Or X₂₀Is a glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid) ]]Modified lysine, or in the side chain on the epsilon-amino group [2 XAAEEAC-gamma-Glu- (N-alpha-fatty diacid)]A modified lysine; x₃₈Is PS (HN)₂) Or SKKKKKKKK (HN)₂)。

3. The GLP-1R activator analog peptide of claim 2 wherein when X₁₆Or X₂₀The structure of the lysine modified by glutamyl fatty acid gamma-Glu (N-alpha-fatty acid) or glutamyl fatty diacid gamma-Glu (N-alpha-fatty diacid) on the side chain epsilon-amino is shown as a chemical formula 1; when X is present₁₆Or X₂₀Is [2 × AEEAC-gamma-Glu- (N-alpha-fatty diacid) on side chain epsilon-amino]Modified lysine, the structure of which is shown in chemical formula 2:

chemical formula 1:

chemical formula 2:

4. a hypoglycemic peptide mimetic homodimer formed by the disulfide linkage of identical monomers according to any one of claims 1 to 3 through cysteine to form an H-GLP-1R activator peptide mimetic homodimer.

5. The dimer of claim 4, wherein the amino acid sequence is any one of:

wherein, X₁₃Is L-alpha-alanine (A) or alpha-aminoisobutyric acid (alpha Aib); x₁₆Or X₂₀Is a glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid) ]]Modified lysine, or in the side chain on the epsilon-amino group [2 XAAEEAC-gamma-Glu- (N-alpha-fatty diacid)]A modified lysine; x₃₈Is PS (HN)₂) Or SKKKKKKKK (HN)₂) (ii) a "|" indicates a disulfide bond formed between two cysteines.

6. The dimer of claim 5, wherein when X is₁₆Or X₂₀Is a glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid) ]]Modified lysine has a structure shown in chemical formula 1; when X is present₁₆Or X₂₀Is [2 × AEEAC-gamma-Glu- (N-alpha-fatty diacid) on side chain epsilon-amino]The structure of modified lysine is shown in chemical formula 2.

7. Use of a GLP-1R activator-like peptide according to any one of claims 1-3, or a homodimer according to any one of claims 4-6, for the manufacture of a medicament for the treatment of a metabolic syndrome disorder.

8. The use of claim 7, wherein the metabolic syndrome disorder comprises hyperglycemia, diabetes, and obesity.

9. A medicament for treating a metabolic syndrome disorder, said medicament comprising a GLP-1R activator-like peptide according to any one of claims 1 to 3, or a homodimer according to any one of claims 4 to 6 and pharmaceutically acceptable salts thereof as an active ingredient.

Technical Field

The invention belongs to the field of medical biology, and particularly relates to molecular modification of GLP-1R activator analog peptide and application of homodimer thereof in treating metabolic diseases.

Background

Exendin-4 is an incretin analogue isolated from saliva of Heloderma subspecium, has 39 amino acids and 53% sequence homology with GLP-1. GIP is a gastrointestinal regulatory peptide of 42 amino acids, which has the functions of regulating the sugar metabolism of the body, promoting the release of insulin from beta cells of the pancreatic islets and reducing body weight. GLP-1 is an incretin-like peptide of 30 amino acid residues, which is released by intestinal L cells upon nutrient uptake. Exendin-4 and GLP-1 are two GLP-1R activators discovered so far. Based on the three active polypeptide amino acid sequences for regulating sugar metabolism, through structural significance changes in recent decades, sugar-reducing GLP-1R activators which are marketed or clinically approved by FDA or SFDA in the united states, such as Liraglutide (marketed in 2011) and Lixisenatide (marketed in 2016), Exenatide (marketed in 2010) once daily and Polyethylene glycol loxetade (marketed in 2019), Albiglutide (marketed in 2014), Dulaglutide (marketed in 2014), Semaglutide (marketed in 2017), tasoglutide (clinical failure in 2015) and Tirzepatide (clinical phase II in 2018), are produced each allosteric. Exenatide, Polyethylene glycol loxatenide and Lixisenatide are allosteric molecules based on the amino acid sequence of the active polypeptide Exendin-4, which have been clinically marketed. Liraglutide, Albiglutide, Dulaglutide, Semaglutide and Taspoglitide are allosteric analogs based on the amino acid sequence of the active polypeptide GLP-1, which are produced by chemical synthesis or a combination of genetic recombination and chemical synthesis. Tirzepatide is a synthetic molecule based on a GIP-Exendin-4 bifunctional receptor activator, which was developed by Lily and has now completed phase II clinics.

Nausea and vomiting are observed in most GLP-1R activators when treated. Since The hypothalamic-pituitary-adrenal axis (HPA or HTPA) is part of The physiological stress response, stimulation of The HPA axis by GLP-1R activators leads to an increase in corticosterone, leading to The development of partial arrhythmias. Therefore, there remains a need for: antagonizing the activation of glucagon and GLP-2 receptors simultaneously when activating the GIP receptor and/or the GLP-1 receptor; ② it is necessary to provide weight loss, antagonism of DPP-4 and other forms of degradation mechanisms by activating GIP receptor and/or GLP-1 receptor effects while maintaining low immunogenicity; and thirdly, the GLP-1R activator still needs to be optimized, because the existing long-acting activator is proved to be not as effective as the natural GLP-1 or Exendin-4 molecules in the aspects of specific activity (blood sugar reducing effect per unit mass), administration dosage, weight reduction and side reaction, and meanwhile, the technological progress is needed to provide a longer-acting blood sugar reducing activator and overcome toxicity.

Disclosure of Invention

The invention aims to provide a GLP-1R activator analog peptide. The GLP-1R activator analog peptide is prepared by performing molecular allosteric treatment on Exendin-4, GIP-Exendin-4 and GLP-1 respectively. On the one hand, the position of cysteine in the peptide chain was investigated with a single Cys → Ser substitution inside the monomeric peptide. On the other hand, fatty acids or fatty acid substituent modifications on the epsilon-amino group of the lysine (K) side chain in the peptide chain are allosterized, and different fatty acids or fatty acid substituents have different effects on the activity of GLP-1R activator-like peptides. The invention also provides a homodimer formed by the GLP-1R activator similar peptide, and finds that the H-type structure dimer (intramolecular single Cys → Ser substitution) formed by cysteine at different positions generates different activities, the continuous hypoglycemic time can reach 22 days at most, and the increase is obvious compared with the current clinical medicine which is used once in 1-7 days.

In order to achieve the purpose, the invention adopts the technical scheme that: a glucagon-like peptide-1 receptor (GLP-1R) activator analog peptide, wherein the GLP-1R activator analog peptide is formed by the modification of a sequence of Exendin-4, GIP-Exendin-4 chimeric peptide or GLP-1 through allosteric modification and a fatty acid chain on a lysine epsilon-amino group. The GLP-1R activator analog peptide takes an amino acid sequence of Exendin-4, GIP-Exendin-4 or GLP-1 peptide as a main chain, Ser on the main chain is replaced by Cys, and the main chain amino acid sequence only contains one Cys; the side chain is the fatty acid chain of the epsilon-amino group of one of the lysines of the backbone.

Preferably, the specific sequence of the GLP-1R activator analogue peptide is any one of the following:

(1)(HN₂)H-X₂-EGTFTCDLS-X₁₂-QMEEEAV-X₂₀-LFIEWL-X₂₇-NGGPSSGAPP-X₃₈or;

(2)(HN₂)H-X₂-EGTFTSDLC-X₁₂-QMEEEAV-X₂₀-LFIEWL-X₂₇-NGGPSSGAPP-X₃₈or;

(3)(HN₂)H-X₂-EGTFTSDLS-X₁₂-QMEEEAV-X₂₀-LFIEWL-X₂₇-NGGPCSGAPP-X₃₈or;

(4)(HN₂)H-X₂-EGTFTSDLS-X₁₂-QMEEEAV-X₂₀-LFIEWL-X₂₇-NGGPSCGAPP-X₃₈or;

(5)(HN₂)Y-X₂-EGTFTCDYSI-X₁₃-LDKIAQ-X₂₀-AFVQWLIAGGPSSGAPP-X₃₈or;

(6)(HN₂)Y-X₂-EGTFTSDYCI-X₁₃-LDKIAQ-X₂₀-AFVQWLIAGGPSSGAPP-X₃₈or;

(7)(HN₂)Y-X₂-EGTFTSDYSI-X₁₃-LDKIAQ-X₂₀-AFVQWLIAGGPCSGAPP-X₃₈or;

(8)(HN₂)Y-X₂-EGTFTSDYSI-X₁₃-LDKIAQ-X₂₀-AFVQWLIAGGPSCGAPP-X₃₈or;

(9)(HN₂)H-X₂-EGTFTSDVSCYLEGQAA-X₂₀-EFIAWLV-X₂₈-GRG(NH₂)；

wherein, X₂Or X₁₃Is L-alpha-glycine or L-alpha-alanine or alpha-aminoisobutyric acid (alpha Aib); x₁₂Or X₂₀Or X₂₇Or X₂₈Is lysine, arginine or glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on side chain epsilon-amino group]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid) ]]Modified lysine, or in the side chain on the epsilon-amino group [2 XAAEEAC-gamma-Glu- (N-alpha-fatty diacid)]A modified lysine; x₃₈Is PS (HN)₂) Or SKKKKKKKK (HN)₂). The capital letter is an abbreviation of L-alpha-amino acid or an amino acid substitution symbol, and the Arabic numerals are an amino acid residue arrangement sequence, NH₂Represents an N-terminal or C-terminal amide group structure.

Preferably, the GLP-1R activator resembles a peptide when X₁₂Or X₂₀Or X₂₇Or X₂₈Is a glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid) ]]Modified lysine has a structure shown in chemical formula 1; when X is present₁₂Or X₂₀Or X₂₇Or X₂₈Is on the side chain epsilon-amino group [2 × AEEAC-gamma-Glu- (N-alpha-fatty diacid)]The structure of modified lysine is shown in chemical formula 2.

The invention also provides a hypoglycemic peptide analogue homodimer, which is formed by connecting the same monomers as in any one of claims 1-3 through a disulfide bond formed by cysteine to form the H-type GLP-1R activator peptide analogue homodimer.

Preferably, the amino acid sequence of the dimer is any one of:

wherein, X₂Or X₁₃Is L-alpha-glycine or L-alpha-alanine or alpha-aminoisobutyric acid (alpha Aib); x₁₂Or X₂₀Or X₂₇Or X₂₈Is lysine, arginine or glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on side chain epsilon-amino group]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid) ]]Modified lysine, or in the side chain on the epsilon-amino group [2 XAAEEAC-gamma-Glu- (N-alpha-fatty diacid)]A modified lysine; x₃₈Is PS (HN)₂) Or SKKKKKKKK (HN)₂) (ii) a "|" indicates a disulfide bond formed between two cysteines.

Preferably, when X₁₂Or X₂₀Or X₂₇Or X₂₈Is a glutamyl fatty acid [ gamma-Glu (N-alpha-fatty acid) on the side chain epsilon-amino]Or glutamyl fatty diacid [ gamma-Glu (N-alpha-fatty diacid) ]]Modified lysine has a structure shown in chemical formula 1; when X is present₁₂Or X₂₀Or X₂₇Or X₂₈Is [2 × AEEAC-gamma-Glu- (N-alpha-fatty diacid) on side chain epsilon-amino]The structure of modified lysine is shown in chemical formula 2.

The invention also provides application of the GLP-1R activator similar peptide or the homodimer in preparing a medicament for treating metabolic syndrome. Preferably, the metabolic syndrome disorder includes hyperglycemia, diabetes, and obesity.

The present invention also provides a medicament for treating a disorder of metabolic syndrome, which comprises the GLP-1R activator-analogous peptide or homodimer described above and a pharmaceutically acceptable salt thereof as an active ingredient.

The invention has the beneficial effects that: the H-type GLP-1R activator analogue homodimer provided by the invention has the advantages that under the condition that the blood sugar reducing strength is not lower than that of the corresponding monomer peptide, the blood sugar reducing action time of the corresponding monomer activator or a GLP-1R activator clinical medicament approved by FDA or SFDA is remarkably prolonged by about 2-3 times, the activity maintaining time of the provided GLP-1R activator analogue homodimer in vivo is as long as 22 days, and the activity maintaining time is remarkably prolonged compared with that of a positive medicament Lixinaglutide (the medicament effect is maintained for 2 days). Compared with a clinical GLP-1R activator, the novel dimer has very obvious structural change, and greatly facilitates the clinical application and the market popularization.

Drawings

FIG. 1 is a graph showing the results of a blood glucose test on a single OGTT.

FIG. 2 is a graph of statistical analysis of body weights of 2G21 treated with T2D model.

FIG. 3 is a graph of a statistical analysis of blood glucose in the T2D model treated with 2G 21.

FIG. 4 is a graph of glycated hemoglobin in a T2D model treated with 2G 21.

FIG. 5 is a graph of statistical analysis of insulin in the T2D model treated with 2G 21.

FIG. 6 is a graph of the statistical analysis of glutamate pyruvate transaminase in the T2D model treated with 2G 21.

FIG. 7 is a graph of the statistical analysis of pancreatic amylase in the T2D model treated with 2G 21.

Detailed Description

In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments and accompanying drawings.

EXAMPLE 1 preparation of monomeric peptides and dimers

Firstly, solid phase chemical synthesis process of monomer peptide: manual solid phase peptide synthesis operation steps.

1. Swelling resin: amino resin (amino resin for C-terminal amidated sequence) (available from Nankai Synthesis science and technology Co., Tianjin) was put into a reaction vessel, and dichloromethane (DCM, Dikma technologies Inc.)15ml/g resin was added and shaken for 30min. SYMPHONY type 12-channel polypeptide synthesizer (SYMPHONY model, software version.201, Protein Technologies Inc.).

2. Grafting with the first amino acid: the solvent was removed by suction filtration through a sand core, 3-fold molar addition of the first Fmoc-amino acid at the C-terminus (all Fmoc-amino acids supplied by Suzhou Tianma pharmaceutical group Fine Chemicals, Inc.), 10-fold molar addition of 4-Dimethylaminopyridine (DMAP) and N, N' -Dicyclohexylcarbodiimide (DCC), and finally addition of Dimethylformamide (DMF) (purchased from Dikma Technologies Inc.) for dissolution and shaking for 30min. Blocking with acetic anhydride.

3. Deprotection: DMF was removed, 20% piperidine-DMF solution (15ml/g) was added for 5min, the solvent was removed by filtration, and 20% piperidine-DMF solution (15ml/g) was added for 15 min. Piperidine is supplied by Shanghai chemical company, national drug group.

4. And (3) detection: the solvent was removed by suction. Taking dozens of resins, washing the resins with ethanol for three times, adding ninhydrin, KCN and phenol solution one drop each, heating the mixture at the temperature of 105 ℃ and 110 ℃ for 5min, and turning dark blue to be a positive reaction.

5. Resin washing: two washes with DMF (10ml/g), two washes with methanol (10ml/g) and two washes with DMF (10ml/g) were performed in sequence.

6. Condensation: depending on the specific synthesis conditions, the following methods may be used alone or in combination in the polypeptide synthesis:

the method a comprises the following steps: three times of protective amino acid and three times of 2- (7-azobenzotriazol) -tetramethyluronium hexafluorophosphate (HBTU, Suzhou Tianma pharmaceutical group fine chemicals Co., Ltd.) were dissolved in DMF as little as possible and added to the reaction vessel. Ten times of N-methylmorpholine (NMM, Suzhou Tianma pharmaceutical group, Fine chemical Co., Ltd.) was added immediately and reacted for 30min, and the detection was negative.

The method b: three times of protective amino acid FMOC-amino acid and three times of 1-hydroxybenzotriazole (HOBt, Suzhou Tianma pharmaceutical group fine chemicals Co., Ltd.) are dissolved by using DMF as little as possible, added into a reaction tube, and immediately added with three times of N, N' -Diisopropylcarbodiimide (DIC) for reaction for 30min, and the detection shows negative.

7. Resin washing: in this order DMF (10ml/g) was washed once, methanol (10ml/g) was washed twice and DMF (10ml/g) was washed twice.

8. The procedure of 2 to 6 steps was repeated, as indicated in Table 1 for GLP-1R activating peptides in which the amino acids have no side chain modification, or GLP-1R activating peptides having side chain modification, and the corresponding amino acids were sequentially linked from right to left. With K₁₂Or K₂₀Or K₂₇Or K₂₈Modified, synthesized as follows 9.

9. Synthesis of K { N- ε - [ γ -Glu- (N- α -fatty acid or fatty diacid) ] }: adding 10ml 2% hydrazine hydrate to react for 30min to remove the protecting group Dde of Fmoc-Lys (Dde) -OH, exposing the side chain amino group, alternately washing with DMF and methanol for six times, and detecting ninhydrin as blue. 550mg of Fmoc-Glu-OTBU and 250mg of HOBT are weighed, dissolved in DMF, added with 0.3ml of DIC, mixed evenly, added into a reactor to react with lysine side chain amino for 1h, pumped to dry, washed 4 times by DMF, and detected as colorless by ninhydrin. 5ml of 20% piperidine DMF solution was added to the reactor for reaction for 20min to remove the amino protecting group Fmoc of Fmoc-Glu-OTBU, followed by six washes with DMF and methanol alternately, and ninhydrin was detected as blue. Weighing 300mg of fatty acid or fatty diacid and HOBT 250mg, dissolving with DMF, adding 0.3ml of DIC, mixing uniformly, adding into a reactor, reacting for 1h, draining, washing with DMF for 4 times, detecting the ninhydrin to be colorless, washing with methanol for 2 times, and draining.

Synthesis of K { N- ε - [2 × AEEAC- γ -Glu- (N- α -fatty diacid) ] }: after the Dde-Lys (Fmoc) group is removed, 2mM of Fmoc-AEEAC-OH and 2mM of benzotriazole-1-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 45mM of HOBt are added, the mixture is dissolved in DMF, 0.375mM of N, N' -Diisopropylethylamine (DIPEA) is added to the mixture under ice water bath for activation for 3min, and the reaction column is added for reaction for 2h, and the end point of the experiment is judged by the detection of the ninhydrin method. After the reaction was complete, Fmoc was removed from 20% piperidine-DMF solution (15ml/g) and washed 6 times with DMF. Fmoc-AEEAC-OH, Fmoc-Glu-OtBu and fatty diacid chain groups were coupled again in the same manner. Reacting with 2% hydrazine hydrate for 30min to remove the sequence lysine protecting group Dde, and grafting on the epsilon amino group of the lysyl side chain through the step 8.

10. The polypeptide after condensation was passed twice through DMF (10ml/g), twice DCM (10ml/g) and twice DMF (10ml/g) and dried by suction for 10 min. Ninhydrin test negative.

11. Removing FMOC protecting group of final N-terminal amino acid of the peptide chain, detecting to be positive, and draining the solution for later use.

12. The resin was washed twice with DMF (10ml/g), twice with methanol (10ml/g), twice with DMF (10ml/g) and twice with DCM (10ml/g) and dried by suction for 10 min.

13. Cleavage of the polypeptide from the resin: preparing cutting fluid (10 ml/g): TFA 94% (j.t. baker Chemical Company), water 2.5%, ethanediithiol (EDT, Sigma-Aldrich Chemistry) 2.5% and trisisopyrophyllane (TIS, Sigma-Aldrich Chemistry) 1%. Cutting time: and (4) 120 min.

14. Drying and washing: the lysate is blown dry as much as possible with nitrogen, washed six times with ether and then evaporated to dryness at normal temperature.

15. The polypeptides were HPLC purified, identified and stored at-20 ℃ in the dark as follows.

Secondly, the inspection method comprises the following steps:

1. purification of the polypeptide by HPLC: the crude peptide was dissolved in pure water or with a small amount of acetonitrile and purified according to the following conditions: high performance liquid chromatography (analytical; software Class-VP. service System; manufacturer Japan SHIMADZU) and Venusi MRC-ODS C18 column (30X 250mm, Tianjin Bonna-Agela Technologies). Mobile phase A liquid: 0.1% trifluoroacetic acid aqueous solution, mobile phase B liquid: 0.1% trifluoroacetic acid + 99.9% acetonitrile (purchased from acetonitrile Fisher Scientific). Flow rate: 1.0ml/min, a loading volume of 30. mu.l, a detection wavelength of 220 nm. Elution procedure: 0-5 min: 90% of solution A and 10% of solution B; 5-30 min: 90% solution A/10% solution B → 20% solution A/80% solution B.

2. Finally, the purified effective solution was lyophilized (Freezone Plus 6 model of lyophilizer, LABCONCO Co., Ltd.) to obtain the final product.

3. And (3) identification: a small amount of finished polypeptide is respectively taken and analyzed by HPLC for purity: high performance liquid chromatography (Shimadzu, Japan) and Venusi MRC-ODS C18 column (4.6X150mm, Tianjin Bonna-Agela Technologies). Mobile phase A liquid: 0.1% trifluoroacetic acid in water, mobile phase B liquid: 99.9% acetonitrile + 0.1% trifluoroacetic acid solution, flow rate: 1.0ml/min, a loading volume of 10. mu.l, a detection wavelength of 220 nm. Elution procedure: 0-5 min: 100% of solution A; 5-30 min: 100% solution A → 20% solution A/80% solution B. The purity required to be determined is greater than 95%. See the patent (Chinese patent ZL201410612382.3) granted by us for a specific method.

MS method for identifying molecular weight of polypeptide: adding water into polypeptide with qualified purity for dissolving, adding 5% acetic acid, 8% acetonitrile and 87 water for dissolving, testing electrospray ionization mass spectrometry to determine molecular weight, and the specific method is disclosed in the patent (Chinese patent ZL 201410612382.3).

4. Sealing and packaging the powdery polypeptide, and storing at-20 deg.C in dark.

And thirdly, forming a dimer: monomeric peptides with a single cysteine inside the peptide chain at a concentration of 1mg/ml were incubated overnight at 37 ℃ in aqueous disodium hydrogen phosphate at a pH of 9.5 to form homodimeric peptides. For the slightly less soluble dimer peptide, the dimer peptide was centrifuged at 4000 rpm for 20 minutes, and the precipitate was taken out as a pure dimer peptide, and dissolved in NaCl-PB solution (pH of physiological saline adjusted to 8.0 with disodium hydrogen phosphate) for use. The solubilized dimeric peptide or the above-mentioned centrifugation supernatant was separated and identified by Sephadex G-25 chromatography (using NaCl-PB solution as a flow term, the dimeric component as a first peak and the residual impurity component as a subsequent peak under a 2X 60cm G-25 column and natural flow rate). The dimeric peptides can be identified by electrophoresis or mass spectrometry of the peptides without thiol reducing agents, as described in our granted patent (chinese patent ZL 201410612382.3).

Four, GLP-1R activator-like peptide monomers and dimers thereof were synthesized by the present research laboratory or entrusted commercial companies, and the inventors confirmed their structures by HPLC purity, ESI or laser flight mass spectrometry, and cysteine oxidation. The GLP-1R activator monomer synthesized by the invention is shown in Table 1, and the amino acid sequence of the homodimer peptide is shown in Table 2.

Table 1: the amino acid sequence of the GLP-1R activator analog peptide monomer synthesized by the invention and the continuous hypoglycemic activity of the GLP-1R activator analog peptide monomer with the same dosage and single injection

Note: in the table, Tirzepatide is a chimeric peptide of the GIP-Exendin-4 peptide; lixisenatide is also an allosteric to Exendin 4; CFA (carbon fat acid) or CFDA (carbon fat diacid) is carbon fatty acid or carbon fatty diacid; the K [ N-epsilon- (gamma-Glu-N-alpha-CFA or CFDA) ], K [ N-epsilon- (2 xAEEAC-gamma-Glu-N-alpha-CFDA) ] represents fatty acyl or fatty diacid monoacyl glutamyl modification of lysine K side chain epsilon-amino, and the specific structure is shown as formula 1 or 2.

Example 2 duration study of hypoglycemic Effect of GLP-1R activator of the present invention

1. The experimental method comprises the following steps: normal KM mice were purchased at the Guangdong province animal center for glucose tolerance assay (OGTT): the glucose tolerance assay of normal Kunming mice is used to screen hypoglycemic activity and persistence of drugs. Male kunming mice (5 weeks old) were divided into groups (NaCl-PB group, Lixisenatide group, monomer G9-G12 series, and dimer 2G9-2G12 series) (n ═ 6) based on undifferentiated fasting blood glucose. After an adaptation period of two 14-hour feeding-10-hour fasting runs, KM mice were subjected to glucose tolerance measurements immediately after each 10-hour fasting. Injecting medicine or monomer (dissolved by physiological saline pH6.5, and physiological saline is used as blank control) or dimer peptide (dissolved by NaCl-PB solution pH8.0, and NaCl-PB solution is generally used as blank control) subcutaneously at back for 30min, and then gavage the mouse with 5% glucose solution on time, and accurately measuring the blood sugar value of the rat tail after gavage. The glucometer and the blood glucose test paper are products of Bayer Heatchcare LLC company. Taking the average blood sugar values of each group as a judgment standard: when the average blood sugar value of the OGTT of each group is continuously higher than the average blood sugar value of a blank control group on the same day twice, the measurement is stopped, and the duration time of the blood sugar of the OGTT is shorter than the duration time of the blood sugar of the blank group and is the duration time of the drug effect.

2. Results of the experiment

2.1 oral glucose tolerance test

The blood glucose was measured by collecting tail blood of mice 30 minutes after single administration before (0min) and 10, 20, 40, 60, and 120min after single oral administration of glucose, and physiological saline was used in the blank control group (fig. 1). The results show that: at 10min, a significant glucose increase (P <0.05) occurred in group 2G33 compared to the Lixisenatide and G21 groups. At 20min, a significant decrease (P <0.05 or 0.01) occurred in the Lixisenatide and G21 groups compared to the blank control group. Significant glucose increases (P <0.05) occurred in group 2G21 compared to the Lixisenatide and G21 groups. At 40min, a significant decrease (P <0.05 or 0.01) occurred in the Lixisenatide, G21, and G33 groups compared to the blank control group. Significant glucose reduction or increase occurred in group 2G33 (P <0.05) compared to the blank, Lixisenatide or G33 groups. At 60min, a significant decrease (P <0.05) occurred in the G21 group compared to the blank control group; significant glucose increases (P <0.05) occurred in groups 2G21 or 2G33 compared to the Lixisenatide and G21 groups. The results show that at the same time the blood glucose level of the dimer is significantly higher than that of the monomeric peptide, indicating that the dimer significantly delays absorption.

OGTT test (migtt) continued for multiple days after a single administration: 30min after the subcutaneous injection of the drug or the monomer or the dimer peptide on the back, the mice are timely gazed with 5 percent glucose solution, and the blood sugar value of the rat tail is accurately measured 35min after the gazing. The average value of blood sugar is used as a judgment standard, the duration of the blood sugar reduction of the Lixisenatide positive drug is 2 days, the G9 series is maintained for 2-9 days, the G10 series is 2-11 days, the G11 series is 2-11 days, and the G12 series is 7-9 days. The dimer 2G9 series was maintained for 4-21 days, the 2G10 series for 4-22 days, the 2G11 series for 6-21 days, and the 2G12 series for 16-20 days. Each monomer set (shown in table 1) was approximately 1/2 duration for its corresponding dimer set (shown in table 2). In comparison, the dimer peptide (preferably 20-carbon fatty acid or fatty diacid) which forms a disulfide bond at the 11 th or 12 th position and is modified by lysine epsilon-amino side chain fatty acid at the 20 th position has obvious weight reduction and obviously increases the duration of the blood sugar reduction. Comparison shows that the 11 th peptide in the 2G9 series, the 20 th and 21 st peptides in the 2G10 series, and the 33 rd and 34 th dimer peptides in the 2G11 series last for 21-22 days at most. The 21 st peptide (2G21) in the 2G10 series was an Exendin 4 allosteric, and was highly homologous to the sequence of the Lixisenatide positive control and modified with the same side chain fatty acids, so the 2G21 peptide was selected for treatment of type II diabetes (T2D) in vivo and for subsequent experiments. The mOGTT experiment needs to be accurately implemented, otherwise, the result may have jump swing.

TABLE 2 GLP-1R activator dimer sequence and duration of single subcutaneous injection hypoglycemic activity at the same dose

Note: CFA (carbon fat acid) or CFDA (carbon fat diacid) in the table is carbon fatty acid or carbon fatty diacid; the K [ N-epsilon- (gamma-Glu-N-alpha-CFA or CFDA) ], K [ N-epsilon- (2 xAEEAC-gamma-Glu-N-alpha-CFDA) ], represents fatty acyl or fatty diacid monoacyl glutamyl modification of a K side chain epsilon-amino, and the specific structure is shown as a formula 1 or 2. The dimer is connected by a disulfide bond formed between cysteines in the monomer peptide to form an H-type dimer; wherein "|" indicates that a disulfide bond is formed between two cysteine residues between the monomers.

EXAMPLE 3 therapeutic Effect of dimers on type II diabetes model

Firstly, constructing a type II diabetes (T2D) mouse model

C57Bl6/J mice were placed in a standard diet in an environment of SPF rating with free access to water. All experimental operations are conducted according to the ethical and use system guiding principles of experimental animals. After a day of feeding according to the standard diet, 5-week-old C57B16/J male mice were divided into 6 groups: NaCl-PB group, Placebo group (model control group), Lixisenatide group, and low-medium-high dose dimer peptide 2G21 group. The NaCl-PB group was a blank control and Placebo was a T2D model control, which were injected with NaCl-PB solution. All T2D model groups were fed 60 kcal% high fat diet (D12492, venture rat-mouse two biotechnology limited, venture, china) until the end of the experiment, and the blank control group kept the standard diet until the end of the experiment. The method for establishing the diabetes model comprises the following steps: after 4 weeks of high-fat feeding, mice were intraperitoneally injected with 75mg/kg streptozotocin (STZ, Sigma chemical Co., USA), 3 days later, re-intraperitoneally injected with a 50mg/kg dose of STZ, and after 3 weeks, mice having a blood sugar of 11mM or more were considered as diabetic mice. These diabetic groups were treated on a high fat diet for an additional 35 days.

II, treatment effect on II type diabetes

Solubility of peptide: for the monomeric peptide containing a fatty acid modified structure in a Lys side chain and not containing Aib amino acid, the monomeric peptide is in a suspension state in water, and the corresponding homodimeric peptide is completely dissolved in the water; for monomeric peptides containing a fatty acid modified structure in the Lys side chain, the Aib amino acid or/and C-terminally amidated structure containing monomeric peptides showed complete solubility in water, while their corresponding homodimeric peptides were poorly soluble in water. For those without Lys side chain fatty acid modification, both monomeric and dimeric peptides were completely soluble in water. All dimer peptides were separately dissolved and injected with NaCl-PB (pH8.0), and the homodimer 2G21 at different doses, low, medium and high, was separately dissolved in NaCl-PB solution [ Na ]₂HPO₄Buffered saline solution (pH8.0)]For animal injection. The monomeric peptide was dissolved in a physiological saline solution for injection (pH6.5 or so).

Setting the administration concentration: our preliminary experiments show that the aging relationship of multiple OGTT over multiple days is easily observed with a single subcutaneous injection of 0.624nmol/100 μ l of Lixisenatide peptide. Therefore, in all glucose tolerance experiments, normal Kunming mice were injected subcutaneously in the buttocks with a single dose of 0.624nmol/100 μ l of Lixisenatide or monomeric or dimeric peptide, and blood glucose was measured and weighed every day by 9-point tail-snip in the morning. For OGTT, the dosing, gavage and blood glucose-taking times for each animal need to be accurate to seconds. In the T2D animal experiment, Lixisenatide is selected as a positive control and the administration mode (subcutaneous administration) of the Lixisenatide.

Lixisenatide 0.624nmol/100 μ l induced postprandial blood glucose levels of 8-11mM in the T2D diabetes model (up to 20mM postprandial blood glucose). At this critical value, the effect-dose relationship of the positive drug Lixisenatide to the GLP-1R dimer was readily observed. In the T2D treatment study, T2D model mice were injected subcutaneously in the buttocks at a dose of 100. mu.l each within 30min, and blood glucose values were measured in the experimental mice every five days, and the entire test was completed within 40 min. The high, medium and low doses of dimer 2G21 peptide were 1.873, 0.624 and 0.208nmol/100 μ L, respectively, and the dose of the positive drug Lixisenatide was 0.624nmol/100 μ L (crude drug synthesized by commercial companies), and the injection was performed once a day until the end of the 35-day experiment.

1. Body weight change after T2D treatment: before administration, there was no significant difference in body weight of the T2D model group. After the experiment, the Placebo group, the Lixisenatide group, the L-2G21 group (low dose), and the M-2G21 group (medium dose) showed a significant increase in body weight (P <0.05 or 0.001) compared to the NaCl-PB group, but the H-2G21 group (high dose) was not different from the normal group, indicating that the treatment was effective. The weight loss was significant in the Lixisenatide and H-2G21 groups compared to the Placebo group (P < 0.05). The body weight was dose-dependently reduced in each of the 2G21 groups, and the M-2G21 group was similarly changed to the Lixisenatide group (FIG. 2).

2. Hypoglycemic effects in T2D treatment: the fasting blood glucose levels of Placebo, Lixisenatide, L-and M-2G21 groups were significantly higher than those of NaCl-PB group (FIG. 3) (P)<0.05 or 0.001), or Placebo, Lixisenatide, L-and M-, H-2G21 groups with significant hyperglycosylated hemoglobin (HbA)_1c) (FIG. 4) (P)<0.001), showing that the T2D model was successful. Lixisenatide and H-2G21 groups compared to Placebo groupA significant reduction in fasting plasma glucose (P <0.05 or 0.01), or a HbA of the groups Lixisenatide, M-2G21 and H-2G21_1cThe significance is reduced (P < 0.05). Compared with the Lixisenatide group, the fasting blood glucose of the L-2G21 group is obviously increased (P is less than 0.05). After the peptide is injected, the blood sugar level is reduced in a dose-dependent manner, and the effect is better along with the more times of administration. The blood glucose changes in the M-2G21 group were similar to those in the Lixisenatide group. HbA_1cSimilar changes in blood glucose values occur in T2D treatment.

3. Biochemical indicators of blood detection during T2D treatment: after the T2D treatment trial, fasting insulin levels were significantly lower in Placebo, Lixisenatide, or L-2G21 groups than in NaCl-PB groups (P <0.01 or 0.001). The fasting insulin in each group 2G21 showed a dose-dependent increase, and the insulin content in the group M-2G21 was increased 2-3 fold (P <0.05 or 0.01) compared to the Lixisenatide group and the group L-2G 21. The insulin content of H-2G21 group was increased 2-4 fold (P <0.05 or 0.001) compared to Placebo, the Lixisenatide group or the L-2G21 group (FIG. 5), showing that 2-3 fold insulin secretion was induced by equimolar concentrations of dimer peptide. The glutamic-pyruvic transaminase (ALT) of the 2G21 group decreased in a dose-dependent manner, and the ALT levels of the H-2G21 group were all lower than those of NaCl-PB or Placebo, L-2G21 group (P <0.05) (FIG. 6). Serum amylase in each group 2G21 showed a dose-dependent decrease, but was not statistically different (P >0.05) from the blank control or Placebo, Lixisenatide (fig. 7).

From the above examples, the following conclusions can be drawn: the homodimer series we developed can significantly increase the duration of drug effect. The research shows that the dimer sequence shows the most promising application prospect to the rodent T2D model, such as the hypoglycemic effect with the longest duration and the weight loss effect.

The structure-activity relationship shows that the dimer without fatty acid modification has the best solubility in water, and the dimer with fatty acid modification peptide contains Aib amino acid structural dimer, even has a C-terminal amidated structure, and has slightly poor solubility in water, which are important data for pharmaceutical preparation research and are also the fundamental reasons of different sugar-reducing activity durations of GLP-1R activators with different structures in the research, namely different spatial conformations, different physicochemical properties are formed, and different sugar-reducing durations are generated.

Single acting homogeneous agentDose administration, results of a single OGTT experiment show that a longer hypoglycemic effect is produced due to slow absorption of the dimer. Multiple OGTT experimental results with a single identical dose administration indicate that longer duration effects are related to allosteric correlations of dimer amino acid 2, disulfide position, symmetric fatty acid or fatty diacid modified Lys and C-terminal amidation. Table 2 shows that the longest active dimer structure contains²αAib、¹¹Or¹²Cys-Cys disulfide bond, symmetric 20 fatty acyl or fatty diacid monoacyl-L-gamma-glutamyl-²⁰Lysine or 20-carbon fatty diacid acyl-gamma-Glu-2 × AEEAC-²⁰Lys and C-terminal amidation. These modifications are characterized as follows: (1) α Aib →²Ala substitutions resulted in longer activity; (2) lys [2 XAAEEAC-gamma-Glu- (N-alpha-20 carbon fatty diacid) compared to other fatty acid modifications]The best result is achieved by modification; (3) c-terminal amidation significantly prolongs activity; (4) the disulfide bond structure at position 12 or 11 in the dimer molecule showed the best activity. The monomeric peptide activity was only 1/2 for the dimer.

In the T2D treatment experiment, 2G21 groups of diabetes model HbA_1cOr has obvious reduction of fasting blood glucose (FPG) value and obvious blood glucose reduction effect, and the 2G21 peptide and the Lixisenatide with the same molar concentration have the effect of treating PPG (postprandial blood glucose) or FPG and HbA_1cThere is a similar reduction.

The weight of the 2G21 group decreased dose-dependently, and the M-2G21 group and the Lixisenatide group were consistent in weight loss. Improved dimer peptide structure for weight loss¹¹Or¹²Cys-Cys disulfide bond, symmetry²⁰Lys [2 × AEEAC-gamma-Glu-20-carbon fatty acid]Modification and C-terminal amidation.

2G21 showed a dose-dependent reduction in alanine Aminotransferase (ALT), indicating that the drug is very protective for the liver.

In the T2D treatment experiment, the 2G21 group showed dose-dependent increase in insulin, and compared with Placebo, Lixisenatide and L-2G21, the M-or H-2G21 group induced 2-4 times higher insulin level, so that 2G21 had better hypoglycemic effect.

In conclusion, the dimeric peptide of the present invention can induce more insulin release, thereby producing better hypoglycemic effect.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.