A novel GLP-1R peptide agonist and its application

Abstract

The present invention provides a novel GLP‑1R peptide agonist, which is a mutant polypeptide of Exendin‑4, characterized in that the first two amino acids at the N-terminus of Exendin‑4 are mutated to obtain the GLP‑1R peptide agonist. The novel GLP‑1R peptide agonist obtained by the present invention has a comparable or even better technical effect than Exendin‑4. The novel GLP‑1R peptide agonist obtained by the present invention has an agonistic effect on GLP‑1R, and in the case of acute administration, effectively regulates intraperitoneal glucose tolerance; in the case of chronic administration, it plays a role in weight loss, hypoglycemia, especially reducing fasting blood sugar, improving glycated hemoglobin and glucose tolerance. Further, the novel GLP‑1R peptide agonist obtained by the present invention has lower side effects than Exendin‑4, and the appetite suppression effect caused by it can be relieved faster than Exendin‑4, and gastrointestinal side effects (including nausea and visceral discomfort) are significantly suppressed.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a novel GLP-1R peptide agonist, in particular to the improvement of Exendin-4. Background Art

[0002] Glucagon-like peptide receptor (GLP-1R) is a typical class B G protein-coupled receptor (GPCR). Its endogenous ligand GLP-1 is a polypeptide hormone secreted by intestinal L cells after food stimulation, which stimulates pancreatic β cells to secrete insulin and participates in blood sugar regulation. Natural GLP-1 is easily degraded by dipeptidyl peptidase-4 (DPP-4) in the body and loses its activity, so researchers have developed a series of other GLP-1R agonists. There are currently more than ten GLP-1R agonist drugs on the market worldwide. This type of drug has performed well in the field of diabetes and the market size is growing.

[0003] The GLP-1R agonists currently on the market can effectively promote the secretion of postprandial insulin and have a clear hypoglycemic effect. It can also reduce food intake, which is also considered to be one of the mechanisms of the weight loss effect of GLP-1R agonists. However, the appetite suppressant effects of current GLP-1R agonist drugs are accompanied by side effects of nausea and vomiting. Evidence-based medicine studies have shown that nausea and vomiting are the most important side effects of this type of drug. A targeted study published in the Journal of the American Medical Association in October 2023 showed that compared with the control group, the risk of GLP-1 agonists (semaglutide / liraglutide) inducing gastrointestinal adverse reactions and related diseases increased by 3-9 times. Therefore, there is an urgent need to develop a new type of GLP-1R agonist drug that can significantly reduce gastrointestinal adverse reactions while effectively exerting a hypoglycemic effect. Summary of the invention

[0004] The purpose of the present invention is to provide a novel GLP-1R peptide agonist that can effectively exert a hypoglycemic effect while significantly reducing gastrointestinal adverse reactions.

[0005] The present invention provides a novel GLP-1R peptide agonist, which is a mutant of Exendin-4. The first two amino acids at the N-terminus of Exendin-4 are mutated to obtain the GLP-1R peptide agonist.

[0006] Preferably, the GLP-1R peptide agonist is Ex4-AS, and the polypeptide sequence of Ex4-AS is the amino acid sequence shown in SEQ ID NO: 1.

[0007] Preferably, in any of the above items, the GLP-1R peptide agonist is Ex4-AP, and the amino acid sequence of Ex4-AP is shown in SEQ ID NO: 2.

[0008] Preferably, in any of the above items, the GLP-1R peptide agonist is obtained by chemical synthesis or recombinant expression.

[0009] Preferably, in any of the above items, the GLP-1R peptide agonist is Ex4-AS, and the nucleotide sequence expressing Ex4-AS is shown in SEQ ID NO:3.

[0010] Preferably, in any of the above items, the GLP-1R peptide agonist is Ex4-AP, and the nucleotide sequence expressing Ex4-AP is as shown in SEQ ID NO:4.

[0011] Preferably, any of the above items is that the dosage of the GLP-1R peptide agonist in animals is 0.1 μg / kg to 10 μg / kg, and more preferably 0.1 μg / kg, 1 μg / kg, 10 μg / kg and the range therebetween.

[0012] Preferably, in any of the above items, the dosage of the GLP-1R peptide agonist at the cellular level is not less than 0.1 nM, and more preferably 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM and the range therebetween.

[0013] The present invention also provides use of any of the above-mentioned GLP-1R peptide agonists in the preparation of hypoglycemic drugs.

[0014] The present invention also provides the use of any of the above-mentioned GLP-1R peptide agonists in the preparation of weight loss drugs or fat reduction drugs.

[0015] The present invention proves through experiments that the GLP-1R peptide agonist significantly inhibits fat differentiation, and chronic administration of Ex4-AS can inhibit fat differentiation of white adipose tissue, which has important application prospects in the field of drugs for inhibiting fat differentiation.

[0016] The present invention also provides a GLP-1R agonist function reporter system for screening and detecting the GLP-1R peptide agonists. Specifically: First, a piggyBac transposon plasmid that simultaneously expresses a cAMP reporter element and GLP-1R is constructed, and the plasmid name is pPB-CRE-EGFP / P2A / Puro-EF1α-hGLP1R-Bsd. The plasmid is co-transfected with a plasmid expressing piggyBac transposase purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd. (plasmid name: pRP-CAG-hyPBase) into HEK293T cells, so that the expression element is inserted into the TTAA sequence position of the cell genome, and a stable cell line that simultaneously expresses the cAMP reporter element and GLP-1R is obtained by blasticidin (Bsd) drug screening. When GLP-1R agonists are present in the culture system, they bind to the overexpressed GLP-1R on the cell membrane to generate the second messenger cAMP, activate the downstream PKA pathway, and initiate the expression of green fluorescent protein (GFP) and puromycin resistance gene (Puro R) through the cAMP response element (CRE). Therefore, the activation state of GLP-1R can be reflected by the cells producing puro resistance and expressing green fluorescent protein.

[0017] The present invention also provides a plasmid for constructing a GLP-1R agonist function reporter system, the plasmid pPB-CRE-EGFP / P2A / Puro-EF1α-hGLP1R-Bsd, and the plasmid sequence is the nucleotide sequence shown in SEQ ID NO: 5.

[0018] The present invention also provides a method for screening and identifying GLP-1R peptide agonists using the GLP-1R agonist function reporter system.

[0019] The beneficial effects of the present invention are as follows: the novel GLP-1R peptide agonist obtained by the present invention has a comparable or even better technical effect than Exendin-4. The novel GLP-1R peptide agonist obtained by the present invention has an agonistic effect on GLP-1R, and in the case of acute administration, effectively regulates intraperitoneal glucose tolerance; in the case of chronic administration, it plays a role in weight loss, hypoglycemia, especially lowering fasting blood sugar, improving glycated hemoglobin and glucose tolerance. Furthermore, the novel GLP-1R peptide agonist obtained by the present invention has lower side effects than Exendin-4, and the appetite suppression effect caused by it can be relieved faster than Exendin-4, and gastrointestinal side effects (including nausea and visceral discomfort) are significantly suppressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a schematic diagram of the GLP-1R agonist function reporting system in preferred embodiment 1 of the present invention.

[0021] Figure 2 These are representative pictures of GFP expression detected by flow cytometry when Exendin-4 acts on the GLP-1R functional reporter cell line for different durations in the preferred embodiment 1 of the present invention.

[0022] Figure 3 This is a statistical graph of GFP detected by flow cytometry when Exendin-4 acts on the GLP-1R function reporter cell line for different durations in the preferred embodiment 1 of the present invention.

[0023] Figure 4 These are representative pictures of GFP expression detected by flow cytometry when different concentrations of Exendin-4 acted on the GLP-1R functional reporter cell line in the preferred embodiment 1 of the present invention.

[0024] Figure 5 This is a statistical graph of GFP detected by flow cytometry when different concentrations of Exendin-4 acted on the GLP-1R function reporter cell line in the preferred embodiment 1 of the present invention.

[0025] Figure 6 This is the strategy for screening novel GLP-1R peptide agonists using a high-abundance peptide library of mammalian cells in the preferred embodiment 2 of the present invention.

[0026] Figure 7 This is the process of screening new GLP-1R peptide agonists using a high-abundance peptide library of mammalian cells in preferred embodiment 2 of the present invention.

[0027] Figure 8 Representative pictures of the expression plasmid corresponding to the candidate peptide sequence in the preferred embodiment 2 of the present invention being transfected into the GLP-1R functional reporter cell line and the GFP expression being detected by flow cytometry.

[0028] Fig. 9 The expression plasmid corresponding to the candidate peptide sequence in the preferred embodiment 2 of the present invention is transfected into the GLP-1R function reporter cell line, and the GFP statistical graph is detected by flow cytometry.

[0029] Fig.10 In the preferred embodiment 2 of the present invention, different concentrations of Ex4-AP or Ex4-AS were added to the GLP-1R function reporter cell line, and the GFP positive rate was detected by flow cytometry and the EC50 values ​​were calculated respectively.

[0030] Fig.11 This is the competitive inhibitory effect of Ex4 (9-39) on the stimulating effect of Ex4-AP or Ex4-AS polypeptide on GLP-1R in the preferred embodiment 2 of the present invention.

[0031] Fig.12 This is a statistical graph of the changes in blood glucose levels of lean mice in the intraperitoneal glucose tolerance test (10 micrograms per kilogram) and the area under the curve of each group in the preferred embodiment 3 of the present invention.

[0032] Fig.13 This is a statistical graph of the changes in blood glucose levels of lean mice in the intraperitoneal glucose tolerance test (1 microgram per kilogram) and the area under the curve of each group in the preferred embodiment 3 of the present invention.

[0033] Fig.14 This is a statistical graph of the changes in blood glucose levels of lean mice in the intraperitoneal glucose tolerance test (0.1 micrograms per kilogram) and the area under the curve of each group in the preferred embodiment 3 of the present invention.

[0034] Fig.15 This is a statistical graph of the changes in blood insulin levels in lean mice during an intraperitoneal glucose tolerance test (1 microgram per kilogram) and the area under the curve of each group in the preferred embodiment 3 of the present invention.

[0035] Fig.16 This is a statistical graph of the changes in blood glucose levels of mice in the intraperitoneal glucose tolerance test (1 microgram per kilogram) of the type 2 diabetic mouse model in the preferred embodiment 3 of the present invention and the area under the curve of each group.

[0036] Fig.17 This is an evaluation of the weight loss effect of chronic administration of the Ex4-AS polypeptide in the type 2 diabetes mouse model in the preferred embodiment 4 of the present invention.

[0037] Fig.18 This is an evaluation of the effect of chronic administration of Ex4-AS polypeptide on improving fasting blood glucose in the type 2 diabetes mouse model in the preferred embodiment 4 of the present invention.

[0038] Fig.19 This is an evaluation of the effect of chronic administration of Ex4-AS polypeptide on improving intraperitoneal glucose tolerance in the type 2 diabetes mouse model in the preferred embodiment 4 of the present invention (1 microgram per kilogram).

[0039] Fig. 20 This is an evaluation of the effect of chronic administration of Ex4-AS polypeptide on improving intraperitoneal glucose tolerance in the type 2 diabetes mouse model in the preferred embodiment 4 of the present invention (10 micrograms per kilogram).

[0040] Fig.21 This is an evaluation of the improvement effect of chronic administration of Ex4-AS polypeptide on glycated hemoglobin in the type 2 diabetes mouse model in the preferred embodiment 4 of the present invention.

[0041] Fig. 22 The effect of the Ex4-AS polypeptide in the preferred embodiment 5 of the present invention on the food intake of mice in different time intervals

[0042] Fig.23This is the taste preference experiment in the preferred embodiment 5 of the present invention to evaluate the degree of visceral discomfort caused by the Ex4-AS polypeptide in mice.

[0043] Fig.24 This is the effect of the Ex4-AS polypeptide in the preferred embodiment 5 of the present invention on the feeding latency of mice.

[0044] Fig.25 The effect of Ex4-AS polypeptide on the pica behavior of mice was evaluated by the kaolin intake in the preferred embodiment 5 of the present invention.

[0045] Fig.26 This is the transcriptomic analysis of differentially expressed genes and KEGG pathway enrichment analysis of epididymal white adipose tissue after chronic administration of Ex4-AS polypeptide in the type 2 diabetes mouse model in the preferred embodiment 6 of the present invention (1 microgram per kilogram).

[0046] Fig. 27 The transcriptomic analysis of the expression of adipose differentiation-related genes in epididymal white adipose tissue after chronic administration of Ex4-AS polypeptide in the type 2 diabetes mouse model in the preferred embodiment 6 of the present invention (1 microgram per kilogram). DETAILED DESCRIPTION

[0047] The present invention is further described in detail below through the accompanying drawings and specific embodiments.

[0048] Example 1

[0049] Establishment and validation of GLP-1R agonist function reporting system:

[0050] First, a piggyBac transposon plasmid that simultaneously expresses a cAMP reporter element and GLP-1R was constructed, the plasmid name is pPB-CRE-EGFP / P2A / Puro-EF1α-hGLP1R-Bsd, and the complete sequence is shown in SEQ NO: 5. This plasmid was co-transfected into HEK293T cells with a plasmid expressing piggyBac transposase purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd. (plasmid name: pRP-CAG-hyPBase (product number VB200615-1126gxw, product name pRP[Exp]-CAG>hyPBase)), so that the expression element was inserted into the TTAA sequence position of the cell genome, and a stable cell line that simultaneously expresses the cAMP reporter element and GLP-1R was obtained by blasticidin (Bsd) drug screening. When GLP-1R agonists are present in the culture system, they bind to the overexpressed GLP-1R on the cell membrane, generate the second messenger cAMP, activate the downstream PKA pathway, and start the expression of green fluorescent protein (GFP) and puromycin resistance gene (Puro R) through the cAMP response element (cAMPResponse Element, CRE). Therefore, the activation state of GLP-1R can be reflected by the cells producing puro resistance and expressing green fluorescent protein ( Figure 1 , wherein A. Schematic diagram of the key structure of the PiggyBac transposon plasmid that simultaneously expresses the cAMP reporter element and GLP-1R; B. GLP-1R activator is added to the culture system of the HEK293T stable cell line that simultaneously expresses the cAMP reporter element and GLP-1R. The agonist binds to the overexpressed GLP-1R on the cell membrane of the stable cell line to generate the second messenger cAMP that activates the downstream PKA pathway and initiates the expression of GFP and Puro R through CRE).

[0051] In order to verify the effectiveness of the constructed GLP-1R reporter system, 1 nM of Exendin-4 (hereinafter referred to as Ex4), a known GLP-1R agonist, was added to the GLP-1R functional reporter cell line culture system, and Ex4 (9-39) (Ex4 (9-39) amino acid sequence is shown in SEQ ID NO: 6) which has no activation effect on GLP-1R was used as a negative control. Flow cytometry was used to detect the expression of GFP at different time points, as shown in FIG. Figure 2 The following are representative images of GFP expression detected by flow cytometry. The results show that Exendin-4 can activate the GLP-1R agonist function reporter system and make the stable cell line express green fluorescent protein. As the action time increases, the GFP positive rate and average fluorescence intensity increase. However, Ex4 (9-39), which has no activation effect on GLP-1R, cannot increase the expression level of green fluorescent protein in the stable cell line. Figure 3 As shown, A is a statistical graph of GFP positive rate detected by flow cytometry, and B is a statistical graph of GFP mean fluorescence intensity (MFI) detected by flow cytometry.

[0052] Different concentrations of agonist Exendin-4 were added to the GLP-1R function reporter cell line. After 24 hours, the expression of GFP was detected by flow cytometry. Representative flow cytometry images are shown in Figure 4 The results show that -5 In the concentration range of ~0.1nM, the GFP positive rate and average fluorescence intensity of the GLP-1R function reporter cell line increased with the increase of Exendin-4 concentration, while Exendin-4 concentration greater than 0.1nM could not further increase the GFP positive rate and average fluorescence intensity. Figure 5 As shown, A is a statistical graph of GFP positive rate detected by flow cytometry, and B is a statistical graph of GFP mean fluorescence intensity (MFI) detected by flow cytometry.

[0053] The above series of results demonstrate the effectiveness of the GLP-1R function reporting system constructed in the present invention.

[0054] Example 2

[0055] Using the high-abundance peptide library of mammalian cells, a new GLP-1R peptide agonist Ex4-AS was screened and obtained:

[0056] (1) Exendin-4 (Ex4) is a 39-amino acid GLP-1R peptide agonist that has been marketed as a diabetes treatment drug (exenatide). Structural biology studies have shown that Exendin-4 binds to and activates GLP-1R in two steps: first, the C-terminus of Ex4 binds to the N-terminal extracellular domain of GLP-1R, and then the N-terminus of Ex4 binds to the receptor core region (TMD domain) of GLP-1R and activates GLP-1R. Based on the above structural features, the present invention retains the C-terminal structure of Ex4, Exendin (9-39), to ensure the binding ability of the peptide segment to GLP-1R, and at the same time randomizes the first two amino acids of the N-terminus in order to screen for new agonist peptides.

[0057] An X2 linear random peptide library is formed at the N-terminus of Exendin (3-39) and connected to the N-terminus of PDGFR-TM (platelet-derived growth factor receptor transmembrane domain, also referred to as PDGFR transmembrane region in the present invention). Paired linear double-stranded DNA is introduced into the established GLP-1R functional reporter cells to form an "AND gate" logic gene circuit, and the "random peptide library X2-Exendin (3-39)" is displayed on the cell surface. GLP-1R activated cells (expressing Puro resistance gene) are screened out through Puro drug screening, and RNA is extracted from these cells, cDNA is synthesized, and PCR amplification is performed. After 3 rounds of screening, the GLP-1R candidate peptide agonist sequence is finally obtained through high-throughput sequencing.

[0058] like Figure 6 The figure shows the strategy of using high-abundance peptide libraries of mammalian cells to screen new GLP-1R peptide agonists: an X2 linear random peptide library is formed at the N-terminus of Exendin (3-39), connected to the N-terminus of PDGFR-TM, and paired linear double-stranded DNA is introduced into the established GLP-1R functional reporter cells to form an "AND gate" logic gene circuit, and the "random peptide library X2-Exendin (3-39)" is displayed on the cell surface, and GLP-1R activated cells are screened out through Puro drug screening.

[0059] like Figure 7 The figure shows the process of using the high-abundance peptide library of mammalian cells to screen new GLP-1R peptide agonists: after Puro drug screening, RNA is extracted from the GLP-1R activated cells expressing the Puro resistance gene, and the next round of screening is carried out after the RNA extraction - cDNA synthesis - PCR amplification process. After three rounds of screening, the GLP-1R candidate peptide agonist sequence is obtained by high-throughput sequencing.

[0060] After three rounds of screening, high-throughput sequencing was performed to select the 6 candidate peptide sequences with the highest enrichment degree to construct an expression plasmid. The target fragment was inserted between the Kpn I and Xba I restriction sites of the pcDNA3.0 vector to obtain the expression plasmid. As shown in the following table, there are six candidate peptide expression plasmids, an Ex4 expression plasmid (positive control), and an Ex4 (9-39) expression plasmid (negative control) sequences. The target fragments inserted into the pcDNA3.0 vector respectively include the nucleotide sequence for expressing Ex4 as shown in SEQ ID NO: 8, the nucleotide sequence for expressing Ex4 (9-39) as shown in SEQ ID NO: 10, the nucleotide sequence for expressing Ex4-AK as shown in SEQ ID NO: 12, the nucleotide sequence for expressing Ex4-AS as shown in SEQ ID NO: 3, the nucleotide sequence for expressing Ex4-AD as shown in SEQ ID NO: 15, the nucleotide sequence for expressing Ex4-AP as shown in SEQ ID NO: 4, the nucleotide sequence for expressing Ex4-AT as shown in SEQ ID NO: 18, and the nucleotide sequence for expressing Ex4-AE as shown in SEQ ID NO: 20; the inserted target fragments also include a KOZAK sequence with a nucleotide sequence of gccacc; an IL-2 signaling sequence as shown in SEQ ID NO: 21, which is used to guide the expression product out of the cell; an HA tag as shown in SEQ ID NO: 22; and a nucleotide sequence as shown in SEQ ID NO: 23. The PDGFR transmembrane region shown in NO: 23 fixes the expression product on the outside of the cell membrane.

[0061]

[0062]

[0063] The candidate peptide expression plasmids were respectively transferred into HEK293T cells containing the GLP-1R functional reporter system, and the GFP positive ratio was detected by flow cytometry. It was found that Ex4-AP (amino acid sequence shown in SEQ ID NO: 2 (N-terminus-C-terminus): APEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS) and Ex4-AS (amino acid sequence shown in SEQ ID NO: 1 (N-terminus-C-terminus): ASEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS) had an activation effect on GLP-1R comparable to that of the known agonist Exendin-4 (amino acid sequence (N-terminus-C-terminus): HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS). Figure 8Shown are representative images of the expression plasmid corresponding to the candidate peptide sequence transfected into the GLP-1R functional reporter cell line and detected by flow cytometry for GFP expression. Fig. 9 Shown are diagrams of the expression plasmid corresponding to the candidate peptide sequence transfected into the GLP-1R function reporter cell system, wherein A is a statistical diagram of GFP positivity detected by flow cytometry; B is a statistical diagram of GFP mean fluorescence intensity (MFI) detected by flow cytometry.

[0064] In addition, replacing the nucleotide sequence expressing the Ex4-AS polypeptide in the pcDNA3.0-Ex4AS-L2 plasmid with the nucleotide sequence shown in SEQ ID NO: 24 can also effectively express the Ex4-AS polypeptide and achieve the same technical effect.

[0065] (2) Ex4-AP, Ex4-AS and Ex4 peptides were chemically synthesized, and different concentrations of Ex4-AP or Ex4-AS (or Ex4) were added to the GLP-1R functional reporter cell line. The expression of green fluorescent protein was detected by flow cytometry to confirm that Ex4-AP and Ex4-AS peptides did have an agonist effect on GLP-1R, and the EC50 ( Fig.10 shown).

[0066] By adding Ex4-AP, Ex4-AS or Ex4 (concentrations equivalent to their respective EC80) and different concentrations of non-activating Ex4 (9-39) to the GLP-1R function reporter cell line for competitive binding, the expression ratio of green fluorescent protein was detected by flow cytometry to observe the competitive inhibition of Ex4 (9-39) on the GLP-1R stimulating effect of Ex4-AP or Ex4-AS polypeptide. It is proved that the stimulating effect of Ex4-AP or Ex4-AS on GLP-1R can be blocked by Ex4 (9-39). Fig.11 The figure shows the competitive inhibition of Ex4(9-39) on the agonism of GLP-1R by Ex4-AP or Ex4-AS polypeptide.

[0067] Example 3

[0068] Evaluation of the acute administration effect of Ex4-AS peptide:

[0069] (1) Ex4-AP or Ex4-AS polypeptide (or Ex4 polypeptide as a positive control) was intraperitoneally injected into 6-8 week old male C57 lean mice at different doses to evaluate their effects on intraperitoneal glucose tolerance test.

[0070] The specific operation steps are as follows: after fasting the test mice for 4 hours, blood was collected from the tail of the test mice and the blood glucose of the test mice was measured using a Roche ACCU-CHEK blood glucose meter, and this time was recorded as time zero. A mixed solution of different types of polypeptides of corresponding doses prepared with a mixed solution of 10% DMSO + 40% PEG-300 + 5% Tween-80 + 45% saline and 20% glucose (mouse injection glucose dose: 2g / kg body weight) was quickly injected into the test mice by intraperitoneal injection. After that, blood was collected from the tail of the test mice at 6 time points of 15 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes and 90 minutes to measure the blood glucose of the mice. A plot was drawn and the area under the blood glucose curve of each mouse was calculated. The results are shown in the figure. Figure 12-14 As shown. In the three dose groups of 10μg / kg, 1μg / kg and 0.1μg / kg, the blood glucose area under the curve of mice injected with Ex4-AP, Ex4-AS or Ex4 polypeptide was significantly decreased compared with that of mice in the control group; in the two dose groups of 10μg / kg and 0.1μg / kg, the area under the curve of mice injected with Ex4-AS polypeptide was significantly decreased compared with that of mice injected with Ex4 polypeptide; in the three dose groups, there was no significant difference in the area under the curve between mice injected with Ex4-AP polypeptide and mice injected with Ex4 polypeptide.

[0071] (2) During the intraperitoneal glucose tolerance test of the 1 μg / kg administration group, blood was collected from the tail of the mice at 0, 5, 10, 15, 30, 45 and 60 minutes, and the blood insulin level was measured using the EZassay Mouse Insulin Enzyme-Linked Immunoassay (ELISA) Kit (Shenzhen Yizhi Biotechnology Co., Ltd.). The plot was drawn and the area under the insulin curve of each mouse was calculated ( Fig.15 ). The results showed that the area under the insulin curve of mice injected with Ex4-AS peptide was significantly increased compared with that of control mice, while there was no significant difference in the area under the insulin curve of mice injected with Ex4-AP peptide compared with that of control mice. Since the Ex4-AS peptide showed better hypoglycemic and insulin-raising effects than the Ex4-AP peptide in the intraperitoneal glucose tolerance test of lean mice, the subsequent experiments were mainly based on the Ex4-AS peptide.

[0072] (3) An acute administration experiment of Ex4-AS peptide was conducted on a type 2 diabetic mouse model.

[0073] A high-fat diet + streptozotocin (hereinafter abbreviated as STZ)-induced type 2 diabetes mouse model was constructed. The specific construction strategy was to purchase 6-8 week old male C57 mice, and after 1 week of adaptive feeding, they were fed with a high-fat diet (60% fat energy high-fat diet, Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., catalog number XTHF60) for 12 weeks, and then given an intraperitoneal injection of STZ 40 mg / kg for 4 consecutive days. Fasting blood glucose was measured starting from the 9th day after the injection. A successful modeling was considered when the fasting blood glucose was higher than 11.1 mmol / L for 4 consecutive days.

[0074] The Ex4-AS peptide was acutely administered to the type 2 diabetic mouse model to evaluate its effect on intraperitoneal glucose tolerance. The specific operation method was the same as that of lean mice (except that the fasting time was 8 hours, which was different from the 4 hours of lean mice), and the dosage was 1 μg / kg. The results showed that in the type 2 diabetic mouse model, the area under the blood glucose curve of mice injected with Ex4-AS peptide or Ex4 peptide was significantly decreased compared with that of the control mice ( Fig.16 ).

[0075] The above series of experimental results show that under acute administration, the regulatory effect of Ex4-AS polypeptide on the intraperitoneal glucose tolerance of lean mice is better than that of Ex4 polypeptide, and the regulatory effect on the intraperitoneal glucose tolerance of type 2 diabetic mouse model is not inferior to that of Ex4 polypeptide.

[0076] Example 4

[0077] Evaluation of the weight loss and hypoglycemic effect of chronic administration of Ex4-AS peptide on type 2 diabetic mouse model:

[0078] A high-fat diet + STZ-induced type 2 diabetes mouse model was constructed. The specific construction strategy was to purchase 6-8 week old male C57 mice, and after 1 week of adaptive feeding, they were fed with a high-fat diet (60% fat energy supply high-fat diet, Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., catalog number XTHF60) for 12 weeks, and then given an intraperitoneal injection of STZ 40 mg / kg for 4 consecutive days. Fasting blood glucose was measured starting from the 9th day after the injection. A successful modeling was considered if the fasting blood glucose was higher than 11.1 mmol / L for 4 consecutive days.

[0079] The model-successful mice were intraperitoneally injected with different doses of Ex4-AS peptide or Ex4 peptide for 21 consecutive days. The weight, fasting blood glucose and glycosylated hemoglobin levels of the mice were measured at the end of the experiment. The mice were also subjected to an intraperitoneal glucose tolerance test to evaluate the therapeutic effect. The results showed that compared with the control group, intraperitoneal injection of 1 μg / kg Ex4-AS peptide could significantly reduce the weight of the mice, while intraperitoneal injection of the same dose of Ex4 peptide had no significant effect on reducing the weight of the mice ( Fig.17-A shows the evaluation of the weight loss effect of chronic administration of Ex4-AS peptide in a type 2 diabetic mouse model (1 μg / kg); when injected with a dose of 10 μg / kg, both Ex4-AS and Ex4 peptide could significantly reduce the body weight of mice, with no significant difference between the two ( Fig.17 -B shows the weight loss effect evaluation of chronic administration of Ex4-AS peptide in the type 2 diabetic mouse model (10 μg / kg). Ex4-AS or Ex4 peptide in different dose groups can significantly reduce the fasting blood glucose of the type 2 diabetic mouse model ( Fig.18 The figure shows the effect of chronic administration of Ex4-AS peptide on improving fasting blood glucose in a type 2 diabetes mouse model, where A is 1 microgram per kilogram and B is 10 micrograms per kilogram) and improving intraperitoneal glucose tolerance in mice ( Figure 19-20 10 μg / kg Ex4-AS was more effective than Ex4 in reducing fasting blood glucose. Intraperitoneal injection of 1 μg / kg Ex4-AS or Ex4 peptide could not significantly reduce the glycated hemoglobin level in type 2 diabetic mice ( Fig.21 -A shows the evaluation of the improvement effect of chronic administration of Ex4-AS peptide on glycated hemoglobin in a type 2 diabetic mouse model (1 microgram per kilogram). 10μg / kg of Ex4-AS or Ex4 peptide can significantly reduce the level of glycated hemoglobin, and there is no significant difference between the two ( Fig.21 -B shows the evaluation of the improvement effect of chronic administration of Ex4-AS peptide on glycated hemoglobin in a type 2 diabetic mouse model (10 micrograms per kilogram).

[0080] The above series of experimental results show that when administered in medium doses, Ex4-AS polypeptide has a better weight loss effect on the type 2 diabetes mouse model than Ex4 polypeptide; when administered in large doses, Ex4-AS polypeptide has a better fasting blood glucose reduction effect on the type 2 diabetes mouse model than Ex4 polypeptide; Ex4-AS polypeptide has a similar effect as Ex4 polypeptide in improving glycated hemoglobin and glucose tolerance in the type 2 diabetes mouse model.

[0081] Example 5

[0082] Evaluation of the appetite suppressant effect and gastrointestinal side effects of Ex4-AS peptide:

[0083] Example 5 further evaluated the appetite suppressant effect and gastrointestinal side effects (including nausea and visceral discomfort) of the Ex4-AS polypeptide.

[0084] After fasting for 12 hours overnight, different doses of Ex4-AS peptide or Ex4 peptide were intraperitoneally injected into lean mice, and the food intake of mice in different time periods after administration was specifically evaluated using metabolic cages. The results showed that after intraperitoneal injection of 1 μg / kg Ex4-AS peptide, the average food intake of each mouse at 0-1 hour and 0-2 hours was comparable to that of mice injected with the same dose of Ex4 peptide, while the average food intake at 0-4 hours, 0-8 hours and 0-24 hours was significantly higher than that of mice injected with the same dose of Ex4 peptide ( Fig. 22 -A shows the effect of Ex4-AS peptide on the food intake of mice in different time intervals (1 μg / kg); after intraperitoneal injection of 10 μg / kg Ex4-AS peptide, the average food intake of each mouse at 0-1 hour, 0-2 hours, and 0-4 hours was comparable to that of mice injected with the same dose of Ex4 peptide, while the average food intake at 0-8 hours and 0-24 hours was significantly higher than that of mice injected with the same dose of Ex4 peptide ( Fig. 22 -B shows the effect of Ex4-AS peptide on the food intake of mice in different time intervals (10 μg / kg). This set of results suggests that the appetite suppression effect caused by Ex4-AS peptide can be relieved faster than that of Ex4 peptide.

[0085] The taste preference experiment was further used to evaluate the visceral discomfort caused by the Ex4-AS polypeptide. First, water bottles containing drinking water were placed at the same height on both sides of the mouse cage. Drinking water was provided at a fixed time of 1.5 hours every day, and the water bottles were removed at other times for a total of 3 days, so that the experimental mice could adapt to the drinking pattern, and the mice with obvious preference for drinking water on one side were eliminated; on the 4th day, a water bottle containing saccharin aqueous solution prepared with cherry-flavored Kool-Aid granules was placed in the same position at the same drinking time. After the drinking time was over, different doses of Ex4-AS polypeptide or Ex4 polypeptide were immediately injected intraperitoneally; one day later, drinking water and saccharin aqueous solution prepared with cherry-flavored Kool-Aid granules were provided in water bottles on both sides of the mouse cage at the same drinking time for the experimental mice to choose. After the drinking time was over, the proportion of saccharin water consumed by each mouse in the total liquid intake was counted. The results showed that the proportion of saccharin water intake of mice injected intraperitoneally with Ex4-AS polypeptide in different dose groups was higher than that of mice injected intraperitoneally with the same dose of Ex4 polypeptide, indicating that the visceral discomfort caused by Ex4-AS polypeptide was weaker than that of Ex4 polypeptide ( Fig.23 ).

[0086] Determined by the natural neuroanatomical structure, the experimental mice do not have vomiting behavior. Laboratory studies mostly use pica behavior, that is, the ingestion of non-food substances to evaluate the nausea reaction of mice, and kaolin is often used as a non-food substance. After the experimental mice were fasted for 14 hours overnight (water is not prohibited), 1μg / kg Ex4-AS polypeptide or Ex4 polypeptide was intraperitoneally injected, and feed, drinking water and kaolin bait blocks prepared with 10g kaolin + 4ml 2% gum arabic were immediately provided. The latency of eating (that is, the time from the time of providing feed to the first eating behavior) and the amount of kaolin consumed within the first 1.5 hours were recorded. The results showed that the latency of eating of the experimental mice injected intraperitoneally with the Ex4-AS polypeptide was shorter than that of the experimental mice injected intraperitoneally with the Ex4 polypeptide ( Fig.24 ), the amount of kaolin consumed was significantly less than that of the experimental mice injected with Ex4 peptide intraperitoneally ( Fig.25 ).

[0087] The above series of experimental results show that the appetite suppression effect caused by Ex4-AS polypeptide can be relieved faster than that of Ex4 polypeptide, and the gastrointestinal side effects including visceral discomfort and nausea are weaker than those of Ex4 polypeptide.

[0088] Example 6

[0089] Evaluation of the effect of chronic administration of Ex4-AS peptide on white adipose tissue

[0090] A high-fat diet + STZ-induced type 2 diabetes mouse model was constructed. The specific construction strategy was to purchase 6-8 week old male C57 mice, and after 1 week of adaptive feeding, they were fed with a high-fat diet (60% fat energy supply high-fat diet, Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., catalog number XTHF60) for 12 weeks, and then given an intraperitoneal injection of STZ 40 mg / kg for 4 consecutive days. Fasting blood glucose was measured starting from the 9th day after the injection. A successful modeling was considered if the fasting blood glucose was higher than 11.1 mmol / L for 4 consecutive days.

[0091] The modeled mice were intraperitoneally injected with 1 μg / kg of Ex4-AS peptide or Ex4 peptide for 21 consecutive days, and then RNA was extracted from the epididymal white adipose tissue (eWAT) of the mice for transcriptomic analysis (RNA-Seq). The results showed that there were 1663 differentially expressed genes in the Ex4-AS group compared with the control group, and 1429 differentially expressed genes in the Ex4 group compared with the control group, of which 567 were shared. We performed KEGG pathway enrichment analysis on the 1096 differentially expressed genes unique to the Ex4-AS group, with the PPAR signaling pathway ranking first, followed by the AMPK signaling pathway, the adipocyte lipolysis signaling pathway, etc. ( Fig.26). The PPAR signaling pathway is essential for adipocyte differentiation, which plays a key role in diet-induced obesity. RNA-Seq results showed that the transcriptional levels of Pparg and Cebpa, key late transcription factors for adipocyte differentiation, were significantly downregulated in the Ex4-AS treatment group, but not in the Ex4 treatment group ( Fig. 27 A), and the transcriptional levels of its downstream genes Plin1, Plin5, Adipoq, Fabp4, Sorbs1 and Angptl4 were more significantly downregulated in the Ex4-AS treatment group than in the Ex4 group ( Fig. 27 B).

[0092] The above experimental results show that chronic administration of Ex4-AS can inhibit the fat differentiation of white adipose tissue.

[0093] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A novel GLP-1R peptide agonist, which is a mutant polypeptide of Exendin-4, characterized in that: The first two amino acids at the N-terminus of Exendin-4 are mutated to obtain the GLP-1R peptide agonist, wherein the GLP-1R peptide agonist is Ex4-AS, and the polypeptide sequence of the Ex4-AS is the amino acid sequence shown in SEQ ID NO: 1; or, the GLP-1R peptide agonist is Ex4-AP, and the amino acid sequence of the Ex4-AP is shown in SEQ ID NO:

2.

2. The GLP-1R peptide agonist according to claim 1, characterized in that The GLP-1R peptide agonist is obtained by chemical synthesis or recombinant expression.

3. The GLP-1R peptide agonist according to claim 2, characterized in that The GLP-1R peptide agonist is Ex4-AS, and the nucleotide sequence expressing Ex4-AS is shown in SEQ ID NO:

3.

4. The GLP-1R peptide agonist according to claim 2, characterized in that The GLP-1R peptide agonist is Ex4-AP, and the nucleotide sequence expressing Ex4-AP is shown in SEQ ID NO:

4.

5. The GLP-1R peptide agonist according to claim 2, characterized in that The dosage of the GLP-1R peptide agonist in animals is 0.1 μg / kg to 10 μg / kg.

6. The GLP-1R peptide agonist according to claim 2, characterized in that The dosage of the GLP-1R peptide agonist at the cellular level is not less than 0.1 nM.

7. Use of the GLP-1R peptide agonist Ex4-AS according to any one of claims 1 to 6 in the preparation of hypoglycemic drugs.

8. Use of the GLP-1R peptide agonist Ex4-AS according to any one of claims 1 to 6 in the preparation of weight loss drugs.

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