GLP-1 variants and uses thereof

Abstract

The present invention relates to degradation of glucagon-like peptide-1 (GLP-1) as well as variants and uses of GLP-1. The invention specifically relates to the discovery that GLP-1 can be degraded by insulin degrading enzyme (IDE) and degradation sites, designs a GLP-1 variant with enhanced stability, and improves the secretion level and stability of GLP-1 cells by regulating IDE.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and protein engineering, and in particular to glucagon-like peptide-1 (GLP-1) variants and applications thereof. Background Art

[0002] Obesity and diabetes have become a major global health crisis. According to the World Health Organization (WHO), more than 2.5 billion adults in the world are overweight in 2022, of which more than 890 million are obese (https: / / www.who.int / news-room / fact-sheets / detail / obesity-and-overweight). Obesity is closely related to type 2 diabetes and many other diseases that threaten human health. According to data released by the International Diabetes Federation (IDF), more than 10.5% of adults and 540 million people in the world suffer from diabetes in 2021 (https: / / idf.org / about-diabetes / diabetes-facts-figures / ), and this number will reach 643 million by 2030. Obesity and diabetes not only increase the risk of cardiovascular disease, cancer and other health problems, but also impose a huge burden on the medical system. It is particularly noteworthy that my country is also the country with the largest number of adult diabetes patients. In the past decade (2011-2021), the number of diabetes patients in my country has increased by 56%. Therefore, how to treat diabetes and obesity more effectively has become an urgent problem that needs to be solved.

[0003] At present, the drugs for the treatment of diabetes mainly include the following categories: insulin, biguanides, SGLT-2 inhibitors, DPP-4 inhibitors and GLP-1 receptor agonists (GLP-1RA). Among them, GLP-1 receptor agonists, as an emerging drug, have shown strong clinical efficacy in weight loss and improvement of diabetes, bringing significant benefits to patients. Human glucagon-like peptide-1 (GLP-1) is a short peptide secreted by L cells in the intestine and is mainly released after eating. Its precursor protein proglucagon undergoes a series of processing and is converted into an active form. The main effects of GLP-1 include stimulating insulin secretion, inhibiting glucagon secretion, slowing gastric emptying, and reducing appetite. These functions make GLP-1 an important molecule for the treatment of type 2 diabetes and obesity. In addition to diabetes and obesity, GLP-1 has shown positive therapeutic effects in many aspects in recent years. For example, in terms of neuroprotection, it can improve cognitive function and play a role in neurodegenerative diseases such as Alzheimer's disease. In addition, GLP-1 also has anti-inflammatory properties, improves liver function and cardiovascular health, and has potential benefits for mental illnesses. The wider application of GLP-1 is being actively explored.

[0004] Although the activity of GLP-1 is of great significance for regulating the body, GLP-1 has a very short biological half-life in the human body, which is only about 1 to 2 minutes, which limits its effective action time in the body. Therefore, enhancing the half-life of GLP-1 has become an important strategy for the development of related drugs. At present, the half-life of GLP-1 is mainly enhanced by inhibiting its degradation enzymes (such as dipeptidyl-peptidase IV (DPP4)) to improve the stability of endogenous GLP-1, or by taking GLP-1RA with a longer half-life to achieve a better hypoglycemic effect and duration. Among them, DPP4 inhibitors include Sitagliptin, which was approved by the FDA in 2006, and subsequent Saxagliptin, Linagliptin, Alogliptin, etc. GLP-1RA polypeptide drugs include Exenatide, which was approved by the FDA for marketing in 2005, and subsequent Liraglutide, Dulaglutide, Semaglutide, etc. At present, the above drugs have become important choices for the treatment of type 2 diabetes. Due to its outstanding weight loss effect, the US FDA approved GLP-1RA (Wegovy) for clinical use in weight loss in 2014.

[0005] In summary, studying the degradation pathway of GLP-1 and discovering new degradation proteases and their degradation mechanisms are of great significance for developing methods that can enhance the half-life of GLP-1RA and improve its hypoglycemic effect. Summary of the invention

[0006] The present invention finds that insulin degrading enzyme (IDE) is related to the degradation pathway of human glucagon-like peptide-1 (GLP-1). In addition, GLP-1 is transformed by chemical or molecular biological methods, and the corresponding sites are modified or replaced, thereby reducing the degradation effect of IDE molecules on GLP-1 and improving the stability of GLP-1.

[0007] The present invention provides use of IDE, its coding sequence, a nucleic acid construct comprising the coding sequence, a cell expressing IDE, a cell comprising the coding sequence, or a cell comprising the nucleic acid construct in screening for GLP-1 variants with improved stability, wherein the IDE sequence is as shown in SEQ ID NO: 1 or has at least 90% sequence identity thereto.

[0008] The present invention also provides a use of insulin degrading enzyme (IDE) for degrading glucagon-like peptide-1 (GLP-1), wherein the IDE sequence is as shown in SEQ ID NO: 1 or has at least 90% sequence identity therewith. The GLP-1 sequence is as shown in SEQ ID NO: 2, positions 7-37.

[0009] In one or more embodiments, the degradation occurs at positions 18-19 and / or 25-26 in SEQ ID NO:2.

[0010] The present invention also provides a method for screening candidate substances capable of regulating the interaction between IDE and GLP-1, comprising the steps of:

[0011] (1) contacting the candidate substance with a system comprising IDE and GLP-1, and

[0012] (2) Detecting the GLP-1 content and comparing it with the GLP-1 content in the control.

[0013] If the content of GLP-1 is less than the control, the candidate substance upregulates the interaction between IDE and GLP-1, that is, promotes the degradation of GLP-1 by IDE; if the content of GLP-1 is greater than the control, the candidate substance downregulates the interaction between IDE and GLP-1, that is, inhibits the degradation of GLP-1 by IDE.

[0014] In one or more embodiments, the control is the same system without the candidate substance.

[0015] In one or more embodiments, the system is a solution system, a cell system, a tissue system or an animal model. The animal can be a rat, a mouse, a rabbit or a monkey.

[0016] The present invention also provides a method for screening GLP-1 variants with changed stability to IDE relative to wild-type GLP-1, the method comprising: obtaining a GLP-1 variant having a mutation or modification at one or more amino acid positions, and testing the change in stability of the GLP-1 variant to IDE relative to wild-type GLP-1.

[0017] In one or more embodiments, the method comprises the steps of:

[0018] (1) contacting a GLP-1 variant with a system comprising IDE, wherein the GLP-1 variant has a mutation or modification at one or more amino acid positions,

[0019] (2) detecting the content of the GLP-1 variant and comparing it with the content of GLP-1 in the control,

[0020] If the content of the GLP-1 variant is less than the content of GLP-1 in the control, the stability of the GLP-1 variant decreases; if the content of the GLP-1 variant is greater than the content of GLP-1 in the control, the stability of the GLP-1 variant increases.

[0021] In one or more embodiments, the GLP-1 variant has a mutation or modification at one or more of Ser18, Tyr19, Ala25, Lys26.

[0022] In one or more embodiments, the control is the same system with the GLP-1 variant replaced with GLP-1.

[0023] In one or more embodiments, the system is a solution system, a cell system, a tissue system or an animal model. The animal can be a rat, a mouse, a rabbit or a monkey.

[0024] In one or more embodiments, the sequence of GLP-1 is as shown in SEQ ID NO:2, positions 7-37.

[0025] The present invention also provides an agent for specifically inhibiting the degradation of GLP-1 by IDE, wherein the agent is a fragment of GLP-1 or a variant of the fragment, wherein the fragment at least comprises one or more amino acid residues of Ser18, Tyr19, Ala25, and Lys26 of GLP-1. The sequence of GLP-1 is shown in SEQ ID NO: 2, positions 7-37.

[0026] In one or more embodiments, the starting position of the fragment is any amino acid in positions 7-18, preferably any amino acid in positions 10-16. In one or more embodiments, the ending position of the fragment is any amino acid in positions 26-37.

[0027] In one or more embodiments, the fragment comprises Ser18. In one or more embodiments, the fragment comprises Ser18 and Tyr19. In one or more embodiments, the fragment comprises Ser18, Tyr19 and Ala25. In one or more embodiments, the fragment comprises Ser18, Tyr19, Ala25 and Lys26. In one or more embodiments, the starting position of the fragment is any amino acid at positions 7-18, preferably any amino acid at positions 10-16, more preferably amino acid at position 15 or 16. In one or more embodiments, the ending position of the fragment is any amino acid at positions 19-37, preferably any amino acid at positions 21-30, more preferably amino acid at position 21 or 22.

[0028] In one or more embodiments, the fragment comprises Tyr19. In one or more embodiments, the fragment comprises Tyr19 and Ala25. In one or more embodiments, the fragment comprises Tyr19, Ala25 and Lys26. In one or more embodiments, the starting position of the fragment is any amino acid in positions 7-19. In one or more embodiments, the ending position of the fragment is any amino acid in positions 20-37.

[0029] In one or more embodiments, the fragment comprises Ala25. In one or more embodiments, the fragment comprises Ala25 and Lys26. In one or more embodiments, the starting position of the fragment is any amino acid in positions 7-25. In one or more embodiments, the ending position of the fragment is any amino acid in positions 26-37.

[0030] In one or more embodiments, the fragment comprises Lys26. In one or more embodiments, the starting position of the fragment is any amino acid in positions 7-26. In one or more embodiments, the ending position of the fragment is any amino acid in positions 27-37.

[0031] In one or more embodiments, the fragment is 4-37 amino acids in length. Preferably, the fragment is 6-20 amino acids in length.

[0032] In one or more embodiments, the variant of the fragment is mutated to other amino acids or has modifications at one, two, three or four positions among Ser18, Tyr19, Ala25, Lys26.

[0033] In one or more embodiments, the fragment is mutated to other amino acids at a position selected from any of the following groups:

[0034] (1) Ser18, (2) Tyr19, (3) Ala25, (4) Lys26, (5) Ser18 and Tyr19, (6) Ser18 and Ala25, (7) Ser18 and Lys26, (8) Tyr19 and Ala25, (9) Tyr19 and Lys26, (10) Ala25 and Lys26, (11) Ser18, Tyr19 and Ala25, (12) Ser18, Tyr19 and Lys26, (13) Ser18, Ala25 and Lys26, (14) Tyr19, Ala25 and Lys26, (15) Ser18, Tyr19, Ala25 and Lys26.

[0035] In one or more embodiments, the additional amino acids are non-positively charged amino acids.

[0036] In one or more embodiments, the additional amino acid is an unnatural amino acid.

[0037] In one or more embodiments, the other amino acids are 2-aminoisobutyric acid (Aib), glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), methionine (Met, M), proline (Pro, P), asparagine (Asn, N) and glutamine (Gln, Q), lysine (Lys, K), arginine (Arg, R) or histidine (His, H).

[0038] In one or more embodiments, the modification is selected from any of the following: non-natural peptide bonds, fatty acylation, acetylation and other acylation modifications, methylation, glycosylation, phosphorylation, ubiquitination, hydroxylation, carbonylation, alkylation, fatty acid, hydroxyl modification, PEGylation, polypropylene glycolation (PPGylation).

[0039] In one or more embodiments, the fragment of GLP-1 is a polypeptide having a sequence of VSSYLE (SEQ ID NO: 10), QAAKEF (SEQ ID NO: 11), DVSPYLEG (SEQ ID NO: 12), GQAWKEFI (SEQ ID NO: 13), DVSYYLEG (SEQ ID NO: 14), DVSRYLEG (SEQ ID NO: 15), DVSMYLEG (SEQ ID NO: 16), GQASKEFI (SEQ ID NO: 17), GQAYKEFI (SEQ ID NO: 18), or GQANKEFI (SEQ ID NO: 19).

[0040] The present invention also provides a variant of GLP-1 or a derivative thereof, wherein the variant has a mutation or modification at one, two, three or four positions of Ser18, Tyr19, Ala25 and Lys26. The sequence of GLP-1 is shown in SEQ ID NO: 2, positions 7-37.

[0041] In one or more embodiments, the variant has a mutation or modification at a position selected from any one of the following groups: (1) Ser18, (2) Tyr19, (3) Ala25, (4) Lys26, (5) Ser18 and Tyr19, (6) Ser18 and Ala25, (7) Ser18 and Lys26, (8) Tyr19 and Ala25, (9) Tyr19 and Lys26, (10) Ala25 and Lys26, (11) Ser18, Tyr19 and Ala25, (12) Ser18, Tyr19 and Lys26, (13) Ser18, Ala25 and Lys26, (14) Tyr19, Ala25 and Lys26, (15) Ser18, Tyr19, Ala25 and Lys26.

[0042] In one or more embodiments, the derivative is Exenatide, Liraglutide, Lixisenatide, Dulaglutide, Semaglutide, Noiigliutide, Pemvidutide, Cafraglutide, Survodutide, Tirzepatide, VK-2735, Survodutide, DD01, Dapiglutide. The sequence of Semaglutide is shown in SEQ ID NO:8.

[0043] In one or more embodiments, the mutations of Ser18, Tyr19, Ala25, and Lys26 are each independently mutations to non-negatively charged amino acids.

[0044] In one or more embodiments, the mutations of Ser18, Tyr19, Ala25, and Lys26 are each independently mutated to glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), methionine (Met, M), proline (Pro, P), asparagine (Asn, N), glutamine (Gln, Q), lysine (Lys, K), arginine (Arg, R), or histidine (His, H).

[0045] In one or more embodiments, the mutation is selected from any one or more of the following groups:

[0046] (1) Any one of S18G, S18A, S18V, S18L, S18I, S18T, S18C, S18M, S18N, S18Q, S18D, S18E, S18K, S18R, S18H, S18F, S18Y, S18W, and S18P,

[0047] (2) any one of Y19G, Y19A, Y19V, Y19L, Y19I, Y19T, Y19C, Y19M, Y19N, Y19Q, Y19D, Y19E, Y19K, Y19R, Y19H, Y19F, Y19S, Y19W, and Y19P,

[0048] (3) Any one of A25G, A25Y, A25V, A25L, A25I, A25T, A25C, A25M, A25N, A25Q, A25D, A25E, A25K, A25R, A25H, A25F, A25S, A25W, and A25P,

[0049] (4) Any one of K26G, K26Y, K26V, K26L, K26I, K26T, K26C, K26M, K26N, K26Q, K26D, K26E, K26A, K26R, K26H, K26F, K26S, K26W, and K26P.

[0050] In one or more embodiments, the mutation is selected from S18P, S18D, S18K, S18N, A25H, A25C, A25F, A25G, A25Q, A25W.

[0051] In one or more embodiments, the variant also has a mutation or modification at Ala8.

[0052] In one or more embodiments, the mutations of Ala8, Ser18, Tyr19, Ala25, and Lys26 are each independently mutations to unnatural amino acids.

[0053] In one or more embodiments, the mutations of Ala8, Ser18, Tyr19, Ala25, and Lys26 are each independently mutated to a D-amino acid or mutated to Aib.

[0054] In one or more embodiments, the mutations of Ala8, Ser18, Tyr19, Ala25, Lys26 are each independently mutations to the D-form of the original amino acid residue.

[0055] In one or more embodiments, the mutation comprises a mutation selected from any one or more of the following: Ser18 mutates to D-Ser, Tyr19 mutates to D-Tyr, Ala25 mutates to D-Ala, and Lys26 mutates to D-Lys. In one or more embodiments, the mutation comprises a mutation selected from any one of the following groups: (1) Ser18 mutates to D-Ser, and Tyr19 mutates to D-Tyr, (2) Ser18 mutates to D-Ser, and Ala25 mutates to D-Ala, (3) Ser18 mutates to D-Ser, and Lys26 mutates to D-Lys, (4) Tyr19 mutates to D-Tyr, and Ala25 mutates to D-Ala, (5) Tyr19 mutates to D-Tyr, and Lys26 mutates to D-Lys, (6) Ala25 mutates to D-Ala, and Lys26 mutates to D-Lys, (7) Ser18 mutates to D -Ser, Tyr19 mutates to D-Tyr, and Ala25 mutates to D-Ala, (8) Ser18 mutates to D-Ser, Tyr19 mutates to D-Tyr, and Lys26 mutates to D-Lys, (9) Ser18 mutates to D-Ser, Ala25 mutates to D-Ala, and Lys26 mutates to D-Lys, (10) Tyr19 mutates to D-Tyr, Ala25 mutates to D-Ala, and Lys26 mutates to D-Lys, (11) Ser18 mutates to D-Ser, Tyr19 mutates to D-Tyr, Ala25 mutates to D-Ala, and Lys26 mutates to D-Lys. In one or more embodiments, the mutation further comprises: Ala8 mutates to Aib.

[0056] In one or more embodiments, the modification is selected from any of the following: non-natural peptide bonds, fatty acylation, acetylation and other acylation modifications, methylation, glycosylation, phosphorylation, ubiquitination, hydroxylation, carbonylation, alkylation, fatty acid, hydroxyl modification, PEGylation, polypropylene glycolation (PPGylation).

[0057] In one or more embodiments, the sequence of the variant comprises a sequence as shown in SEQ ID NO:3-7 or 9.

[0058] In one or more embodiments, the variant retains the biological activity of GLP-1.

[0059] In one or more embodiments, the variant is more stable toward IDE than GLP-1.

[0060] Another aspect of the present invention provides a polynucleotide, wherein the sequence of the polynucleotide comprises one or more selected from the following:

[0061] (a) a nucleotide sequence encoding (1) a fragment of GLP-1 or a variant of the fragment, or (2) a variant of GLP-1 or a derivative thereof as described herein,

[0062] (b) a nucleotide sequence having at least 80% (preferably at least 90%) identity with the sequence shown in (a), and

[0063] (c) The complementary sequence of (a) or (b).

[0064] Another aspect of the present invention provides a nucleic acid construct comprising the polynucleotide encoding the gene described herein.

[0065] In one or more embodiments, the nucleic acid construct is a vector. Preferably, the nucleic acid construct is an expression vector or a homologous recombination vector.

[0066] Another aspect of the present invention provides a host cell, wherein:

[0067] (1) comprising, expressing or secreting a variant of GLP-1 or a derivative thereof as described herein,

[0068] (2) containing the polynucleotide described herein, or

[0069] (3) Containing the nucleic acid construct described herein.

[0070] In one or more embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0071] In one or more embodiments, the host cell is a prokaryotic cell, such as Escherichia coli, Bacillus, or lactic acid bacteria. In one or more embodiments, the host cell is a eukaryotic cell, such as yeast or filamentous fungi.

[0072] The present invention also provides a method for improving the stability of GLP-1 or its derivatives, comprising mutating or modifying one or more positions among Ser18, Tyr19, Ala25, and Lys26. The sequence of GLP-1 is shown in SEQ ID NO:2, positions 7-37.

[0073] In one or more embodiments, the mutation or modification comprises a mutation or modification at a position selected from any one of the following groups: (1) Ser18, (2) Tyr19, (3) Ala25, (4) Lys26, (5) Ser18 and Tyr19, (6) Ser18 and Ala25, (7) Ser18 and Lys26, (8) Tyr19 and Ala25, (9) Tyr19 and Lys26, (10) Ala25 and Lys26, (11) Ser18, Tyr19 and Ala25, (12) Ser18, Tyr19 and Lys26, (13) Ser18, Ala25 and Lys26, (14) Tyr19, Ala25 and Lys26, (15) Ser18, Tyr19, Ala25 and Lys26.

[0074] In one or more embodiments, the derivative is Exenatide, Liraglutide, Lixisenatide, Dulaglutide, Semaglutide, Noiigliutide, Pemvidutide, Cafraglutide, Survodutide, Tirzepatide, VK-2735, Survodutide, DD01 or Dapiglutide. The sequence of Semaglutide is shown in SEQ ID NO:8.

[0075] In one or more embodiments, the mutations of Ser18, Tyr19, Ala25, and Lys26 are each independently mutations to non-negatively charged amino acids.

[0076] In one or more embodiments, the mutations of Ser18, Tyr19, Ala25, and Lys26 are each independently mutated to glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), methionine (Met, M), proline (Pro, P), asparagine (Asn, N), glutamine (Gln, Q), lysine (Lys, K), arginine (Arg, R), or histidine (His, H).

[0077] In one or more embodiments, the variant also has a mutation or modification at Ala8.

[0078] In one or more embodiments, the mutations of Ala8, Ser18, Tyr19, Ala25, and Lys26 are each independently mutations to unnatural amino acids.

[0079] In one or more embodiments, the mutations of Ala8, Ser18, Tyr19, Ala25, and Lys26 are each independently mutated to a D-amino acid or mutated to Aib.

[0080] In one or more embodiments, the mutations of Ala8, Ser18, Tyr19, Ala25, Lys26 are each independently mutations to the D-form of the original amino acid residue.

[0081] In one or more embodiments, the modification is selected from any of the following: non-natural peptide bonds, fatty acylation, acetylation and other acylation modifications, methylation, glycosylation, phosphorylation, ubiquitination, hydroxylation, carbonylation, alkylation, fatty acylation, hydroxyl modification, PEGylation, polypropylene glycolation (PPGylation).

[0082] The present invention also provides a use of a reagent in preparing a kit for regulating GLP-1 content or stability, regulating animal blood sugar, treating metabolic diseases, or treating diseases that benefit from GLP-1 content regulation or stability regulation, wherein the reagent comprises:

[0083] (1) an agent for regulating the expression or activity of IDE,

[0084] (2) Agents that regulate the interaction between IDE and GLP-1,

[0085] (3) A variant of GLP-1 or a derivative thereof, wherein the variant has a mutation or modification at one, two, three or four positions of Ser18, Tyr19, Ala25 and Lys26.

[0086] In one or more embodiments, the interaction is an interaction between IDE and GLP-1 at one or more of Ser18, Tyr19, Ala25, Lys26, which interaction allows IDE to degrade GLP-1.

[0087] In one or more embodiments, the disease is a metabolism-related disease or a neurological disease.

[0088] In one or more embodiments, the metabolism-related disease is diabetes, obesity, non-alcoholic fatty liver disease, insulin resistance syndrome.

[0089] In one or more embodiments, the neurological disease is Alzheimer's disease and Parkinson's syndrome.

[0090] In one or more embodiments, the use is: use of an agent that downregulates the expression or activity of IDE and / or an agent that downregulates the interaction between IDE and GLP-1 in the preparation of a kit for upregulating the level or stability of GLP-1, downregulating blood sugar in animals, treating diabetes, treating obesity, or treating diseases that benefit from unchanged or increased GLP-1 levels or enhanced GLP-1 stability.

[0091] In one or more embodiments, the use is: the use of a variant of GLP-1 or its derivatives described in any embodiment of the present invention in the preparation of a kit for up-regulating GLP-1 content or stability, down-regulating animal blood sugar, treating diabetes, treating obesity, or treating a disease that benefits from unchanged or increased GLP-1 content or enhanced GLP-1 stability. In one or more embodiments, the disease is a metabolic-related disease or a nervous system disease. In one or more embodiments, the metabolic-related disease is diabetes, obesity, non-alcoholic fatty liver disease, insulin resistance syndrome. In one or more embodiments, the nervous system disease is Alzheimer's disease and Parkinson's syndrome.

[0092] In one or more embodiments, the agent that downregulates the expression of IDE is an inhibitory molecule that specifically interferes with the transcription and / or expression of the IDE gene. In one or more embodiments, the inhibitory molecule uses the IDE gene or its transcript as an inhibition target. In one or more embodiments, the inhibitory molecule is: (1) a dsRNA, antisense nucleic acid, small interfering RNA, microRNA, shRNA, reRNA, sgRNA that uses the IDE gene or its transcript as an inhibition or silencing target, or (2) a construct that can express or form (1).

[0093] In one or more embodiments, the agent that downregulates the activity of IDE is an IDE inhibitor, such as IDE inhibitor 6bk, or an inhibitory antibody. Examples of inhibitory antibodies include Abcam (ab133561), Abcam (ab109538), Abcam (ab233020), Abcam (ab247897), Abcam (ab317428), Abcam (ab201836), Abcam (ab273669), Thermo (GT143), Thermo (GT286), Thermo (JJ0949), Thermo (OTI4E5), proteintech (3E10G1).

[0094] In one or more embodiments, the agent that downregulates the activity of IDE is an activity-downregulated IDE protein variant or its coding sequence.

[0095] In one or more embodiments, the diseases that benefit from unchanged or increased GLP-1 content or enhanced GLP-1 stability include: diabetes, obesity, non-alcoholic fatty liver disease, and insulin resistance syndrome.

[0096] In one or more embodiments, the agent that downregulates the interaction of IDE with GLP-1 competes with the agent that inhibits IDE with GLP-1.

[0097] In one or more embodiments, the agent that downregulates the interaction of IDE with GLP-1 is a fragment of GLP-1 comprising one or more amino acid residues of Ser18, Tyr19, Ala25, Lys26.

[0098] In one or more embodiments, the fragment is 4-37 amino acids in length. Preferably, the sequence is 6-20 amino acids in length.

[0099] In one or more embodiments, the agent that downregulates the interaction of IDE with GLP-1 is a fragment of GLP-1 or a variant of the fragment as described in any of the embodiments herein.

[0100] In one or more embodiments, the agent that downregulates the interaction of IDE with GLP-1 is a polypeptide of VSSYLE (SEQ ID NO: 10), QAAKEF (SEQ ID NO: 11), DVSPYLEG (SEQ ID NO: 12), and GQAWKEFI (SEQ ID NO: 13), DVSYYLEG (SEQ ID NO: 14), DVSRYLEG (SEQ ID NO: 15), DVSMYLEG (SEQ ID NO: 16), GQASKEFI (SEQ ID NO: 17), GQAYKEFI (SEQ ID NO: 18), or GQANKEFI (SEQ ID NO: 19).

[0101] In one or more embodiments, the use is: use of an agent that upregulates the expression or activity of IDE and / or an agent that upregulates the interaction between IDE and GLP-1 in the preparation of a kit for downregulating the level or stability of GLP-1, upregulating animal blood glucose, or treating diseases that benefit from reduced GLP-1 levels or reduced GLP-1 stability.

[0102] In one or more embodiments, diseases that benefit from reduced GLP-1 levels or reduced GLP-1 stability include: hypoglycemia, muscle loss, and decreased bone density.

[0103] In one or more embodiments, the agent that upregulates the expression or activity of IDE includes an expression vector capable of expressing IDE.

[0104] The present invention also provides a method for regulating the content or stability of GLP-1, comprising:

[0105] (1) regulating the expression or activity of IDE, or

[0106] (2) Regulate the interaction between IDE and GLP-1.

[0107] In one or more embodiments, the method is a method of upregulating GLP-1 level or stability, comprising:

[0108] (1) downregulating the expression or activity of IDE, or

[0109] (2) Downregulate the interaction between IDE and GLP-1.

[0110] In one or more embodiments, the downregulation of IDE protein expression or activity comprises:

[0111] (1) Specific interference with IDE gene transcription and / or expression,

[0112] (2) inhibiting the activity of IDE using an IDE inhibitor, such as 6bk or an inhibitory antibody or ligand,

[0113] (3) Using a downregulated IDE protein variant to inhibit IDE activity.

[0114] In one or more embodiments, (1) the interference is interference with the transcription of the IDE gene or the translation of its transcript.

[0115] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the IDE gene is selected from the group consisting of: (1) antisense nucleic acid, microRNA, siRNA, RNAi, dsRNA, sgRNA, shRNA, or a combination thereof, and (2) a nucleic acid construct that can express or form (1).

[0116] In one or more embodiments, the sequence of the shRNA is shown in SEQ ID NO: 20-25.

[0117] In one or more embodiments, (2) comprises in a cell:

[0118] (i) expressing specific antibodies or ligands of IDE protein that can downregulate the activity of IDE protein (e.g. inhibitory antibodies), or

[0119] (ii) introducing a nucleic acid sequence encoding the antibody or ligand and / or a nucleic acid construct capable of expressing (i).

[0120] In one or more embodiments, (3) includes expressing an IDE protein with reduced activity in the cell.

[0121] In one or more embodiments, down-regulating the interaction between IDE and GLP-1 comprises: causing the interaction between IDE and GLP-1 to be competitively inhibited. In one or more embodiments, (4) comprises introducing a competitive inhibitor into the cell.

[0122] In one or more embodiments, the agent that downregulates the interaction of IDE with GLP-1 is a fragment of GLP-1 or a variant of the fragment as described in any of the embodiments herein.

[0123] In one or more embodiments, the method is a method of down-regulating GLP-1 level or stability, comprising:

[0124] (1) Upregulating the expression or activity of IDE, or

[0125] (2) Upregulate the interaction between IDE and GLP-1.

[0126] In one or more embodiments, the amino acid sequence of the GLP-1 protein is as shown in SEQ ID NO: 1 or a sequence having at least 80% identity thereto.

[0127] The present invention also provides an RNA that specifically interferes with IDE expression, comprising any one or more sequences of SEQ ID NOs: 20-25.

[0128] Another aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and:

[0129] (1) a variant of GLP-1 or a derivative thereof and / or its coding sequence as described in any embodiment herein, or

[0130] (2) A fragment of GLP-1 or a variant of the fragment and / or its coding sequence as described in any embodiment of the present invention.

[0131] In one or more embodiments, the disease is a metabolism-related disease or a neurological disease.

[0132] In one or more embodiments, the metabolism-related disease is diabetes, obesity, non-alcoholic fatty liver disease, insulin resistance syndrome.

[0133] In one or more embodiments, the neurological disease is Alzheimer's disease and Parkinson's syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] Figure 1GLP-1 is the degradation substrate of IDE. Among them, A is the intact GLP-1 detected by mass spectrometry, and IDE can rapidly degrade GLP-1; B is that the rate of IDE degradation of GLP-1 is much faster than that of DPP4, and both IDE and DPP4 can degrade GLP-1; C is that small molecule inhibitors of IDE and DPP4 can inhibit the degradation of GLP-1; D is that GLP-1 and IDE can interact with each other; E is the site where IDE and GLP-1 (7-37) are cross-linked.

[0135] Figure 2 Determination of the degradation site of GLP-1 by IDE. Wherein, A is the degradation site of GLP-1 by IDE, which is concentrated on Ser 18 -Tyr 19 and Ala 25 -Lys 26 ; B and C are short peptide inhibitors that can competitively inhibit the degradation of GLP-1; D is the inhibitory effect of two short peptide inhibitors added simultaneously on the degradation of GLP-1 is similar to that of 6bk.

[0136] Figure 3 GLP-1 is degraded by IDE secreted by JHH-7 cells. A indicates that GLP-1 is rapidly degraded in the culture medium; B indicates that the degradation sites of GLP-1 in the culture medium are mainly concentrated on Ser 18 -Tyr 19 and Ala 25 -Lys 26 ; C: The expression of IDE in the culture medium is reduced, and GLP-1 is almost not degraded; D: The IDE content is reduced, and the degradation site of GLP-1 in the culture medium is no longer concentrated on Ser 18 -Tyr 19 and Ala 25 -Lys 26 ; E shows that the secreted GLP-1 content in the culture medium increased after IDE was knocked down in the NCI-H716 cell line.

[0137] Figure 4 The stability of GLP-1 after mutation. A is the sequence information of GLP-1 after mutation; B is the degradation of GLP-1 after mutation by IDE; C is the comparison of the cleavage sites of IDE to the new receptor agonist and GLP-1 at the 15-minute time point.

[0138] Figure 5It is the stability of GLP-1 in different microenvironments after mutation. Among them, A is the expression of IDE in JHH7 culture medium, mouse plasma and peritoneal cavity; BC is the stability and cleavage site of different GLP-1 receptor agonists in cell culture medium; DE is the stability and cleavage site of different GLP-1 receptor agonists in mouse plasma; FG is the stability and cleavage site of different GLP-1 receptor agonists in mouse peritoneal fluid.

[0139] Figure 6 is the agonist activity of GLP-1 after mutation.

[0140] Figure 7 To compare the hypoglycemic effects of mutated GLP-1, GLP-1 and Liraglutide.

[0141] Figure 8 is the effect of the mutant peptide on the reaction with IDE and the degradation of GLP-1 as a competitive inhibitor. 18 The stability of the short peptide DVSXYLEG after reaction with IDE, B is the mutant Ala 25 The stability of the short peptide GQAXKEFI after reaction with IDE, C is the mutant Ser 18 The inhibitory effect of the short peptide DVSXYLEG as a competitive inhibitor on the degradation of GLP-1 by IDE, where D is the mutant Ala 25 The inhibitory effect of the short peptide GQAXKEFI on IDE degradation of GLP-1 as a competitive inhibitor, E is a mutant Ser 18 The short peptide DVSXYLEG and the mutant Ala 25 The short peptide GQAXKEFI was added simultaneously as a competitive inhibitor to inhibit the degradation of GLP-1 by IDE.

[0142] Fig. 9 It is the stability of the mutated GLP-1 in the environment of simulated intestinal and gastric fluid. Among them, A is the degradation trend of GLP-1 and mutant GLP-1 in the simulated intestinal fluid over time, and B is the degradation trend of GLP-1 and mutant GLP-1 in the simulated gastric fluid over time.

[0143] Fig.10 is the expression of IDE and DPP4 in mouse brain tissue or monkey cerebrospinal fluid. Among them, A is the expression of IDE and DPP4 in mouse brain tissue, and B is the expression of IDE and DPP4 in monkey cerebrospinal fluid.

[0144] Fig.11 The effect of mutant GLP-1 on food intake, blood glucose and body weight in high-fat diet-induced obese (DIO) mice.18 -Semaglutide's effect on blood glucose in mice, B is a comparison of Semaglutide and D-Ser 18 -Semaglutide's effect on the food intake of mice, C is a comparison of Semaglutide and D-Ser 18 -Effect of semaglutide on body weight in mice.

[0145] Fig.12 The knockdown effect of different shRNAs on IDE expression in JHH7 cells. DETAILED DESCRIPTION

[0146] The inventors revealed for the first time that insulin degrading enzyme (IDE) can degrade glucagon-like peptide-1 (GLP-1) and discovered its corresponding degradation site. They also revealed that the cellular secretion level and stability of GLP-1 can be increased by regulating the expression level and activity of IDE.

[0147] As used herein, "glucagon-like peptide-1" is an incretin secreted by intestinal L cells, which has multiple physiological functions, including promoting insulin synthesis and secretion in a glucose-dependent manner, inhibiting glucagon secretion to regulate blood sugar, and delaying gastric emptying and increasing satiety. GLP-1 can promote the synthesis and secretion of insulin. When blood sugar rises, GLP-1 acts on receptors on pancreatic β cells to stimulate the release of insulin, and this stimulation is glucose-dependent. Only when blood sugar rises will GLP-1 promote insulin secretion, thereby effectively avoiding the occurrence of hypoglycemia. For example, after eating, the intestine secretes GLP-1, which can accurately adjust the amount of insulin secretion according to the degree of blood sugar increase after food digestion and absorption, so that blood sugar is maintained at a normal level. At the same time, GLP-1 can also inhibit the secretion of glucagon, which prompts the liver to release glucose into the blood and increase blood sugar. GLP-1 reduces the output of glucose in the liver by inhibiting the secretion of glucagon, thereby lowering blood sugar. GLP-1 receptor agonist drugs can be used to treat type 2 diabetes. Compared with traditional hypoglycemic drugs, they have additional benefits such as lowering blood sugar, reducing weight, and lowering blood pressure. They are mostly injected subcutaneously, with a low risk of hypoglycemia, and are also of interest in the field of weight loss. GLP-1 is composed of 30 or 31 amino acids and can bind specifically to receptors, but will be rapidly degraded by dipeptidyl peptidase-4 (DPP-4), which has also prompted the development of GLP-1 receptor agonist DPP-4 inhibitors in drug research and development to prolong their duration of action.

[0148] Insulin-degrading enzyme (IDE) is a metalloproteinase that mainly exists in cells and can specifically recognize and degrade insulin. IDE is a zinc metalloproteinase with zinc ions in its active center. This metal ion is essential for the catalytic activity of the enzyme and is involved in the binding and hydrolysis of the substrate. The molecular structure of IDE is relatively complex and usually consists of multiple domains. These domains play different roles in substrate recognition, binding, and enzyme activity regulation. IDE has a specific substrate binding region that can recognize the specific structure of substrate molecules such as insulin, thereby initiating the degradation process. The sophistication of its structure allows it to act highly specifically on certain peptides and proteins without causing unnecessary degradation effects on other molecules.

[0149] For example, IDE selectively uses the size and charge distribution of the substrate binding cavity to trap peptides with diverse structures. Since the IDE cavity carries a high positive charge, the absence of a significant positive charge at the C-terminus of the peptide avoids charge repulsion between the IDE and the C-terminus, and such peptides are better IDE substrates. The size of the catalytic center of IDE is only large enough to accommodate peptides with less than 50 amino acids, and peptides that are too long will slow down the IDE cleavage rate.

[0150] IDE inhibitors are a class of compounds that can inhibit the activity of insulin degrading enzyme (IDE). They prevent IDE from degrading its substrates (such as insulin, amyloid β-peptide, etc.) normally by binding to the active site or other related sites of IDE. The mode of action can be competitive inhibition, that is, the inhibitor competes with the substrate for the active site of IDE; or non-competitive inhibition, where the inhibitor binds to other sites of IDE, changes the conformation of the enzyme, and thus indirectly affects the enzyme's ability to degrade the substrate.

[0151] In the examples herein, the IDE inhibitor 6bk was used as a control. 6bk was screened and optimized based on the molecule 6b to obtain the molecule with the lowest IC50. 6bk has the advantages of high specificity, good inhibitory effect, and wide inhibition range. X-ray experiments show that 6bk exerts its inhibitory effect by binding to a site far away from the catalytic pocket of the IDE enzyme. At present, the known IDE substrates include insulin, glucagon, amylin, etc. 6bk can inhibit the degradation of insulin, glucagon, and amylin by IDE.

[0152] The inventors have found that IDE can interact with GLP-1. Therefore, the present invention provides a method for screening a candidate substance that can regulate the interaction between IDE and GLP-1, comprising the steps of: (1) contacting the candidate substance with a system containing IDE and GLP-1, and (2) detecting the content of GLP-1 and comparing it with the content of GLP-1 in a control. If the content of GLP-1 is less than that of the control, the candidate substance upregulates the interaction between IDE and GLP-1, i.e., promotes the degradation of GLP-1 by IDE; if the content of GLP-1 is greater than that of the control, the candidate substance downregulates the interaction between IDE and GLP-1, i.e., inhibits the degradation of GLP-1 by IDE. In one or more embodiments, the control is the same system without the candidate substance.

[0153] The present invention also provides a method for screening a GLP-1 variant with a change in stability with respect to IDE, comprising the steps of: (1) contacting a GLP-1 variant with a system containing IDE, wherein the GLP-1 variant has a mutation at one or more amino acid positions, and (2) detecting the content of the GLP-1 variant and comparing it with the content of GLP-1 in a control, wherein if the content of the GLP-1 variant is less than the content of GLP-1 in the control, the stability of the GLP-1 variant is reduced; if the content of the GLP-1 variant is greater than the content of GLP-1 in the control, the stability of the GLP-1 variant is improved. Specifically, the GLP-1 variant has a mutation at one or more positions of Ser18, Tyr19, Ala25, and Lys26. In one or more embodiments, the control is the same system in which the GLP-1 variant is replaced with GLP-1.

[0154] The system described herein can be a solution system, a cell system, a tissue system or an animal model. The animal can be a rat, a mouse, a rabbit or a monkey.

[0155] In this article, the "interaction" between IDE and GLP-1 mainly refers to the degradation of GLP-1 by IDE. 18 -Tyr 19 Between and Ala 25 -Lys 26 In this article, by regulating the interaction between IDE and GLP-1, it can play a beneficial role in metabolic-related diseases or nervous system diseases, such as diabetes, obesity, non-alcoholic fatty liver disease, insulin resistance syndrome, Alzheimer's disease or Parkinson's syndrome.

[0156] "GLP-1 encoded protein" and "GLP-1 protein" refer to polypeptides having GLP-1 activity, including but not limited to variant forms of the polypeptide. Herein, GLP-1 is 7-37 of SEQ ID NO: 1, and the amino acid numbering of GLP-1 is referred to the amino acid numbering of SEQ ID NO: 2. For example, Ser18 of GLP-1 is Ser18 of SEQ ID NO: 2. Variant forms of polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants.

[0157] The present invention also relates to derivatives of GLP-1. In one or more embodiments, the derivative is Exenatide, Liraglutide, Lixisenatide, Dulaglutide, Semaglutide, Noiigliutide, Pemvidutide, Cafraglutide, Survodutide, Tirzepatide, VK-2735, Survodutide, DD01 or Dapiglutide. Semaglutide is a GLP-1 receptor agonist, a synthetic peptide compound, mainly used to treat type 2 diabetes, and also has a certain effect in weight loss and cardiovascular protection. Its chemical structure is modified based on GLP-1. For example, on the basis of natural GLP-1, the peptide chain is modified, and the amino acids at positions 8 and 34 are replaced, etc., so that it is not easily degraded quickly. The lysine of semaglutide contains a modified structure of C18 fatty diacid, and there is also an arm modification composed of glutamic acid and 2-(2-(2-aminoethoxy)ethoxy)acetic acid dimer between the modified structure and lysine. This structural change prolongs the half-life of the drug in the body, thereby achieving a treatment regimen that only requires administration once a week. The sequence of semaglutide is shown in SEQ ID NO:8.

[0158] The term "variant" or "mutant" refers to a peptide or polypeptide whose amino acid sequence is changed by insertion, deletion or substitution of one or more amino acids compared to a reference sequence but retains at least one biological activity. The mutant described in any embodiment herein includes an amino acid sequence having at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the reference sequence and retaining the biological activity of the reference sequence. The sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. Mutants also include amino acid sequences that have one or more mutations (insertions, deletions or substitutions) in the amino acid sequence of the reference sequence while still retaining the biological activity of the reference sequence. The multiple mutations generally refer to within 1-30, such as 1-10, 1-8, 1-4 or 1-2. For example, in the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide.

[0159] Amino acids with similar properties often refer to amino acid families with similar side chains, which have been clearly defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative amino acid substitutions for many common known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on comparisons of their physical properties with the properties of genetically encoded amino acids. For another example, adding one or more amino acids to the amino terminus and / or carboxyl terminus generally does not change the function of a polypeptide or protein.

[0160] In this article, by designing a short peptide of GLP-1 or mutating or modifying the short peptide, it can be used as an IDE inhibitor to regulate the content and stability of GLP-1. For example, in the sequence of SEQ ID NO: 10-19, the two short peptides DVSPYLEG and GQAWKEFI can not only resist the IDE cleavage effect, but also inhibit the IDE cleavage of GLP-1 as a short peptide competitive inhibitor. Other short peptides, such as DVSYYLEG, DVSRYLEG, DVSMYLEG, GQASKEFI, GQAYKEFI and GQANKEFI, can also better protect GLP-1 from being cleaved by IDE in experiments as competitive inhibitors. Therefore, the present invention also provides a reagent that specifically inhibits the degradation of GLP-1 by IDE, wherein the reagent is a fragment of GLP-1 or a variant of the fragment, wherein the fragment contains at least one or more amino acid residues of Ser18, Tyr19, Ala25, and Lys26, and the length of the fragment is 6-20 amino acids. In one or more embodiments, the fragment is mutated to other amino acids or has modifications at one, two, three or four positions among Ser18, Tyr19, Ala25, Lys26.

[0161] In this article, by mutating or modifying GLP-1 or its derivatives, the biological activity of GLP-1 can be retained and its stability against IDE can be increased. Therefore, the present invention also provides variants of GLP-1 or its derivatives, which have mutations or modifications at one, two, three or four positions of Ser18, Tyr19, Ala25, and Lys26. For example, the commercialized GLP-1 derivative Semaglutide is also mutated herein, including mutating the 18th position of the sequence shown in SEQ ID NO:9 to a D-type amino acid. In addition, the mutated or modified GLP-1 or its derivatives can be further mutated or modified (for example, at the Ala8 position) to increase its stability against other proteases (for example, DPP4).

[0162] The term "GLP-1 receptor (GLP-1R)" belongs to the G protein-coupled receptor (GPCR) family. It is a transmembrane protein composed of 7 transmembrane α-helical domains connected by intracellular and extracellular loops. This structure enables it to transmit extracellular signals to the cell on the cell membrane. Its extracellular region is mainly responsible for binding to ligands such as GLP-1, while the intracellular region interacts with signal transduction molecules such as G proteins. GLP-1 receptors are widely distributed in multiple tissues and organs of the human body. In the pancreas, it is mainly present on the surface of pancreatic β cells, which is essential for regulating insulin secretion. When GLP-1 binds to the GLP-1 receptor on the pancreatic β cells, it can stimulate the secretion of insulin, thereby participating in the regulation of blood sugar. It is also distributed in gastrointestinal tissues, such as the mucosal cells of the stomach and small intestine. The activation of GLP-1 receptors can affect gastrointestinal motility and digestive juice secretion and other functions, such as delaying gastric emptying, prolonging the residence time of food in the stomach, making people feel full and reducing food intake. GLP-1 receptors are also distributed in certain areas of the brain, such as the hypothalamus. Their role in the central nervous system is mainly to regulate appetite and energy balance, and to affect the body's eating behavior through nerve signal conduction. The activation of the GLP-1 receptor is divided into two steps. The first step is that the C-terminus of GLP-1 recognizes GLP-1R and binds to the NTD domain of GLP-1R. The second step is that the N-terminus of GLP-1 activates GLP-1R.

[0163] The present invention also relates to a polynucleotide sequence encoding the GLP-1 variant of the present invention or a variant thereof. The polynucleotide may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0164] Variants of polynucleotides may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it. A "polynucleotide encoding a polypeptide" may be a polynucleotide that includes the polypeptide encoding the polypeptide, or may be a polynucleotide that also includes additional coding and / or non-coding sequences.

[0165] The nucleotide sequence or fragment of GLP-1 or its variant of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available DNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. Usually, it is cloned into a vector, then transferred into cells, and then the relevant sequence is isolated from the proliferated host cells by conventional methods.

[0166] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is shorter. Usually, by synthesizing multiple small fragments first and then connecting them, very long fragments of the sequence can be obtained. At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0167] Those skilled in the art can use well-known methods to construct expression vectors containing the gene of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. When using the gene of the present invention to construct a recombinant expression vector, any one of an enhancing, constitutive, tissue-specific or inducible promoter can be added before the transcription initiation nucleotide.

[0168] The vector including the gene of the present invention can be used to transform appropriate host cells so that the host expresses the protein. The host cell can be a prokaryotic cell, such as Escherichia coli, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell, preferably a Malvaceae plant cell, more preferably a Gossypium hirsutum cell. Those skilled in the art are aware of how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism, it can be treated with the CaCl2 method or electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Transformation of plants can also be carried out using methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, young embryo transformation method, flower bud soaking method, etc. For transformed plant cells, tissues or organs, conventional methods can be used to regenerate plants to obtain transgenic plants. When the polynucleotide is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to increase transcription of genes.

[0169] The present invention also relates to nucleic acid constructs. The term "nucleic acid construct" or "polynucleotide construct" refers to one or more single-stranded or double-stranded nucleic acid molecules that are isolated from naturally occurring genes or modified to contain nucleic acid fragments in a manner that does not exist in nature. The term "nucleic acid molecule" primarily refers to the physical nucleic acid molecule, and the term "nucleic acid sequence" primarily refers to the nucleotide sequence on the nucleic acid molecule, but the two terms are used interchangeably, especially with respect to nucleic acid molecules, or nucleic acid sequences, that can encode proteins or protein domains. The nucleic acid construct contains the polynucleotide sequences described herein, and one or more regulatory sequences operably linked to these sequences. The techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0170] The polypeptides described herein can be expressed or produced using the polynucleotide sequences of the present invention by conventional recombinant DNA techniques. In general, the following steps are involved: (1) transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding the polypeptide of the present invention, or with an expression vector containing the polynucleotide; (2) culturing the host cell in a suitable culture medium; (3) isolating and purifying the protein from the culture medium or cells. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid or vector can be used as long as it can replicate and be stable in the host.

[0171] The vector containing the above-mentioned appropriate DNA sequence and the appropriate promoter or control sequence can be used to transform the appropriate host cell to enable it to express the protein. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells, animal cells, etc. Those skilled in the art are aware of how to select appropriate vectors, promoters, enhancers and host cells.

[0172] The present invention also provides the use of a reagent in preparing a kit for regulating the content or stability of GLP-1, regulating animal blood sugar, treating metabolic diseases, or treating diseases that benefit from the regulation of GLP-1 content or stability, the kit comprising (1) a reagent for regulating the expression or activity of IDE, (2) a reagent for regulating the interaction between IDE and GLP-1, (3) a variant of GLP-1 or its derivative, wherein the variant has a mutation or modification at one, two, three or four positions of Ser18, Tyr19, Ala25, Lys26, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device. Unless otherwise defined, the practice of the present invention will employ conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, which are all within the technical scope of the art.

[0173] The kit can play a beneficial effect in metabolic-related diseases (eg, diabetes, obesity, non-alcoholic fatty liver disease, insulin resistance syndrome) or neurological diseases (eg, Alzheimer's disease and Parkinson's syndrome).

[0174] The present invention provides the use of an agent for downregulating the expression or activity of IDE and / or an agent for downregulating the interaction between IDE and GLP-1 and / or a variant of GLP-1 or its derivative as described in any embodiment herein in the preparation of a kit for upregulating the level or stability of GLP-1, downregulating blood sugar in animals, treating diabetes, treating obesity, or treating diseases that benefit from unchanged or increased GLP-1 level or enhanced GLP-1 stability.

[0175] The present invention provides the use of an agent that upregulates the expression or activity of IDE and / or an agent that upregulates the interaction between IDE and GLP-1 in preparing a kit for downregulating the content or stability of GLP-1, upregulating blood glucose in animals, or treating diseases that benefit from reduced GLP-1 content or reduced GLP-1 stability. In one or more embodiments, IDE (e.g., an agent that upregulates the expression or activity of IDE and / or an agent that upregulates the interaction between IDE and GLP-1) can be used in combination with DDP4 to achieve faster degradation of GLP-1; or an IDE inhibitor (e.g., an agent that downregulates the expression or activity of IDE and / or an agent that downregulates the interaction between IDE and GLP-1) can be used in combination with a DDP4 inhibitor to achieve the purpose of stabilizing or increasing the content of GLP-1 or alleviating the decrease in the content of GLP-1.

[0176] The term "pharmaceutical composition" contains a GLP-1 variant described herein and / or its coding sequence, or a polynucleotide, nucleic acid construct, host cell described herein, and a pharmaceutically acceptable excipient, including but not limited to a diluent, a carrier, a solubilizer, an emulsifier, a preservative and / or an adjuvant. The excipient is preferably non-toxic to the recipient at the dose and concentration employed. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, the pH, osmotic properties, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. These substances are known in the prior art. The optimal pharmaceutical composition can be determined depending on the intended route of administration, mode of delivery, and desired dosage.

[0177] Pharmaceutical compositions for in vivo administration are usually provided in the form of sterile preparations. Sterilization is achieved by filtering through a sterile filtration membrane. When the composition is freeze-dried, this method can be used for sterilization before or after freeze-drying and rehydration. The pharmaceutical composition of the present invention can be selected for parenteral delivery. The composition for parenteral administration can be stored in a freeze-dried form or in a solution. For example, it is prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are usually placed in a container with a sterile access hole, such as an intravenous solution strip or a vial with a stopper that can be pierced by a hypodermic injection needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as oral). The preparation of the pharmaceutically acceptable composition is within the technology of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Technologies for preparing a variety of other sustained or controlled delivery methods (such as liposome carriers, bioerodible microparticles or porous beads and accumulated injections) are also known to those skilled in the art.

[0178] Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or in the form of a dehydrated or lyophilized powder. The formulation can be stored in a ready-to-use form or in a form of rehydration before administration (e.g., lyophilization). The present invention also provides a kit for producing a single-dose administration unit. The kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0179] The present invention also provides a method for treating a patient (especially a patient with a disease related to blood sugar disorders) by administering a binding molecule or a pharmaceutical composition thereof as described in any embodiment of the present invention. Herein, the terms "patient", "subject", "individual", and "object" are used interchangeably herein, including any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits, etc.), and most preferably a human. "Treatment" refers to the subject using the treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., diabetes, hypoglycemia, obesity, cardiovascular disease, non-alcoholic fatty liver disease, insulin resistance syndrome, Parkinson's syndrome, Alzheimer's disease). The treatment regimen that effectively treats the patient can vary according to a variety of factors (such as the patient's disease state, age, weight, and the ability of the therapy to stimulate the subject's anti-disease response).

[0180] The present invention also provides a method for regulating the secretion level and stability of GLP-1 cells, comprising: (1) down-regulating the expression or activity of IDE protein in cells, thereby promoting GLP-1 secretion or maintaining stability; or (2) up-regulating the expression or activity of IDE protein in cells, thereby inhibiting GLP-1 secretion or reducing stability.

[0181] In one or more embodiments, (1) includes: transferring the coding sequence of IDE protein into cells to obtain transformed cells, (2) includes: (a) specifically interfering with the transcription and / or expression of IDE-related genes, (b) down-regulating the activity of IDE protein, (c) expressing IDE protein with reduced activity in cells, or (d) introducing inhibitors to competitively bind to IDE protein. In one or more embodiments, the interference in (a) is interference with the transcription of IDE gene or the translation of its transcript, and the down-regulation of IDE protein expression or activity in (b) includes: (i) expressing in cells specific antibodies or ligands (such as inhibitory antibodies) against IDE protein that can down-regulate the activity of IDE protein, or (ii) introducing nucleic acid sequences encoding (i) and / or nucleic acid constructs that can express (i).

[0182] Other aspects of the present invention are obvious to those skilled in the art due to the disclosure of this article. The present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0183] Example

[0184] Example 1, IDE degradation of GLP-1

[0185] 1. Experimental results

[0186] First, 20 μM GLP-1 and 200 nM IDE were co-incubated in 20 mM Tris-HCl, pH 7.7 in vitro. It was found that the abundance of GLP-1 decreased with time, proving that GLP-1 is a substrate of IDE ( Figure 1 .A). Moreover, under the same enzyme concentration, the degradation rate of GLP-1 by IDE is much faster than that of the currently known GLP-1 degrading enzyme DPP4 ( Figure 1 .B). When the IDE inhibitor 6bk or DPP4 inhibitor Sitagliptin at the same concentration as the protease is added to the reaction system, the degradation of GLP-1 is inhibited to a certain extent ( Figure 1 .C).

[0187] We added equal amounts of IDE and GLP-1(7-37) to the pure protein system and added 1mM BS3 cross-linker, such as Figure 1 As shown in Figure .D, the band position of GLP-1 (7-37) in the cross-linker group shifted from the original 3.7KD to about 110KD. Therefore, when 1mM BS3 cross-linker was added to equal amounts of IDE and GLP-1, it can be seen that GLP-1 has a significant mass shift, which also proves that there is indeed an interaction between IDE and GLP-1 ( Figure 1 .D).

[0188] We cut the band that was shifted and detected it by mass spectrometry. We found that the 34th lysine (K) of GLP-1 (7-37) and the 899th lysine (K) of IDE produced a cross-linked fragment ( Figure 1 E) This site is the binding site for ATP. It has been reported that mutating the K at position 899 to C will increase the catalytic activity of IDE by 40 times.

[0189] 2. Experimental Methods

[0190] (1) Mass spectrometry to collect the abundance of peptide fragments after GLP-1 degradation:

[0191] a. 1mg GLP-1 (purchased from Sangon Biotech, T510039-0001) was dissolved in 1ml of a solution containing 25% DMSO. The in vitro reaction system was 50μL, in which the concentration of GLP-1 was 20μM, the concentration of IDE was 200nM, and the solution components of the reaction system were: 20mM Tris-HCl, 100mM NaCl, pH 7.7. The reaction solution was incubated at 37°C and 750rpm with shaking. 10μL was taken at 0 minutes, 15 minutes, 30 minutes and 60 minutes to detect the abundance of GLP-1 and its degradation peptides, and three sets of replicates were set at each time point. 90μL of 0.1% FA solution was added to each sample to terminate the reaction for subsequent desalting.

[0192] b. Take a small amount of C18 filler and fill it into a 200μL pipette tip. First, activate the C18 column with 100μL acetonitrile (ACN), then add 100μL 0.1% FA (0.1% formic acid) solution to balance the C18 column. Then inject the sample obtained in step (a) into the C18 column and repeat 3 times. After desalting with 100μL 0.1% FA 3 times, add 100μL 90% ACN + 0.1% FA to elute the peptides adsorbed on the C18 column. Repeat the above operation once to obtain a total of 200μL eluent. After vacuum drying at 30°C, add 20μL 0.1% FA to redissolve and detect by mass spectrometry.

[0193] c. GLP-1 and its degraded peptides were detected by Thermo Q-Exactive HF, and the liquid phase was EASY-nLC 1000. The length of the analytical column was 15 cm, the inner diameter was 100 μm, and the filler was 1.9 μm C18. The mobile phase A was 98% H2O, 2% ACN, 0.1% FA, and the mobile phase B was 98% ACN, 2% H2O, 0.1% FA. Each sample was collected for 30 minutes, and the mobile phase gradient was: 0min: 8%, 2min: 20%, 5min: 45%, 9min: 55%, 28min: 80%, 30min: 95%. The liquid phase flow rate was 300nl / min. The mass spectrometry method was collected in DDA (data dependent acquisition) mode, and the top 5 parent ions were selected for the collection of daughter ions. MS1 was detected by Orbitrap, with an acquisition range of 250-1500m / z and a resolution of 60000. AGC was set to 3e6, the maximum injection time was 20ms, HCD was used for fragmentation, the fragmentation energy was 32, MS2 was detected by Orbitrap, the initial ion mass-to-nuclear ratio was 120m / z, the resolution was 30000, AGC was set to 1e5, and the maximum injection time was 45ms.

[0194] d. Data analysis: The obtained mass spectrometry data raw files were searched with Maxquant 1.6.0.1 software and matched with the sequence of GLP-1(7-37). The mass accuracy of MS1 was set to 20ppm and that of MS2 was set to 0.1Da. No fixed modification or variable modification was set, and the enzyme was set to unspecific.

[0195] (2) Detection of GLP-1 degradation based on the staining method:

[0196] 1mg GLP-1 was dissolved in 1ml of a solution containing 25% DMSO, and the in vitro reaction system was 15μL, wherein the concentration of GLP-1 was 20μM, the concentration of IDE was 200nM, the concentration of 6bk was 200nM, the concentration of DPP4 (purchased from Abcam, ab79138) was 200nM, and the concentration of Sitagliptin was 200nM. The solution components of the reaction system were: 20mM Tris-HCl, 100mM NaCl, PH 7.7. When adding the inhibitor group, the inhibitor and enzyme needed to be incubated in advance for 30 minutes, the reaction solution was shaken and incubated at 37°C, 750rpm, and 5μL of 5xloading (with DTT) was added at 15 minutes and heated at 95°C for 5 minutes to terminate the reaction. Each group of samples was added to the sample well of 12.5% ​​SDS-polyacrylamide gel, and the voltage was first 60V, and the voltage was kept constant for 30 minutes, then the voltage was changed to 120V and kept constant for 90 minutes. Then, the samples were stained with Coomassie Brilliant Blue until the GLP-1 band appeared, and then decolorized with water until the band was clear.

[0197] (3) Crosslink detects the interaction between IDE and GLP-1:

[0198] a. 1 mg GLP-1 was dissolved in 1 ml of a solution containing 25% DMSO. The in vitro reaction system was 50 μL, in which the amount of GLP-1 was 10 μg, the amount of IDE was 10 μg, the amount of BS3 was 1 mM, and the solution composition of the reaction system was: H2O. The reaction solution was incubated at 37°C and 750 rpm with shaking. After 60 minutes of reaction, 1 M Tris-HCl PH6.8 was added to a final concentration of 100 mM to terminate the reaction, and 5x Loading (DTT) was added to dilute it to 1x, and heated at 95°C for 10 minutes.

[0199] b. Add 10 μL of each sample to the loading well of 7.5% SDS-polyacrylamide gel, first use 60V voltage, keep constant voltage for 30 minutes, then switch the voltage to 120V and keep constant voltage for 90 minutes.

[0200] c. For Coomassie staining, use Coomassie Brilliant Blue to stain directly, and then decolorize with water until the bands are clear. For WB, the PVDF membrane needs to be activated with methanol first, and then placed on filter paper. Then the gel is placed on the PVDF membrane and covered with a layer of filter paper to form a "sandwich". The transfer conditions are constant voltage 25V, 0.3A, 60 minutes.

[0201] d. After blocking with 5% BSA solution for one hour, add primary antibody for overnight incubation, wash the membrane three times with TBST, five minutes each time, add secondary antibody for one hour, wash the membrane three times with TBST, and develop after five minutes each time.

[0202] Example 2: Determination of GLP-1 cleavage sites by IDE and design of inhibitory short peptides

[0203] 1. Experimental results

[0204] In order to clarify the cleavage site of GLP-1 by IDE, we detected the product of the reaction between 200nM IDE and 20μM GLP-1 substrate by mass spectrometry and found that the degradation of GLP-1 by IDE mainly occurred at Ser 18 -Tyr 19 and Ala 25 -Lys 26 Two sites ( Figure 2 .A). Therefore, we synthesized a hexapeptide containing these two cleavage sites (VSSYLE and QAAKEF), added 1mM to the reaction system as a competitive inhibitor of the substrate for co-incubation, and found that in a short period of time, the addition of short peptides containing the two sites can effectively inhibit the degradation of GLP-1 by IDE ( Figure 2 .B,C), the inhibitory effect is similar to that of 6bk. In addition, this type of short peptide inhibitor has the function of specifically inhibiting IDE from degrading GLP-1, while 6bk is not selective and may inhibit the function of IDE from degrading other substrates such as Aβ.

[0205] 2. Experimental Methods

[0206] (1) Mass spectrometry to collect GLP-1 and its degradation peptide abundance:

[0207] a. 1mg GLP-1 (purchased from Sangon Biotech, T510039-0001) was dissolved in 1ml of a solution containing 25% DMSO. The in vitro reaction system was 50μL, in which the concentration of GLP-1 was 20μM, the concentration of IDE was 200nM, and the solution components of the reaction system were: 20mM Tris-HCl, 100mM NaCl, pH 7.7. The reaction solution was incubated at 37°C and 750rpm with shaking. 10μL was taken at 0 minutes, 15 minutes, 30 minutes and 60 minutes to detect the abundance of GLP-1 and its degradation peptides, and three sets of replicates were set at each time point. 90μL of 0.1% FA solution was added to each sample to terminate the reaction for subsequent desalting.

[0208] b. Take a small amount of C18 filler and fill it into a 200μL pipette tip. First, activate the C18 column with 100μL acetonitrile (ACN), then add 100μL 0.1% FA (0.1% formic acid) solution to balance the C18 column. Then inject the sample obtained in step (a) into the C18 column and repeat 3 times. After desalting with 100μL 0.1% FA 3 times, add 100μL 90% ACN + 0.1% FA to elute the peptides adsorbed on the C18 column. Repeat the above operation once to obtain a total of 200μL eluent. After vacuum drying at 30°C, add 20μL 0.1% FA to redissolve and detect by mass spectrometry.

[0209] c. GLP-1 and its degraded peptides were detected by Thermo Q-Exactive HF, and the liquid phase was EASY-nLC 1000. The length of the analytical column was 15 cm, the inner diameter was 100 μm, and the filler was 1.9 μm C18. The mobile phase A was 98% H2O, 2% ACN, 0.1% FA, and the mobile phase B was 98% ACN, 2% H2O, 0.1% FA. Each sample was collected for 30 minutes, and the mobile phase gradient was: 0min: 8%, 2min: 20%, 5min: 45%, 9min: 55%, 28min: 80%, 30min: 95%. The liquid phase flow rate was 300nl / min. The mass spectrometry method was collected in DDA (data dependent acquisition) mode, and the top 5 parent ions were selected for the collection of daughter ions. MS1 was detected by Orbitrap, with an acquisition range of 250-1500m / z and a resolution of 60000. AGC was set to 3e6, the maximum injection time was 20ms, HCD was used for fragmentation, the fragmentation energy was 32, MS2 was detected by Orbitrap, the initial ion mass-to-nuclear ratio was 120m / z, the resolution was 30000, AGC was set to 1e5, and the maximum injection time was 45ms.

[0210] d. Data analysis: The obtained mass spectrometry data raw files were searched with Maxquant 1.6.0.1 software and matched with the sequence of GLP-1(7-37). The mass accuracy of MS1 was set to 20ppm and that of MS2 was set to 0.1Da. No fixed modification or variable modification was set, and the enzyme was set to unspecific.

[0211] (2) Detection of GLP-1 degradation based on the staining method:

[0212] 1mg GLP-1 was dissolved in 1ml of a solution containing 25% DMSO. The in vitro reaction system was 15μL, in which the concentration of GLP-1 was 20μM, the concentration of IDE was 200nM, and the concentration of two hexapeptides (VSSYLE and QAAKEF) was 1mM. The solution components of the reaction system were: 20mM Tris-HCl, 100mM NaCl, pH 7.7. The reaction solution was incubated at 37°C, 750rpm, and 5μL of 5xloading (with DTT) was added at 15 minutes and heated at 95°C for 5 minutes to terminate the reaction. Each group of samples was added to the loading well of a 12.5% ​​SDS-polyacrylamide gel, and a voltage of 60V was applied for 30 minutes, and then the voltage was changed to 120V and the voltage was applied for 90 minutes. Then, the samples were stained with Coomassie Brilliant Blue until the GLP-1 band appeared, and decolorized with water until the band was clear.

[0213] Example 3: Degradation of GLP-1 by intracellular and cell-secreted IDE

[0214] 1. Experimental results

[0215] After proving the degradation effect of IDE on GLP-1 in vitro, we collected the cell culture medium of JHH7 and concentrated it with a 3k filter membrane, added 5μM GLP-1 exogenously, and incubated it at 37℃ for a certain period of time, and then detected its degradation in the culture medium. First, we detected the presence of IDE protease in the cell culture medium of JHH7, and found that exogenously added GLP-1 would be degraded in the concentrated culture medium over time ( Figure 3 .A). Analysis of the degradation site of GLP-1 in the culture medium revealed that the degradation site of GLP-1 in the JHH7 conditioned medium was still at Ser 18 -Tyr 19 and Ala 25 -Lys 26 superior( Figure 3.B). Based on this, we speculated that IDE in the culture medium played a major role in the degradation of GLP-1. Therefore, we knocked down IDE in JHH7 to reduce the expression of IDE in the culture medium. We found that GLP-1 was almost not degraded under this condition, and the restoration of IDE expression would lead to the re-degradation of GLP-1 ( Figure 3 .C). Mass spectrometry results also showed that knocking down the expression of IDE could reduce the expression of GLP-1 at Ser 18 -Tyr 19 and Ala 25 -Lys 26 The amount of enzyme-cut fragments produced was significantly reduced, which also verified our hypothesis. In addition to detecting the effect of IDE in the cell culture medium on the degradation of exogenously added GLP-1, we also selected the cell line NCI-H716 that can secrete GLP-1 by itself to further study whether IDE will have an effect on the cell line that can secrete GLP-1 by itself. Therefore, we knocked down the expression of IDE in this cell line, collected the culture medium of this cell, and used a C18 column to adsorb the GLP-1 secreted by the cells into the culture medium. Through mass spectrometry, we detected that the secretion of GLP-1 in the culture medium was more than that of cells that did not knock down IDE, which also shows that IDE is the enzyme that causes the degradation of GLP-1 expressed and secreted by the cells themselves.

[0216] 2. Experimental Methods

[0217] (1) Mass spectrometry acquisition of GLP-1 and its degradation peptide abundance in the culture medium:

[0218] a. First, replace the JHH7 cell culture medium in a 6 cm dish with 1.5 mL of PBS and place it in a cell culture incubator at 37°C and 5% CO2. After culturing for 1 hour, collect the culture medium and concentrate it to 500 μL using a 3k filter at 12,000 g.

[0219] b. 1 mg GLP-1 was dissolved in 1 ml of a solution containing 25% DMSO. The in vitro reaction system was 100 μL, in which the concentration of GLP-1 was 5 μM. The solution components of the reaction system were: concentrated JHH7 culture medium. The reaction solution was incubated at 37°C and 750 rpm with shaking. 20 μL was taken at 0 minutes and 15 minutes to detect the abundance of GLP-1 and its degradation peptides. Three sets of repetitions were set at each time point. 90 μL of 0.1% FA solution was added to each sample to terminate the reaction for subsequent desalting.

[0220] c. Take a small amount of C18 filler and fill it into a 200μL pipette tip. First, activate the C18 column with 100μL acetonitrile (ACN), then add 100μL 0.1% FA (0.1% formic acid) solution to balance the C18 column. Then inject the sample obtained in step (a) into the C18 column and repeat 3 times. After desalting with 100μL 0.1% FA 3 times, add 100μL 90% ACN + 0.1% FA to elute the peptides adsorbed on the C18 column. Repeat the above operation once to obtain a total of 200μL eluent. After vacuum drying at 30°C, add 20μL 0.1% FA to redissolve and detect by mass spectrometry.

[0221] d. GLP-1 and its degraded peptides were detected by Thermo Q-Exactive HF, and the liquid phase was EASY-nLC 1000. The length of the analytical column was 15 cm, the inner diameter was 100 μm, and the filler was 1.9 μm C18. The mobile phase A was 98% H2O, 2% ACN, 0.1% FA, and the mobile phase B was 98% ACN, 2% H2O, 0.1% FA. Each sample was collected for 30 minutes, and the mobile phase gradient was: 0min: 8%, 2min: 20%, 5min: 45%, 9min: 55%, 28min: 80%, 30min: 95%. The liquid phase flow rate was 300nl / min. The mass spectrometry method was collected in DDA (data dependent acquisition) mode, and the top 5 parent ions were selected for the collection of daughter ions. MS1 was detected by Orbitrap, with an acquisition range of 250-1500m / z and a resolution of 60000. AGC was set to 3e6, the maximum injection time was 20ms, HCD was used for fragmentation, the fragmentation energy was 32, MS2 was detected by Orbitrap, the initial ion mass-to-nuclear ratio was 120m / z, the resolution was 30000, AGC was set to 1e5, and the maximum injection time was 45ms.

[0222] e. Data analysis: The obtained mass spectrometry data raw files were searched with Proteome discoverer 2.1 software and matched with the sequence of GLP-1(7-37). The mass accuracy of MS1 was set to 10ppm and that of MS2 was set to 0.02Da. No fixed modification or variable modification was set, and the enzyme was set to No-enzyme (unspecific).

[0223] (2) WB detection of IDE content in culture medium:

[0224] a. Collect cells and lyse them by sonication with RIPA lysis buffer for 2 minutes at a sonication frequency of 35%.

[0225] b. Measure protein concentration using BCA and quantify to the same concentration.

[0226] c. After collecting the cell culture medium, concentrate it to the same volume using a 3k filter.

[0227] d. Add 10 μL of each sample to the loading well of 12.5% ​​SDS-polyacrylamide gel, first use 60V voltage, keep constant voltage for 30 minutes, then switch the voltage to 120V and keep constant voltage for 90 minutes.

[0228] e. Activate the PVDF membrane with methanol and place it on filter paper. Then place the gel on the PVDF membrane and cover it with a layer of filter paper to form a "sandwich". The transfer conditions are constant voltage 25V, 0.3A, 60 minutes.

[0229] f. After blocking with 5% BSA solution for one hour, add primary antibody and incubate overnight, wash the membrane three times with TBST, each time for five minutes, add secondary antibody and incubate for one hour, wash the membrane three times with TBST, each time for five minutes before developing.

[0230] (3) Construction of JHH7 and NCI-H716 cell lines with knockdown of IDE expression:

[0231] a. First, generate two oligonucleotide chains, the forward oligonucleotide chain is: CCGG-TAGTTTCTGATTCACTATTAG-CTCGAG-CTAATAGTGAATCAGAAACTA-TTTTTG; the reverse oligonucleotide chain is: AATTCAAAAA-TAGTTTCTGATTCACTATTAG-CTCGAG-CTAATAGTGAATCAGAAACTA.

[0232] b. Digest the pLKO.1 vector with EcoRI and AgeI, and connect the two nucleotide chains to this vector to construct the IDE knockdown plasmid.

[0233] c. Transfer it into 293T cells, collect the culture medium, obtain lentivirus and use it to infect JHH7 cells for 3 days, then add 8 μg / ml puromycin to select IDE knockdown cell lines.

[0234] (4) Detection of GLP-1 content in NCI-H716 cell culture medium:

[0235] a. Quantify wild-type NCI-H716 and IDE-knockdown NCI-H716 and plate them in six-well plates, with 2x10 6 cells.

[0236] b. After incubating the cells with 2 ml PBS for 1 hour, the culture medium was collected.

[0237] c. 500 μL of the aliquot was concentrated to 80 μL using a 3K filter, and the IDE content in the culture medium was detected by WB.

[0238] d. Another 1.5 ml of the culture medium was filtered through a C18 column to allow the secreted GLP-1 to be adsorbed onto the C18 column.

[0239] e. Take a small amount of C18 filler and fill it into a 200μL pipette tip. First, activate the C18 column with 100μL acetonitrile (ACN), then add 100μL 0.1% FA (0.1% formic acid) solution to balance the C18 column. Then inject the sample obtained in step (d) into the C18 column and repeat 3 times. After desalting with 100μL 0.1% FA 3 times, add 100μL 90% ACN + 0.1% FA to elute the peptides adsorbed on the C18 column. Repeat the above operation once to obtain a total of 200μL eluent. After vacuum drying at 30°C, add 20μL 0.1% FA to redissolve and detect by mass spectrometry.

[0240] f. GLP-1 and its degraded peptides were detected by Thermo Q-Exactive HF, and the liquid phase was EASY-nLC 1000. The length of the analytical column was 15 cm, the inner diameter was 100 μm, and the filler was 1.9 μm C18. The mobile phase A was 98% H2O, 2% ACN, 0.1% FA, and the mobile phase B was 98% ACN, 2% H2O, 0.1% FA. Each sample was collected for 30 minutes, and the mobile phase gradient was: 0min: 8%, 2min: 20%, 5min: 45%, 9min: 55%, 28min: 80%, 30min: 95%. The liquid phase flow rate was 300nl / min. The mass spectrometry method was collected in DDA (data dependent acquisition) mode, and the top 5 parent ions were selected for the collection of daughter ions. MS1 was detected by Orbitrap, with an acquisition range of 250-1500m / z and a resolution of 60000. AGC was set to 3e6, the maximum injection time was 20ms, HCD was used for fragmentation, the fragmentation energy was 32, MS2 was detected by Orbitrap, the initial ion mass-to-nuclear ratio was 120m / z, the resolution was 30000, AGC was set to 1e5, and the maximum injection time was 45ms.

[0241] g. Data analysis: The obtained mass spectrometry data raw files were searched using Proteome discoverer 2.1 software and matched with the sequence of GLP-1(7-37). The mass accuracy of MS1 was set to 10ppm and that of MS2 to 0.02Da. No fixed modification or variable modification was set, and the enzyme was set to No-enzyme (unspecific).

[0242] Example 4, Stability of GLP-1 after mutation

[0243] 1. Experimental results

[0244] After clarifying the degradation effect of IDE on GLP-1, we modified the main cleavage site of IDE, mutated the two amino acids on the left and right of the cleavage site into D-type amino acids, and replaced the main cleavage site A of DPP4 with the unnatural amino acid Aib (2-aminoisobutyric acid (α-Aminoisobutyric acid), obtaining Aib with mutations at both sites. 8 -D-Ser 18 -Ala 25 -GLP-1(7-37) and Aib with single mutation 8 -D-Ser 18 -GLP-1(7-37), Aib 8 -D-Tyr 19 -GLP-1(7-37), Aib8-D-Ala 25 -GLP-1(7-37), Aib 8 -D-Lys 26 - Five new GLP-1 receptor agonists of GLP-1(7-37) and the Ser 18 Mutated D-Ser 18 -Semaglutide( Figure 4 .A), and the seven GLP-1 receptor agonists were reacted in vitro with 20 μM of each and 200 nM of IDE, and the effects of these substrates on resisting IDE degradation were tested by incubation for 0 min, 5 min, 15 min and 30 min. The experimental results showed that the stability of the GLP-1 receptor agonists after site modification was better than that of GLP-1 itself, and we found that Aib with both sites mutated 8 -D-Ser 18 -Ala 25 -GLP-1(7-37) and D-Ser 18 -Semaglutide is most stable in the presence of IDE ( Figure 4 .B,C).

[0245] In order to clarify whether the modified GLP-1 receptor agonist still has good stability in complex environments with more proteases such as cell culture medium, mouse plasma or mouse peritoneal fluid. We collected the culture medium of JHH7, extracted and diluted mouse blood samples, and injected PBS into the mouse peritoneum, and then collected the perfusion fluid again. First, the presence of IDE can be detected in cell culture medium, mouse plasma and mouse peritoneal fluid ( Figure 5 .A), so we added equal amounts of GLP-1 and other modified GLP-1 receptor agonists to these reaction systems and further compared the Aib with mutations at both sites. 8 -D-Ser1 8 -Ala 25 -GLP-1(7-37) and D-Ser 18 -Semaglutide and GLP-1 and Semaglutide in different microenvironments. After mass spectrometry detection of the products of these substrates in different microenvironments, it was found that Ser 18 -Tyr 19 The site is also one of the main cleavage sites of GLP-1 and Semaglutide, and the mutant Ser 18 -Tyr 19 The GLP-1 receptor agonists after the site are more stable and do not produce a large number of enzyme fragments at this site ( Figure 5 .BG).

[0246] 2. Experimental Methods

[0247] (1) Mass spectrometry acquisition of D-type GLP-1 and its degradation peptide abundance:

[0248] a.4mg Aib 8 -D-Ser 18 -Ala 25 -GLP-1(7-37), Aib 8 -D-Ser 18 -GLP-1(7-37), Aib 8 -D-Tyr 19 -GLP-1(7-37), Aib 8 -D-Ala 25 -GLP-1(7-37), Aib 8 -D-Lys 26 -GLP-1(7-37) and D-Ser 18 -Semaglutide (both synthesized at Nanjing Jiepeptide Biotechnology Co., Ltd.) were dissolved in 1 ml of DMSO solution and then diluted to 1 mg / ml with ddH2O.

[0249] b. IDE degradation in vitro reaction system is 50 μL, in which Aib 8 -D-Ser 18 -Ala 25 -GLP-1(7-37), Aib 8 -D-Ser 18 -GLP-1(7-37), Aib8 -D-Tyr 19 -GLP-1(7-37), Aib 8 -D-Ala 25 -GLP-1(7-37), Aib 8 -D-Lys 26 -GLP-1(7-37) and D-Ser 18 -Semaglutide concentration was 20μM, IDE concentration was 200nM, and the reaction system solution composition was: 20mM Tris-HCl, 100mM NaCl, PH 7.7. The reaction solution was incubated at 37°C, 750rpm, and 10μL was taken at 0 minutes, 15 minutes, 30 minutes and 60 minutes to detect the abundance of GLP-1 and its degradation peptides, and three sets of replicates were set at each time point. 90μL of 0.1% FA solution was added to each sample to terminate the reaction for subsequent desalting.

[0250] c. Cell culture medium reaction system: Replace JHH7 cell culture medium with 1 ml PBS. After culturing for 1 hour, collect the culture medium and concentrate it. The reaction system is 100 μL, Aib 8 -D-Ser 18 -Ala 25 -GLP-1(7-37), Aib 8 -D-Ser 18 -GLP-1(7-37), Aib 8 -D-Tyr 19 -GLP-1(7-37), Aib 8 -D-Ala 25 -GLP-1(7-37), Aib 8 -D-Lys 26 -GLP-1(7-37) and D-Ser 18 The concentration of -Semaglutide was 5 μM and the reaction time was 30 min.

[0251] d. Plasma reaction system: Take 300 μL of mouse plasma and dilute it with PBS at a ratio of 1:4. Reaction system 50 μL, Aib 8 -D-Ser 18 -Ala 25 -GLP-1(7-37), Aib 8 -D-Ser 18 -GLP-1(7-37), Aib 8 -D-Tyr 19 -GLP-1(7-37), Aib 8 -D-Ala25 -GLP-1(7-37), Aib 8 -D-Lys 26 -GLP-1(7-37) and D-Ser 18 The concentration of -Semaglutide was 5 μM and the reaction time was 30 min.

[0252] e. Mouse peritoneal fluid reaction system: Inject 1 ml PBS into the mouse peritoneum and draw it out after two minutes. Reaction system 100 μL, Aib 8 -D-Ser 18 -Ala 25 -GLP-1(7-37), Aib 8 -D-Ser 18 -GLP-1(7-37), Aib 8 -D-Tyr 19 -GLP-1(7-37), Aib 8 -D-Ala 25 -GLP-1(7-37), Aib 8 -D-Lys 26 -GLP-1(7-37) and D-Ser 18 The concentration of -Semaglutide was 5 μM and the reaction time was 2 hours.

[0253] f. Take a small amount of C18 filler and fill it into a 200μL pipette tip. First, activate the C18 column with 100μL acetonitrile (ACN), then add 100μL 0.1% FA (0.1% formic acid) solution to balance the C18 column. Then inject the sample obtained in step (ce) into the C18 column and repeat 3 times. After desalting with 100μL 0.1% FA 3 times, add 100μL 90% ACN + 0.1% FA to elute the peptides adsorbed on the C18 column. Repeat the above operation once to obtain a total of 200μL eluent. After vacuum drying at 30°C, add 20μL 0.1% FA to redissolve and detect by mass spectrometry.

[0254] g.Aib 8 -D-Ser 18 -Ala 25 -GLP-1(7-37), Aib 8 -D-Ser 18 -GLP-1(7-37), Aib 8 -D-Tyr 19 -GLP-1(7-37), Aib 8 -D-Ala 25 -GLP-1(7-37), Aib 8 -D-Lys26 -GLP-1(7-37) and D-Ser 18 -Semaglutide and its degraded peptides were detected by Thermo Q-Exactive HF, and the liquid phase was EASY-nLC1000. The length of the analytical column was 15 cm, the inner diameter was 100 μm, and the filler was 1.9 μm C18. The mobile phase A was 98% H2O, 2% ACN, 0.1% FA, and the mobile phase B was 98% ACN, 2% H2O, 0.1% FA. Each sample was collected for 30 minutes, and the mobile phase gradient was: 0min: 8%, 2min: 20%, 5min: 45%, 9min: 55%, 28min: 80%, 30min: 95%. The liquid phase flow rate was 300nl / min. The mass spectrometry method was collected in DDA (data dependent acquisition) mode, and the top 5 parent ions were selected for the collection of daughter ions. MS1 was detected by Orbitrap, with an acquisition range of 250-1500m / z and a resolution of 60000. AGC was set to 3e6, the maximum injection time was 20ms, HCD was used for fragmentation, the fragmentation energy was 32, MS2 was detected by Orbitrap, the initial ion mass-to-nuclear ratio was 120m / z, the resolution was 30000, AGC was set to 1e5, and the maximum injection time was 45ms.

[0255] h. Data analysis: The obtained mass spectrometry data raw files were searched using Proteome discoverer 2.1 software and matched with the sequence of GLP-1(7-37). The mass accuracy of MS1 was set to 10ppm and MS2 to 0.02Da. The variable modification was set to Aib(+14.016Da,A) and the enzyme was set to No-enzyme(unspecific).

[0256] Example 5, Agonist activity of GLP-1 after mutation

[0257] 1. Experimental results

[0258] In order to verify whether our site-modified GLP-1 receptor agonist is still active, we overexpressed GLP-1R in 293T cells and added new GLP-1 receptor agonists of different concentrations to determine the activity of the GLP-1 receptor agonist based on the amount of cAMP produced by the cells. The results showed that the site-modified GLP-1 receptor agonist still had good agonist activity and did not have much effect on its binding to the receptor, which is consistent with the results reported in previous literature ( Figure 6 ).

[0259] 2. Experimental Methods

[0260] (1) GLP-1 agonist activates receptors to detect cAMP levels:

[0261] a. Transfect 293T cells with a confluence of 70% in a 6 cm dish with the flag-pCMV-GLP-1R vector for 24 hours to overexpress GLP-1R in the cells. Collect the cells and dilute them to 400 cells / μL, and seed them in a 384-well plate at a number of 2000 cells per well.

[0262] b. Aib 8 -D-Ser 18 -Ala 25 -GLP-1(7-37), Aib 8 -D-Ser 18 -GLP-1(7-37), Aib 8 -D-Tyr 19 -GLP-1(7-37), Aib 8 -D-Ala 25 -GLP-1(7-37), Aib 8 -D-Lys 26 -GLP-1(7-37) and D-Ser 18 -Semaglutide agonist concentration range is set between 0.5pM-1μM, 5μL receptor agonist is added to each well, and cultured at 37°C, 5% CO2 for 30 minutes to stimulate cells to produce cAMP.

[0263] c. After the stimulation, d2-cAMP and the corresponding buffer in the cAMP-GSDYNAMIC KIT (purchased from PerkinElmer, 62AM4PEB) were added, and the fluorescence values ​​at 665nm and 620nm were detected under the excitation light of 337nm. The results were calculated as the ratio of 665nm / 620nm.

[0264] Example 6: Short-term hypoglycemic effect of mutated GLP-1 on high-fat diet-induced obese (DIO) mice

[0265] 1. Experimental results

[0266] In order to further verify the effect of our site-modified GLP-1 receptor agonist in mice, we selected 28 high-fat diet-induced obesity (DIO) mice of similar weight for the experiment. The mice were randomly divided into 4 groups, 7 in each group, and the intraperitoneal glucose tolerance test (IPGTT) under drug administration conditions was performed. The specific experimental steps were: normal saline, 50nmol / kg GLP1, 50nmol / kg Liraglutide, 50nmol / kg Aib were injected at -30min. 8 -D-Ser 18 -Ala 25 -GLP-1(7-37), injected 2g / kg glucose at 0min, and then tested the blood glucose fluctuation of mice, and found that Aib 8 -D-Ser 18 -Ala 25 - The GLP-1(7-37) group did have a better glucose-lowering effect than the GLP-1 group, and was similar to the glucose-lowering effect of Liraglutide ( Figure 7 ).

[0267] 2. Experimental Methods

[0268] (1) Induced obesity mouse (DIO) model:

[0269] DIO diet induction. C57BL / 6J mice (Lingchang) were purchased, and mice of similar weight were selected and randomly divided into two groups. At 6 weeks of age, one group was fed with 60% kcal% FAT ultra-high fat diet (Ruidi Biotechnology, D12492), and the other group was fed with normal food as the control group. The mice were allowed to eat freely for 16 weeks. The experiment was then carried out in the last 2 weeks of the mice diet.

[0270] (2) Mouse glucose tolerance test:

[0271] After the mice were fasted for 16 hours, the glucose tolerance test was started. Fasting basal blood glucose measurement. The mice were taken out of the cage and gently placed on the iron grid. The tail of the mouse was cut off about 1-2 mm with scissors. The tail of the mouse was gently squeezed along the tail vein to enrich the blood into one drop. The first drop of blood was discarded and the second drop of blood was used. Fasting blood glucose was measured with a blood glucose meter (Roche, Active) and a blood glucose test strip (Roche, Active). The measured value was the default blood glucose value at 0 min. The glucose concentration of the glucose tolerance test was 2 g / kg, and the drug concentration was 50 nmol / kg. The mouse was gently picked up and injected with glucose solution using a 1 mL syringe according to the standard intraperitoneal injection operation. The injection volume was determined according to the weight of the mouse, and 0.01 mL was injected per gram of body weight. The timing started from the completion of the injection. The blood glucose value of each mouse at each time point was measured at 15 min, 30 min, 45 min, 60 min, 90 min, and 120 min.

[0272] Example 7: Reaction of short peptides with IDE and their effect on GLP-1 degradation as competitive inhibitors

[0273] 1. Experimental results

[0274] After clarifying that the main degradation sites of IDE on GLP-1 are Ser18-Tyr19 and Ala25-Lys26, we synthesized the octapeptides DVSXYLEG and GQAXKEFI containing mutations at these two sites, used them as reaction substrates, added 1 μM and 250 nM IDE to the reaction system, and reacted at 37°C for 15 minutes, hoping to screen out amino acid mutations that can resist IDE degradation. The results showed that mutation of position 18 of Ser18-Tyr19 to Pro, Asp, Lys, and Asn can improve the stability of DVSXYLEG, and mutation of position 25 of Ala25-Lys26 to His, Cys, Phe, Gly, Gln, and Trp can improve the stability of GQAXKEFI ( Figure 8 .A,B), so we speculate that applying these mutations to the Ser18-Tyr19 and Ala25-Lys26 sites of GLP-1 may help GLP-1 resist degradation by IDE.

[0275] In addition, according to the existing data, the addition of hexapeptides VSSYLE and QAAKEF at the two sites of Ser18-Tyr19 and Ala25-Lys26 to the IDE and GLP-1 reaction system can play a role in competitively inhibiting IDE degradation. Therefore, we also added 100μM of the synthesized octapeptide containing two site mutations to the reaction system of 100μl, IDE 200nM, and substrate GLP-1 (Abz / DNP) 10μM in a 96-well plate to detect the luminescence of GLP-1. Since the complete form of GLP-1 (Abz / DNP) has a fluorescent group (Abz) at the N-terminus and a quenching group (DNP) at the C-terminus, it does not emit fluorescence. When cleaved by IDE, the fluorescent group and the quenching are separated, and we can detect the increase in the value at Ex / Em=320nm / 420nm. By comparing the differences in fluorescence values ​​at 20 minutes after adding different peptides, we found that the inhibitory effect of the octapeptide with partial mutations was better than that of DVSSYLEG and GQAAKEFI, and the inhibitory effect of the octapeptide with two sites added at the same time was better than that of the short peptide with only a single site. The inhibitory effect was best when 100 μM of DVSYYLEG and GQANKEFI were added to the reaction system ( Figure 8 .C,D,E).

[0276] From the results of the reaction of the octapeptide after the binding site mutation with IDE alone and the results of the octapeptide as a short peptide competitive inhibitor, the two short peptides DVSPYLEG and GQAWKEFI can not only resist the cleavage effect of IDE, but also effectively inhibit the cleavage of GLP-1 by IDE as short peptide competitive inhibitors. Other short peptides, such as DVSYYLEG, DVSRYLEG, DVSMYLEG, GQASKEFI, GQAYKEFI and GQANKEFI, reacted with IDE alone and showed the characteristics of being more easily cleaved by IDE. In the experiment as a competitive inhibitor, they can better protect GLP-1 from being cleaved by IDE, which may be due to the fact that these short peptides have a stronger affinity with the active pocket of IDE.

[0277] 2. Experimental Methods

[0278] (1) Mass spectrometry acquisition of peptide abundance after IDE degradation of octapeptide:

[0279] a. 1 mg of octapeptide (all synthesized in GenScript) was dissolved in 1 ml of ddH2O, and the in vitro reaction system was 20 μL, in which the concentration of octapeptide was 1 μM, the concentration of IDE was 250 nM, and the solution composition of the reaction system was: 10 mM NH4HCO3. The reaction solution was incubated at 37°C and 750 rpm with shaking, and the time points were set at 0 min and 15 min to detect the abundance of the octapeptide, and three sets of replicates were set at each time point.

[0280] b. The octapeptide and its degraded peptides were detected by Thermo Q-Exactive HF, and the liquid phase was EASY-nLC1000. The length of the analytical column was 15 cm, the inner diameter was 100 μm, and the filler was 1.9 μm C18. The mobile phase A was 98% H2O, 2% ACN, 0.1% FA, and the mobile phase B was 98% ACN, 2% H2O, 0.1% FA. Each sample was collected for 30 minutes, and the mobile phase gradient was: 0min: 8%, 2min: 20%, 5min: 45%, 9min: 55%, 28min: 80%, 30min: 95%. The liquid phase flow rate was 300nl / min. The mass spectrometry method was collected in DDA (data dependent acquisition) mode, and the top 5 parent ions were selected for the collection of daughter ions. MS1 was detected by Orbitrap, with an acquisition range of 250-1500m / z and a resolution of 60000. AGC was set to 3e6, the maximum injection time was 20ms, HCD was used for fragmentation, the fragmentation energy was 32, MS2 was detected by Orbitrap, the initial ion mass-to-nuclear ratio was 120m / z, the resolution was 30000, AGC was set to 1e5, and the maximum injection time was 45ms.

[0281] c. Data analysis: Thermo Xcalibur Qual Browser software was used to extract the peak of the full-length octapeptide and calculate the peak area as the abundance of the full-length octapeptide.

[0282] (2) Fluorescence detection of octapeptide as a competitive inhibitor to protect GLP-1:

[0283] a. 1 mg of octapeptide (both synthesized in GenScript) was dissolved in 1 ml of ddH2O, and 4 mg of GLP-1 (Abz / DNP) (synthesized in GenScript) was dissolved in 1 ml of DMSO.

[0284] b. The in vitro reaction system is 100 μL, in which the concentration of the single-site octapeptide is 200 μM, and the concentration of the two-site octapeptide is 100 μM each of DVSXYLEG and GQAXKEFI. The concentration of IDE is 200 nM, and the solution composition of the reaction system is: 20 mM Tris-HCl, 100 mM NaCl, PH 7.7.

[0285] c. Add the reaction solution to a 96-well plate and use EnSpire TM The multi-label microplate reader detects the fluorescence signal at Ex=320nm, Em=420nm in real time.

[0286] d. The fluorescence signal at 0 min was taken as the basic signal, and the values ​​at other time points were divided by the fluorescence signal value at 0 min. Relitive Fluorescence Intensity (RFI) = FI (x min) / FI (0 min).

[0287] Example 8, Stability of mutated GLP-1 in simulated intestinal and gastric fluid environments

[0288] 1. Experimental results

[0289] In order to study whether the mutated GLP-1 has the possibility of oral administration, we tested the stability of the mutated GLP-1 in an environment simulating intestinal and gastric fluid. After preparing a suspension of simulated intestinal fluid (SIF) and gastric fluid (SGF) with porcine pepsin and porcine pancreatic enzymes, we added 5 μg of GLP-1 and the mutated GLP-1 to 40 μl of the reaction system and incubated at 37°C for 0 minutes, 5 minutes, 15 minutes and 30 minutes. It can be seen that GLP-1 gradually degrades over time in the environment of simulated gastrointestinal fluid, while the mutated D-Ser18-Semaglutide is relatively stable in both environments ( Fig. 9 ).

[0290] 2. Experimental Methods

[0291] (1) Preparation of suspension simulating intestinal and gastric fluid environment:

[0292] a. Intestinal fluid: Dissolve 0.68g KH2PO3 in 25ml water, add sodium hydroxide solution to adjust pH to 6.8. Then, add 1g porcine pancreatic enzyme under gentle stirring, and adjust the volume to 100ml with water to form a good suspension.

[0293] b. Gastric juice: Dissolve 0.2 g NaCl in 50 ml water, adjust the pH to 1.2 by adding hydrochloric acid and add 0.32 g porcine pepsin, fill the volume to 100 ml with water.

[0294] (2) Degradation of GLP-1 and mutated GLP-1 in simulated intestinal gastric fluid environment

[0295] a. First, dilute the prepared intestinal juice and gastric juice to 1 / 500 of the original.

[0296] b. The reaction system was 40 μl, and the concentration of GLP-1 and mutated GLP-1 was 5 μg.

[0297] c. Incubate the sample at 37°C, 750 rpm with shaking, add 10 μl 5x loading (with DTT) at 0 min, 5 min, 15 min, and 30 min, and terminate the reaction by heating at 95°C for 10 min.

[0298] d. Add 10 μL of each sample to the loading well of 12.5% ​​SDS-polyacrylamide gel, first use 60V voltage, keep constant voltage for 30 minutes, then switch the voltage to 120V and keep constant voltage for 90 minutes.

[0299] e. Activate the PVDF membrane with methanol and place it on filter paper. Then place the gel on the PVDF membrane and cover it with a layer of filter paper to form a "sandwich". The transfer conditions are constant voltage 25V, 0.3A, 60 minutes.

[0300] f. After blocking with 5% BSA solution for one hour, add primary antibody and incubate overnight, wash the membrane three times with TBST, each time for five minutes, add secondary antibody and incubate for one hour, wash the membrane three times with TBST, each time for five minutes before developing.

[0301] Example 9, IDE concentration in the brain is higher than DPP-4

[0302] 1. Experimental results

[0303] The Western Blot method was used to simultaneously detect the expression of IDE and DPP-4 in the mouse brain and monkey cerebrospinal fluid, and it was found that the expression of IDE was higher than that of DPP-4 ( Fig.10 A, B).

[0304] 2. Experimental Methods

[0305] (1) WB detection of IDE and DPP-4 expression in mouse brain:

[0306] a. First, five C57 / BL6J mice (Lingchang) were taken, and they were sacrificed and perfused with PBS, and then the whole brain was removed. After weighing and recording the weight of the mouse brain, an equal proportion of PBS (1 g = 1 ml) was added, and then grinding beads were added and ground with a tissue grinder, and then centrifuged at 14000 rpm for 20 minutes and the supernatant was taken.

[0307] b. Measure protein concentration using BCA and quantify to the same concentration.

[0308] c. Mix with 4X Loading Buffer at a ratio of 1:4 and cook at 95 degrees Celsius for 10 minutes.

[0309] d. Add 5 μg of each sample to the loading well of a 10% SDS-polyacrylamide gel, first apply a voltage of 60 V and keep the voltage constant for 30 minutes, then switch the voltage to 120 V and keep the voltage constant for 90 minutes.

[0310] e. Activate the PVDF membrane with methanol and place it on filter paper. Then place the gel on the PVDF membrane and cover it with a layer of filter paper to form a "sandwich". The transfer conditions are constant voltage 25V, 0.3A, 60 minutes.

[0311] f. After blocking with 5% BSA solution for one hour, add primary antibody and incubate overnight, wash the membrane three times with TBST, each time for five minutes, add secondary antibody and incubate for one hour, wash the membrane three times with TBST, each time for five minutes before developing.

[0312] (2) WB detection of IDE and DPP-4 expression in monkey cerebrospinal fluid:

[0313] a. Use BCA to measure the protein concentration in monkey cerebrospinal fluid and quantify it to the same concentration.

[0314] b. Mix with 4X Loading Buffer at a ratio of 1:4 and cook at 95 degrees Celsius for 10 minutes.

[0315] c. Add 2 μg of each sample to the loading well of 10% SDS-polyacrylamide gel, first use 60V voltage, keep constant voltage for 30 minutes, then switch the voltage to 120V and keep constant voltage for 90 minutes.

[0316] d. Activate the PVDF membrane with methanol and place it on filter paper. Then place the gel on the PVDF membrane and cover it with a layer of filter paper to form a "sandwich". The transfer conditions are constant voltage 25V, 0.3A, 60 minutes.

[0317] e. After blocking with 5% BSA solution for one hour, add primary antibody for overnight incubation, wash the membrane three times with TBST, five minutes each time, add secondary antibody for one hour, wash the membrane three times with TBST, and develop after five minutes each time.

[0318] Example 10 Effects of Semaglutide after mutation on food intake, blood glucose and body weight in high-fat diet-induced obese (DIO) mice

[0319] 1. Experimental results

[0320] We randomly divided 21 DIO-C57 / BL6J mice (Biocytogen) of similar body weight into three groups: saline group; Semaglutide (25nmol) and D-Ser18-Semaglutide (25nmol), with 7 mice in each group. Semaglutide and D-Ser18-Semaglutide were injected intraperitoneally once a day with equal concentrations, and the changes in food intake, blood sugar, and body weight of the mice were continuously observed ( Fig.11 .A, B, C). We found that the effect of the reduction in food intake in the D-Ser18-Semaglutide group was significantly greater than that in the Semaglutide group ( Fig.11 C) The main pathway for GLP-1RA to achieve weight loss is to act on the brain to reduce food intake, thereby achieving the goal of weight loss. This shows that the mechanism of IDE degradation of GLP-1 that we discovered has great potential in GLP-1RA weight loss.

[0321] 2. Experimental Methods

[0322] (1) Injection. Twenty-one DIO-C57 / BL6J mice (Biocytogen) were randomly divided into three groups: saline group, Semaglutide (25 nmol), and D-Ser18-Semaglutide (25 nmol), with 7 mice in each group, and intraperitoneally injected once a day for two days.

[0323] (2) The blood glucose, body weight, and food intake of mice were tested every day.

[0324] a. Method for measuring body weight: Place the mouse in a weighing box and weigh the mouse using an electronic balance (range: 0-1000g, sensitivity 0.01g).

[0325] b. Method for measuring blood sugar: Take the mouse out of the cage and gently place it on the iron grid. Cut off about 1-2 mm of the end of the mouse's tail with scissors. Gently squeeze the mouse's tail along the tail vein to allow the blood to accumulate into one drop. Discard the first drop of blood and use the second drop of blood. Use a blood glucose meter (Roche, Active) and blood glucose test strips (Roche, Active) to measure blood sugar.

[0326] c. Method for measuring food intake: At the same time every day, use an electronic balance to weigh the weight of the food in the mouse feeding trough.

[0327] Mouse food intake = weight of food eaten by mice the day before - weight of food eaten by mice today / number of mice in the cage

[0328] (Note: Each group should have at least three cages to reduce errors)

[0329] Example 11: Effect of knocking down IDE expression on the amount of GLP-1 in mouse liver

[0330] We will also use shRNA to knock down the expression of IDE in mouse liver to explore its effect on the amount of GLP-1. The specific principle is: the shRNA knockdown principle is to use the sense and antisense sequences of the target gene contained in the shRNA molecule to degrade the complementary mRNA through the RNA interference (RNAi) pathway, thereby inhibiting the expression of the target gene. We package the plasmid expressing shRNA into a virus, which can specifically recognize liver cells and transfer the shRNA plasmid into mouse liver cells, thereby achieving the effect of gene knockout. The shRNA sequence is shown in SEQ ID NO:20.

[0331] Example 12: Effect of knocking down the expression of IDE on the expression of IDE in JHH7

[0332] We used shRNA to knock down the expression of IDE in human liver cells to explore its effect on the amount of GLP-1. The specific principle is: the shRNA knockdown principle is to use the sense and antisense sequences of the target gene contained in the shRNA molecule to degrade the complementary mRNA through the RNA interference (RNAi) pathway, thereby inhibiting the expression of the target gene. We packaged the plasmid expressing shRNA into a virus, which can specifically recognize the liver cell JHH7, and transferred the shRNA plasmid into the JHH7 cell, thereby achieving the effect of gene knockout.

[0333] The shRNA used for knockdown of IDE in human cells was: 1) CCTGGTCATTATCTTGGTCAT (SEQ ID NO: 21), 2) CCTGAAGACAAGCGAGAATAT (SEQ ID NO: 22), 3) CTTACTATTCATCCAACTTAA (SEQ ID NO: 23), 4) ATAACTGTGGCATCGAGATAT (SEQ ID NO: 24), 5) TAGTTTCTGATTCACTATTAG (SEQ ID NO: 25)

[0334] Method for constructing JHH7 and NCI-H716 cell lines with knockdown of IDE expression:

[0335] a. First generate two oligonucleotide chains, positive and negative.

[0336] The forward oligonucleotide strand is:

[0337] 1)CCGG-TAGTTTTCTGATTCACTATTAG-CTCGAG-CTAATAGTGAATCAGAAACTA-TTTTTG;

[0338] 2)CCGG-CCTGAAGACAAGCGAGAATAT-CTCGAG-ATATTCTCGCTTGTCTTCAGG-TTTTTG;

[0339] 3)CCGG-CTTACTATTCATCCAACTTAA-CTCGAG-TTAAGTTGGATGAATAGTAAG-TTTTTG;

[0340] 4)CCGG-ATAACTGTGGCATCGAGATAT-CTCGAG-ATATCTCGATGCCACAGTTAT-TTTTTG;

[0341] 5)CCGG-TAGTTTTCTGATTCACTATTAG-CTCGAG-CTAATAGTGAATCAGAAACTA-TTTTTG;

[0342] The reverse oligonucleotide strand is:

[0343] 1)AATTCAAAAA-TAGTTTCTGATTCACTATTAG-CTCGAG-CTAATAGTGAATCAGAAACTA;

[0344] 2)AATTCAAAAA-CCTGAAGACAAGCGAGAATAT-CTCGAG-ATATTCTCGCTTGTCTTCAGG;

[0345] 3)AATTCAAAAA-CTTACTATTCATCCAACTTAA-CTCGAG-TTAAGTTGGATGAATAGTAAG;

[0346] 4)AATTCAAAAA-ATAACTGTGGCATCGAGATAT-CTCGAG-ATATCTCGATGCCACAGTTAT;

[0347] 5)AATTCAAAAA-TAGTTTCTGATTCACTATTAG-CTCGAG-CTAATAGTGAATCAGAAACTA.

[0348] b. Digest the pLKO.1 vector with EcoRI and AgeI, and connect the two nucleotide chains to this vector to construct the IDE knockdown plasmid.

[0349] c. Transfer it into 293T cells, collect the culture medium, obtain lentivirus and use it to infect JHH7 cells for 3 days, then add 8 μg / ml puromycin to select IDE knockdown cell lines.

[0350] The knockdown effects of different shRNAs on IDE expression in JHH7 cells Fig.12 shown.

[0351] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the disclosed scope of this application. Applications of some of the basic features may be made within the scope of the following appended claims.

[0352] Part of this article sequence:

[0353] SEQ ID NO: 1: Insulin degrading enzyme

[0354]

[0355] SEQ ID NO:2:GLP-1

[0356] HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

[0357] SEQ ID NO:3:Aib 8 -D-Ser 18 -Ala 25 -GLP-1(7-37)

[0358] His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-D-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

[0359] SEQ ID NO:4:Aib 8 -D-Ser 18 -GLP-1(7-37)

[0360] His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

[0361] SEQ ID NO:5:Aib 8 -D-Tyr 19 -GLP-1(7-37)

[0362] His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-D-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

[0363] SEQ ID NO:6:Aib 8 -D-Ala 25 -GLP-1(7-37)

[0364] His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-D-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

[0365] SEQ ID NO:7:Aib 8 -D-Lys 26 -GLP-1(7-37)

[0366] His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-D-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly

[0367] SEQ ID NO:8:Semaglutide

[0368] His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(ADO-ADO-γE-C18)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly

[0369] SEQ ID NO:9:D-Ser 18 -Semaglutide

[0370] His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(ADO-ADO-γE-C18)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly

[0371] SEQ ID NO:10:

[0372] VSSYLE

[0373] SEQ ID NO:11:

[0374] QAAKEF

[0375] SEQ ID NO:12:

[0376] DVSPYLEG

[0377] SEQ ID NO:13:

[0378] GQAWKEFI

[0379] SEQ ID NO:14:

[0380] DVSYYLEG

[0381] SEQ ID NO:15:

[0382] DVSRYLEG

[0383] SEQ ID NO:16:

[0384] DVSMYLEG

[0385] SEQ ID NO:17:

[0386] GQASKEFI

[0387] SEQ ID NO:18:

[0388] QAYKEFI

[0389] SEQ ID NO:19:

[0390] GQANKEFI

[0391] SEQ ID NO: 20: Mouse IDE knockdown shRNA

[0392] CGGCTGCATATTGAAGCACTT

[0393] SEQ ID NO:21: Human IDE knockdown shRNA1

[0394] CCTGGTCATTATCTTGGTCAT

[0395] SEQ ID NO: 22: Human IDE knockdown shRNA2

[0396] CCTGAAGACAAGCGAGAATAT

[0397] SEQ ID NO: 23: Human IDE knockdown shRNA3

[0398] CTTACTATTCATCCAACTTAA

[0399] SEQ ID NO: 24: Human IDE knockdown shRNA4

[0400] ATAACTGTGGCATCGAGATAT

[0401] SEQ ID NO: 25: Human IDE knockdown shRNA5

[0402] TAGTTTCTGATTCACTATTAG.

Claims

1. A fragment of GLP-1 or a variant of the fragment, characterized in that The fragment contains at least one or more amino acid residues of Ser18, Tyr19, Ala25, and Lys26, Preferably, the fragment is 4-37 amino acids in length, and / or, Preferably, the fragment is mutated to other amino acids or has modifications at one or more positions among Ser18, Tyr19, Ala25, Lys26; more preferably, the other amino acids include: non-positively charged amino acids, non-natural amino acids or D-type amino acids.

2. A variant of GLP-1 or a derivative thereof, wherein: The variant has a mutation or modification at any one, any two, any three or four positions among Ser18, Tyr19, Ala25 and Lys26, Preferably, the mutation is to a non-negatively charged amino acid, an unnatural amino acid or a D-amino acid, More preferably, The mutation is selected from any one or more of the following groups: (1) Any one of S18G, S18A, S18V, S18L, S18I, S18T, S18C, S18M, S18N, S18Q, S18D, S18E, S18K, S18R, S18H, S18F, S18Y, S18W, and S18P, (2) any one of Y19G, Y19A, Y19V, Y19L, Y19I, Y19T, Y19C, Y19M, Y19N, Y19Q, Y19D, Y19E, Y19K, Y19R, Y19H, Y19F, Y19S, Y19W, and Y19P, (3) Any one of A25G, A25Y, A25V, A25L, A25I, A25T, A25C, A25M, A25N, A25Q, A25D, A25E, A25K, A25R, A25H, A25F, A25S, A25W, and A25P, (4) Any one of K26G, K26Y, K26V, K26L, K26I, K26T, K26C, K26M, K26N, K26Q, K26D, K26E, K26A, K26R, K26H, K26F, K26S, K26W, K26P, or The mutation includes any one or more mutations selected from the following: Ser18 mutation to D-Ser, Tyr19 mutation to D-Tyr, Ala25 mutation to D-Ala, Lys26 mutation to D-Lys, or The variant also has a mutation or modification at Ala8, or The modification is selected from any of the following: non-natural peptide bonds, fatty acylation, acetylation and other acylation modifications, methylation, glycosylation, phosphorylation, ubiquitination, hydroxylation, carbonylation, alkylation, fatty acid, hydroxyl modification, PEGylation, polypropylene glycolation (PPGylation).

3. A polynucleotide, the sequence of which comprises one or more selected from the following: (a) a nucleotide sequence encoding (1) the fragment of GLP-1 or a variant of the fragment according to claim 1, or (2) a variant of GLP-1 or a derivative thereof according to claim 2, (b) a nucleotide sequence having at least 80% (preferably at least 90%) identity with the sequence shown in (a), and (c) The complementary sequence of (a) or (b).

4. A nucleic acid construct comprising a polynucleotide encoding the polynucleotide of claim 3, wherein: The nucleic acid construct is a vector, preferably, the nucleic acid construct is an expression vector or a homologous recombination vector.

5. A host cell, wherein The host cell: (1) containing, expressing or secreting the fragment of GLP-1 or a variant of the fragment according to claim 1 or a variant of GLP-1 or a derivative thereof according to claim 2, (2) containing the polynucleotide according to claim 3, or (3) comprising the nucleic acid construct according to claim 4, Preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

6. A method for improving the stability of GLP-1 or its derivatives, wherein: The method comprises performing a mutation or modification as described in claim 2 at one or more positions among Ser18, Tyr19, Ala25, and Lys26.

7. Use of a reagent in preparing a kit for regulating GLP-1 content or stability, regulating blood sugar in animals, treating metabolic diseases, or treating diseases that benefit from GLP-1 content regulation or stability regulation, wherein: The reagents include: (1) an agent for regulating the expression or activity of IDE, (2) Agents that regulate the interaction between IDE and GLP-1, (3) The variant of GLP-1 or its derivative according to claim 2, Preferably, the use is: (1) Use of an agent for downregulating the expression or activity of IDE and / or an agent for downregulating the interaction between IDE and GLP-1 and / or a variant of GLP-1 or a derivative thereof according to claim 2 in the preparation of a kit for upregulating the content or stability of GLP-1, downregulating blood glucose in animals, or treating diseases that benefit from unchanged or increased GLP-1 content or enhanced GLP-1 stability, (2) Use of an agent that upregulates the expression or activity of IDE and / or an agent that upregulates the interaction between IDE and GLP-1 in the preparation of a kit for downregulating the level or stability of GLP-1, upregulating animal blood glucose, or treating diseases that benefit from reduced GLP-1 levels or reduced GLP-1 stability.

8. The use according to claim 7, characterized in that The agent for downregulating the expression of IDE is an inhibitory molecule that specifically interferes with the transcription and / or expression of the IDE gene, and / or The agent that downregulates the activity of IDE is an IDE inhibitor, and / or The agent for downregulating the activity of IDE is an activity-downregulated IDE protein variant or its coding sequence, and / or The reagent for down-regulating the interaction between IDE and GLP-1 and the reagent for competitively inhibiting IDE by GLP-1 are preferably the reagent according to claim 1, and / or The reagent for upregulating the expression or activity of IDE includes an expression vector capable of expressing IDE.

9. A non-therapeutic method for regulating GLP-1 content or stability, comprising: (1) regulating the expression or activity of IDE, or (2) Regulate the interaction between IDE and GLP-1.

10. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and: (1) the variant of GLP-1 or its derivative according to claim 2 and / or its coding sequence, and / or (2) The GLP-1 fragment or a variant of the fragment and / or its coding sequence according to claim 1.

11. Use of IDE, its coding sequence, a nucleic acid construct comprising the coding sequence, a cell expressing IDE, a cell comprising the coding sequence, or a cell comprising the nucleic acid construct in screening for GLP-1 variants with improved stability, Preferably, the amino acid sequence of IDE is as shown in SEQ ID NO: 1 or has at least 90% sequence identity thereto.

12. A method for screening candidate substances capable of regulating the degradation of GLP-1 by IDE, comprising the steps of: (1) contacting the candidate substance with a system comprising IDE and GLP-1, and (2) Detecting the GLP-1 content and comparing it with the GLP-1 content in the control. If the content of GLP-1 is less than the control, the candidate substance promotes the degradation of GLP-1 by IDE; if the content of GLP-1 is greater than the control, the candidate substance inhibits the degradation of GLP-1 by IDE.

13. A method for screening a GLP-1 variant for altered stability to IDE relative to wild-type GLP-1, the method comprising: obtaining a GLP-1 variant having a mutation or modification at one or more amino acid positions, and testing the GLP-1 variant for changes in stability to IDE relative to wild-type GLP-1, Preferably, the method comprises the steps of: (1) contacting a GLP-1 variant with a system comprising IDE, wherein the GLP-1 variant has a mutation or modification at one or more amino acid positions, and (2) detecting the content of the GLP-1 variant and comparing it with the content of GLP-1 in the control, If the content of the GLP-1 variant is less than the content of GLP-1 in the control, the stability of the GLP-1 variant decreases; if the content of the GLP-1 variant is greater than the content of GLP-1 in the control, the stability of the GLP-1 variant increases. Preferably, the GLP-1 variant has a mutation or modification at one or more of Ser18, Tyr19, Ala25, Lys26.

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