CN117603300A

CN117603300A - Small molecule peptide with dipeptidyl peptidase-4 inhibitory activity and application thereof

Info

Publication number: CN117603300A
Application number: CN202311469970.1A
Authority: CN
Inventors: 欧瑜; 孙仲侃
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2023-11-07
Filing date: 2023-11-07
Publication date: 2024-02-27

Abstract

The invention discloses a dipeptidyl peptidase-4 (DPP-4) inhibiting oligopeptide and application thereof, wherein the inhibiting oligopeptide comprises an amino acid sequence IAVP and a functional variant thereof. The invention uses the computer simulation molecule docking technology to virtually screen the binding capacity of peptide segments and DPP-4, thus obtaining the peptide with DPP-4 inhibiting activity and the functional variant thereof. The in vivo activity shows that IAVP and functional variant IAVPGE, IAVPGEVA thereof can obviously improve the oral glucose tolerance curve of mice, and IAVPGE can produce the DPP-4 inhibitory oligopeptide with the characteristics of small molecular weight, convenient artificial synthesis, definite action mechanism, high safety and the like for the type 2 diabetes model mice, thereby being capable of reducing blood sugar, reducing lipid, promoting insulin secretion, improving insulin resistance, and being used for treating and/or preventing DPP-4 mediated diseases such as diabetes, nonalcoholic fatty liver, atherosclerosis and the like.

Description

Small molecule peptide with dipeptidyl peptidase-4 inhibitory activity and application thereof

Technical Field

The invention relates to the technical field of active small molecular peptides, in particular to a small molecular peptide with dipeptidyl peptidase-4 inhibitory activity and application thereof.

Background

Diabetes is a type of metabolic disease that is widespread worldwide. In recent years, the incidence of diabetes has increased year by year. Wherein type 2 diabetes (T2D) accounts for more than 90%. Unlike the absolute deficiency of insulin in type 1 diabetics, type 2 diabetes is manifested by a relative deficiency of insulin, i.e., peripheral tissues are insensitive to insulin action and produce disturbances in glucose, lipid, protein metabolism, with later stages leading to serious diabetic complications. The oral hypoglycemic agents commonly used in clinic at present mainly comprise: insulin secretion promoters (sulfonylureas), insulin sensitizers (biguanides), and carbohydrate modulators (α -glucosidase inhibitors). However, the efficacy of these drugs gradually decreases with the duration of administration, and is accompanied by a number of side effects such as hypoglycemia, gastrointestinal dysfunction, liver injury, etc.

Dipeptidyl peptidase-4 (DPP-4) is a serine protease that hydrolyzes Xaa-Pro or Xaa-Ala structures at the N-terminus of the peptide chain. DPP-4 is expressed in various parts of the whole body of human body, and the enzyme plays an important role in inflammatory reaction and insulin regulation. Endogenous glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic hormone (GIP) in human body can promote insulin secretion of human body, inhibit glucagon secretion, and play an important role in regulating blood glucose balance of human body. However, both amino acids at the amino terminus of GLP-1 and GIP are rapidly cleaved by DPP-4 to lose their physiological function of regulating blood glucose. DPP-4 inhibitors reduce the inactivation of GLP-1 in vivo by inhibiting DPP-4, resulting in elevated levels of endogenous GLP-1. GLP-1 plays a role in lowering blood sugar by stimulating insulin secretion from islet beta cells and inhibiting glucagon secretion from islet alpha cells. In addition, the DPP-4 inhibitor can also reduce the incidence risk of cardiovascular adverse events, reduce the level of total cholesterol and triglyceride, play a certain therapeutic role in diseases such as atherosclerosis, nonalcoholic fatty liver and the like, and is a powerful tool for researching the functions of DPP-4 in the aspects of immune response, ischemia reperfusion injury, heart failure, cancer and the like.

The DPP-4 inhibitor is used alone without increasing the risk of hypoglycemia and weight, but DPP-4 inhibitors such as sitagliptin, vildagliptin and the like have side effects such as liver and kidney injury, gastrointestinal disturbance and the like, so that the research and development of a diabetes drug which is wide in source, low in price, safe and free from toxic and side effects has a very great prospect.

The active small molecular peptide is a peptide compound which is beneficial to the life activities of organisms or has physiological effects, has high oral absorption and utilization rate, and the amino acid of a metabolite basically has no toxicity, so the active small molecular peptide has high safety and is a functional factor with great development prospect. Therefore, the small molecular peptide which is simple and easy to prepare and has DPP-4 inhibitory activity is found to have great significance in preventing and treating diabetes.

Disclosure of Invention

Object of the Invention

The invention aims to disclose a peptide with DPP-4 inhibition activity, wherein the inhibition oligopeptide comprises an amino acid sequence IAVP and a functional variant thereof, has good DPP-4 inhibition activity and can be used for treating and/or preventing DPP-4 mediated diseases, such as diabetes, nonalcoholic fatty liver disease, atherosclerosis and the like.

The technical scheme is as follows:

a DPP-4 inhibitory oligopeptide, comprising the amino acid sequence IAVP and functional variants thereof, wherein the variants are those produced upon addition of any one of the following sequences to the C-terminus of IAVP: G. GE, GEV or GEVA.

In some embodiments, the variant is a variant resulting from one or more conservative substitutions of the GE, GEV, or GEVA moiety added at the C-terminus of the IAVP.

In some embodiments, the conservative substitution is selected from the group consisting of a substitution between D and E and a substitution between V, L and I.

In some embodiments, the variant is a variant that results from the addition of any one of the following sequences to the C-terminus of IAVP: G. GD, GE, GDV, GEV, GDL, GEL, GDI, GEI, GDVA, GEVA, GDLA, GELA, GDIA or GEIA.

The DPP-4 inhibitory oligopeptide is characterized in that the inhibitory oligopeptide is IAVP, IAVPGE or IAVPGEVA

The DPP-4 inhibition oligopeptide can be applied to medicines for treating or preventing DPP-4 mediated diseases.

The use, DPP-4 mediated diseases are diabetes, diabetic complications, or obesity.

A pharmaceutical composition comprising the inhibitory oligopeptide of claim 1 or 2, and one or more pharmaceutically acceptable excipients.

The pharmaceutical composition is characterized in that the dosage form comprises, but is not limited to, tablets, granules, capsules, injections, pills, oral liquids, inhalants, ointments, suppositories or patches.

The invention also provides application of the oligopeptide in preparing products for relieving diabetes, lipid-lowering products and antioxidant products.

The medicine contains the DPP-4 inhibitory oligopeptide and one or more pharmaceutically acceptable auxiliary materials.

The dosage forms of the medicine include, but are not limited to, tablets, granules, capsules, injections, pills and oral liquids.

The pharmaceutically acceptable auxiliary materials comprise diluents, excipients, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants or stabilizers and the like.

The administration mode of the medicine comprises subcutaneous injection, intramuscular injection, intravenous drip and oral administration.

The experimental method comprises the following steps:

the invention utilizes AutoDock Vina software to carry out molecular docking on potential inhibitory oligopeptide fragments and variants thereof generated by adding and/or substituting partial amino acid sequences and DPP-4 active sites, and predicts physicochemical properties, ADMET and other properties. Then, the oligopeptide IAVP and two representative functional variants IAVPGE and IAVPGEVA thereof are synthesized by a solid phase synthesis method, the in vitro DPP-4 inhibition activity is verified, and the inhibition type is determined. Subsequently, ICR mice administered with DPP-4 inhibitory oligopeptides by intragastric administration were subjected to an oral glucose tolerance test to verify their biological activity in vivo. Finally, a functional variant IAVPGE was used to intervene in high-fat diet-induced type 2 diabetes C57BL/6J mice, and its control effect on diabetes was verified.

The beneficial effects are that:

1) The invention performs directional virtual screening by a computer-aided technology, and screens out inhibitory oligopeptides (IAVP, IAVPGE or IAVPGEVA) with hypoglycemic effect by using methods of molecular docking, online database prediction and the like; the oligopeptide IAVPGE has obvious improvement on the physiological index of a type 2 diabetes mouse, has the high-dose group equivalent to the blood glucose reducing effect of sitagliptin, has better blood lipid reducing effect compared with the effect of sitagliptin, has no side effect of the sitagliptin, and is safer, and the method specifically comprises the following steps of:

the oligopeptide IAVPGE was already in the normal fasting blood glucose range after the end of administration in type 2 diabetic mice. The oligopeptide IAVPGE has similar effect to sitagliptin, and can effectively reduce the content of glycosylated hemoglobin. The oligopeptide IAVPGE and sitagliptin can promote insulin secretion, and can improve insulin resistance better. The oligopeptides IAVPGE and sitagliptin can both increase GLP-1 level, thereby playing a beneficial role in promoting insulin secretion and regulating blood glucose balance; the oligopeptide IAVPGE can reduce the liver index, total cholesterol and triglyceride elevation caused by obesity, and has better lipid-lowering effect compared with sitagliptin. The oligopeptide IAVPGE has no hepatotoxicity after long-term administration, and can effectively reduce hepatic cell damage and prevent hepatic injury. The experiment shows that the oligopeptide IAVPGE has almost no hemolytic activity and high safety. After the oligopeptide IAVPGE is used for intervening the type 2 diabetes mice for 4 weeks, the main organ tissues of the mice do not generate lesions on the original basis, and the in vivo and in vitro experiments prove that the oligopeptide IAVPGE has high safety and almost no toxic or side effect.

2) The invention provides a basis for the existence of corresponding functions of variants of the DPP-4 molecular peptide by exploring the binding mode and interaction type of the peptide and the DPP-4 molecular peptide in the butt joint conformation. Finally, peptide segments with potential inhibitory activity and representative variants thereof are selected, the DPP-4 inhibitory activity is verified through in vitro and in vivo experiments, and the inhibition type is consistent with the binding mode of molecular docking.

3) The determination of the inhibition type shows that IAVP and the functional variant thereof are competitive inhibitors of DPP-4, and can be directly combined with the active site of DPP-4 to competitively inhibit the decomposition of endogenous substrate GLP-1, thereby playing a beneficial regulation role of reducing blood sugar.

4) The functional variant IAVPGE of the oligopeptide is used for intervening in a high-fat diet-induced type 2 diabetes mouse model, and the mouse model proves that the mouse model has the effects of reducing blood sugar and blood fat, promoting insulin secretion, improving insulin resistance and the like, and can be applied to the prevention and treatment of diabetes and diabetic complications.

Drawings

FIG. 1 is a diagram showing the molecular docking of oligopeptides with DPP-4; A-C are the binding position and interaction of IAVP, IAVPGE, IAVPGEVA and DPP-4 active cavity respectively;

FIG. 2 is an oligopeptide purity identification; a-C is a high performance liquid chromatogram of IAVP, IAVPGE, IAVPGEVA respectively;

FIG. 3 is an oligopeptide molecular weight identification; a-C are mass spectrograms of IAVP, IAVPGE, IAVPGEVA respectively;

FIG. 4 shows the inhibitory activity of oligopeptides on DPP-4;

FIG. 5 is the type of inhibition of DPP-4 by oligopeptides; A-C are each a double reciprocal graph of IAVP, IAVPGE, IAVPGEVA inhibiting DPP-4; D-F are graphs fitted with competitive inhibition models for IAVP, IAVPGE, IAVPGEVA inhibition DPP-4, respectively;

FIG. 6 is an oral hypoglycemic activity of oligopeptides; (comparison of control group with IAVP group) ^* p＜0.05， ^** p＜0.01， ^*** p is less than 0.001; compared with IAVPGE group ^# p＜0.05， ^## p＜0.01， ^### p is less than 0.001; compared with IAVPGEVA group ^○ p＜0.05， ^○○ p＜0.01， ^○○○ p＜0.001；n＝5)；

FIG. 7 is a therapeutic effect of oligopeptide IAVPGE on high fat diet induced type 2 diabetic C57BL/6J mice; a is the change in body weight of each group of mice before and after dosing; b is the average intake of weight per hundred grams during the administration period of each group of miceThe food amount; c is the average water intake per hundred grams of body weight during the dosing period for each group of mice; d is the change in fasting blood glucose values before and after dosing of each group of mice; e is the level of glycosylated hemoglobin in the blood of each group of mice; f is the level of insulin in the serum of each group of mice; g is HOMA-IR index of each group of mice; h is the level of GLP-1 in the serum of each group of mice; i is the liver index of each group of mice; J-K is the experimental result of oral glucose tolerance test of each group of mice; L-M is the experimental result of the insulin resistance test of each group of mice; N-O is the level of total cholesterol and triglycerides in the plasma of each group of mice; P-Q is the level of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in the serum of each group of mice; r is islet tissue H of mice in each group&E dyeing results with a scale of 100 μm; (compared to the normal group) ^# p＜0.05， ^## p＜0.01， ^### p is less than 0.001; compared with the model group ^* p＜0.05， ^** p＜0.01， ^*** p＜0.001；n＝8)。

FIG. 8 is the safety profile of the oligopeptide IAVPGE; a is the influence of the oligopeptide IAVPGE with different concentrations on the survival rate of normal liver cells L02; b is the influence of the oligopeptide IAVPGE with different concentrations on the survival rate of islet beta cells Rin-m5 f; c is the hemolytic activity of the oligopeptides IAVPGE with different concentrations and different time; d is the H & E staining result of oligopeptide IAVPGE on main organ tissues of high-fat diet induced type 2 diabetes C57BL/6J mice, and the scale is 100 μm.

Detailed Description

The present invention will be described in detail below with reference to the drawings and examples.

Example 1: virtual screening of oligopeptides

1. Molecular docking screening DPP-4 inhibitory oligopeptides

The amino acid sequence of the library of potential bioactive peptides screened in advance is introduced using autodock tools, subjected to pretreatment such as hydrogenation, and selected as a ligand.

A PDB file (PDB ID:1X 70) of DPP-4 downloaded from RCSB Protein Data Bank (https:// www.rcsb.org /) is imported, supplemented with incomplete amino acid residues, dehydrated, hydrogenated, etc., and selected as a receptor. The catalytic triplet site of DPP-4 is Ser630-Asp708-His740, and the three sites are usedThe DPP-4 binding site is generated by taking amino acid as a center, and the Grid Box size of the active site is set as followsThe number of models generated was set to 10 and the original small molecule ligand was deleted so that the oligopeptide was bound to the site.

The bioactive peptide library was docked with the active site of DPP-4 using AutoDock Vina. The result shows that the oligopeptide with the amino acid sequence of IAVP can be well combined with the active site of DPP-4, and the combining affinity is-8.0 kcal/mol. The variant sequences generated by adding G, GD, GE, GDV, GEV, GDL, GEL, GDI, GEI, GDVA, GEVA, GDLA, GELA, GDIA or GEIA to the C-terminal of the amino acid sequence of IAVP were butted by the same method, and the result shows that all the variant sequences have better binding affinity with the active site of DPP-4 (Table 1). Interactions in the docking results were represented using a Pymol plot.

The results show that these four amino acids of IAVP (fig. 1A) can form hydrophobic interactions with Tyr547, his740 in the hydrophobic pocket of DPP-4 active cavity S1, electrostatic attraction with Asp663, tyr666, etc., and hydrogen bonding interactions with Tyr 662. It can also form a salt bridge with Glu205, glu206 in the S2 pocket, forming a hydrogen bond interaction with Arg 125.

Whereas the variant IAVPGE (fig. 1B) of IAVP can also form an electrostatic attraction with Asp663, tyr666 by forming a hydrophobic interaction of the portion of IAVP with Tyr547, his740, tyr662, tyr666 in the S1 pocket. It can also form a salt bridge with Glu205, glu206 in the S2 pocket, forming a hydrogen bond interaction with Ser 209. Whereas the extended GE moiety may additionally form hydrogen bonding interactions with Arg358, tyr585, forming a salt bridge with Arg356, which favors a more stable conformation in its binding to DPP-4.

Similarly, a variant IAVPGEVA of IAVP (fig. 1C) can form a hydrophobic interaction with Tyr631, tyr666 in the S1 pocket, a hydrogen bond interaction with Tyr547, tyr662, asn710, his740, an electrostatic attraction with Glu205 in the S2 pocket, a hydrogen bond interaction with Arg125, and a hydrophobic interaction with Phe357 through IAVP moieties. Whereas the extended GEVA moiety may form hydrogen bond interactions with Arg356, tyr 585. However, in some docking conformations, the last two amino acids of IAVPGEVA are not themselves able to form interactions with the DPP-4 active cavity that are favorable for binding, and may sterically hinder the formation of a conformation with which Glu in the peptide chain is favorable, e.g., glu361 may form negative-negative charge repulsion with Glu that weakens the stability of the conformation, unfavorable for stable binding between the two.

Taken together, the results of in silico molecular docking show that IAVP and variants thereof bind to the active site of DPP-4, and that the binding conformation of variant IAVPGE, IAVPGEVA reveals that the amino acids in the four positions of IAVP are highly conserved with the binding sites of the S1 and S2 pockets of DPP-4 and with the key amino acid residues that interact, which also provides a basis for the potential DPP-4 inhibitory activity of the variants.

Table 1, binding affinity (kcal/mol) of oligopeptides and variants thereof to DPP-4 docking

Amino acid sequence	Binding affinity (kcal/mol)
		IAVP	-8.0
IAVPG	-7.7
		IAVPGD	-7.6
IAVPGE	-7.2
		IAVPGDV	-8.1
IAVPGEV	-7.5
		IAVPGDL	-7.7
IAVPGEL	-7.6
		IAVPGDI	-8.1
IAVPGEI	-7.9
		IAVPGDVA	-8.1
IAVPGEVA	-7.6
		IAVPGDLA	-7.7
IAVPGELA	-7.1
		IAVPGDIA	-7.4
IAVPGEIA	-7.6

2. Physicochemical Properties of oligopeptides, ADMET Property prediction

pI/Mw engineering using ExpasyHaving predicted peptide molecular weight and isoelectric point (https:// web. Expasy. Org/computer_pi /), predicting peptide net charge using Pepdraw (http:// www2.Tulane. Edu/-biochem/WW/Pepdraw /), predicting peptide water solubility using Peptide property calculator (http:// www.innovagen.com/proteomics-tools), predicting ADMET properties using ADMET lab 2.0 (https:// ADMET. Scbdd. Com /), mainly for human intestinal absorption (Human Intestinal Absorption, HIA) to achieve 20% bioavailability (F) _20％ ) And acute oral toxicity are used as investigation indexes. The results show (Table 2) that IAVP and its variant sequences are almost free of oral toxicity, absorbable by the digestive tract, and have good bioavailability. Among them, the sequences produced by merely conservatively substituting the sequences added at the C-terminus of IAVP have almost no difference in all physicochemical properties and ADMET properties from the original sequences, for example, between IAVPGD and IAVPGE, between IAVPGDV, IAVPGEV, IAVPGDL, IAVPGEL, IAVPGDI and IAVPGEI, etc. Among them, IAVPGE is best in water solubility, IAVPGEVA sequences are long and may have interactions that are detrimental to binding, and they are selected as representative variants of IAVP for subsequent validation.

TABLE 2 physicochemical Properties of oligopeptides and ADMET Properties prediction

Example 2: synthesis, purity and molecular weight characterization of oligopeptides

The IAVP, IAVPGE, IAVPGEVA was synthesized by Shanghai Bioengineering Co., ltd using a solid phase synthesis method. IAVP purity was greater than 99% (fig. 2A, table 3), mass spectrum identified molecular weight 398.25Da (fig. 3A). IAVPGE purity was greater than 98% (fig. 2B, table 4), mass spectrum identified molecular weight 584.20Da (fig. 3B). IAVPGEVA purity was greater than 99% (fig. 2C, table 5) and mass spectrum identified molecular weight 753.90Da (fig. 3C). The purity and molecular weight of the oligopeptide synthesized by the solid phase synthesis method meet the requirements.

TABLE 3 IAVP high Performance liquid chromatography peak Table

Peak number	Retention time	Peak area	Peak height	Peak area%	Peak height%
						1	9.938	14320	1303	0.110	0.275
2	10.514	52305	2838	0.403	0.598
						3	11.608	12926816	470508	99.487	99.127
Totals to		12993441	474650	100.000	100.000

TABLE 4 IAVPGE high Performance liquid chromatography peak Table

Peak number	Retention time	Peak area	Peak height	Peak area%	Peak height%
						1	6.828	36557	4494	0.181	0.337
2	7.418	92322	14422	0.458	1.083
						3	8.684	36681	5382	0.182	0.404
4	8.916	66583	7785	0.330	0.585
						5	9.249	46157	5604	0.229	0.421
6	9.592	55028	9225	0.273	0.693
						7	9.878	19784737	1279594	98.169	96.083
8	11.887	35629	5253	0.177	0.394
						Totals to		20153693	1331760	100.000	100.000

TABLE 5 IAVPGEVA high performance liquid chromatography peak tables

Peak number	Retention time	Peak area	Peak height	Peak area%	Peak height%
						1	9.873	10858	2116	0.041	0.141
2	10.731	31826	4186	0.120	0.279
						3	11.918	40331	5948	0.152	0.396
4	12.408	108490	15466	0.408	1.030
						5	12.642	26414073	1472437	99.226	98.011
6	13.948	14593	2166	0.055	0.144
						Totals to		26620171	1502319	100.000	100.000

Example 3: oligopeptide in-vitro DPP-4 inhibition rate determination

1. Preparation of the reagent:

1) Buffer solution: 50mM Tris-HCl buffer, pH 8.0, containing 1mM EDTA.

2) Substrate solution: the fluorescent substrate Gly-Pro-AMC of DPP-4 is dissolved in DMSO in a dark environment to obtain a 25mM concentrated stock solution, and then the concentrated stock solution is diluted to 500 mu M for later use by a buffer solution.

3) Enzyme solution: after the enzyme activity of DPP-4 was measured, the solution was diluted to 1.5mU/mL with a buffer solution, and another part was heated in a water bath at 90℃for 20min to inactivate the enzyme.

4) Sample solution: the lyophilized powders of IAVP, IAVPGE and IAVPGEVA were dissolved in PBS to give 3000. Mu.M mother solution, which was then diluted with buffer solution to 1000. Mu.M, 300. Mu.M, 100. Mu.M, 10. Mu.M and 1. Mu.M, respectively, for use.

2. The specific implementation steps are as follows: in a dark environment, 50. Mu.L of enzyme solution was added to each well of a black flat bottom 96-well plate on ice, 25. Mu.L of IAVP, IAVPGE and IAVPGEVA solutions at concentrations of 1. Mu.M, 10. Mu.M, 100. Mu.M, 300. Mu.M, 1000. Mu.M and 3000. Mu.M, respectively, were each provided with 3 multiplex wells, and finally 50. Mu.L of substrate solution at a concentration of 500. Mu.M was added (buffer solution was used for blank control wells instead of sample solution; inactivated enzyme solution was used for negative control wells) and after 30min of reaction at 37℃the corresponding light intensities RFU (lambda.) were measured for each well using a fluorescent microplate reader _ex ＝380nm，λ _em =460 nm). According toThe DPP-4 inhibition of the peptide was calculated by the following formula:

inhibition ratio = (F _{Blank control} -F _{Sample of} )/(F _{Blank control} -F _{Negative control} )×100％

The inhibition curve of the oligopeptide against DPP-4 is shown in FIG. 4. Fitting the data using a dose-effect-inhibition model in GraphPad Prism 9 nonlinear regression, specifically selecting logarithmic concentration of inhibitors, normalized response and variable slope options, IC ₅₀ The test results are shown in Table 6. The results show that IAVP and its functional variant IAVPGE, IAVPGEVA have good DPP-4 inhibitory activity, and IC is obtained at a final concentration of 200. Mu.M substrate Gly-Pro-AMC ₅₀ 47.90, 54.18 and 150.7 μm respectively. The DPP-4 of IAVP has the strongest inhibitory activity.

TABLE 6 oligopeptide IC ₅₀ Detection result

Example 4: DPP-4 inhibition type assay for oligopeptides

1. Preparation of the reagent:

1) Buffer solution: 50mM Tris-HCl buffer, pH 8.0, containing 1mM EDTA.

2) Substrate solution: the fluorescent substrate Gly-Pro-AMC of DPP-4 is dissolved in DMSO in a dark environment to obtain 25mM concentrated stock solution, and then diluted with buffer solution to 1000. Mu.M, 500. Mu.M, 250. Mu.M, 125. Mu.M, 50. Mu.M and 25. Mu.M respectively for later use.

3) Enzyme solution: after the enzyme activity of DPP-4 was measured, it was diluted to 1.5mU/mL with a buffer solution.

4) Sample solution: the lyophilized powders of IAVP, IAVPGE and IAVPGEVA were dissolved in PBS to give 3000. Mu.M mother liquor, which was then diluted with buffer solution to 300. Mu.M and 100. Mu.M, respectively, for use.

5) Standard solution: AMC was dissolved in DMSO in a dark environment to give 25mM stock solutions, which were then diluted with buffer solution to 320. Mu.M, 160. Mu.M, 80. Mu.M, 40. Mu.M and 20. Mu.M, respectively, for use.

2. The specific implementation steps are as follows: in the light-resistant environment, the light-resistant material is in a light-resistant environment,to each well of a black flat bottom 96-well plate, 50. Mu.L of enzyme solution was added on ice, followed by 25. Mu.L of one sample solution at a concentration of 100. Mu.M or 300. Mu.M, respectively. Finally, 50. Mu.L of substrate solutions at 25. Mu.M, 50. Mu.M, 125. Mu.M, 250. Mu.M, 500. Mu.M and 1000. Mu.M were added to each concentration of the sample solution, respectively, and 3 wells were set for each substrate concentration. The above-described loading process was repeated for different types of sample solutions prepared. The blank group replaced the sample solution with the buffer solution. 125 μl of standard solutions of different concentrations prepared in advance are added to the wells in the same 96-well plate. Immediately after the completion of the sample addition, the reaction was started by placing the sample in a fluorescence microplate reader preheated to 37℃in advance, and the relative fluorescence intensity RFU (. Lamda.) of each well was immediately measured _ex ＝380nm，λ _em =460 nm) and is measured every 5min for the following 30 min.

Standard curves were obtained using linear fitting of standards of different concentrations and their RFU, and RFU of the remaining wells was converted to the corresponding concentration c (μm) according to regression equations. The reaction time is taken as an abscissa, the concentration of the product AMC is taken as an ordinate, and the GraphPad Prism 9 is respectively used for linear fitting to obtain the equation of the product concentration c of different substrate concentrations relative to the reaction time t under a certain concentration of different samples (the final concentration of the samples is 20 mu M or 60 mu M respectively). Wherein the slope of the equation is the initial reaction velocity V ₀ (μM/min)。

For initial substrate concentration at different sample concentrations [ S]And the corresponding initial reaction velocity V ₀ Taking the reciprocal and taking 1/[ S ]]On the abscissa, 1/V ₀ And (3) a regression curve is made for the ordinate, a regression equation is fitted, and the inhibition types of IAVP, IAVPGE and IAVPGEVA are judged according to the double-reciprocal mapping method. Then the initial substrate concentration is carried out by different models in the inhibitor enzyme reaction kinetic equation of GraphPad Prism 9 nonlinear regression [ S ]]And an initial reaction rate V ₀ Fitting is performed to verify the judgment.

Inhibitors can be broadly divided into inhibition types: 1. competitive inhibitors: inhibitors compete with the substrate of the enzyme for binding to the active site of the enzyme, thereby affecting the binding of the enzyme to the substrate; 2: non-competitive inhibitors: the enzyme can be combined with the substrate and the inhibitor simultaneously to cause the conformational change of the enzyme molecule,and results in a decrease in enzyme activity; 3: anti-competitive inhibitors: inhibitors bind only to the intermediate complex of enzyme and substrate and inhibit enzyme activity. According to the double reciprocal plots of IAVP, IAVPGE and IAVPGEVA inhibiting DPP-4 (FIGS. 5A-C), several regression curves approximately intersect at the same point on the ordinate axis, i.e. V, in the absence and presence of different concentrations of inhibitor _max Approximately the same, and K _m As the concentration of inhibitor increases, competitive inhibition is graphically judged. After fitting the data for IAVP, IAVPGE and IAVPGEVA with a competitive inhibition model (FIG. 5D-F), R ² And the values are also more than 0.99, which shows that the fitting performance is good and the conclusion is reliable. Double reciprocal plots of IAVP, IAVPGE and IAVPGEVA on DPP-4 inhibition and R of different fitted curves in competitive inhibition models ² The best fit values for Km and Ki are shown in table 7. Ideally, ki=ic 50/(1+ [ S)]Km), the IC50 of the inhibitor when the substrate concentration is infinitely small, which is a property of the inhibitor itself that does not change with changes in substrate concentration.

The results show that the Ki values of IAVP and IAVPGE are similar and smaller than IAVPGEVA. IAVP and its functional variant IAVPGE, IAVPGEVA are competitive inhibitors of DPP-4 by binding directly to the active site of DPP-4 and thus competitively inhibiting the breakdown of endogenous substrates, since the sequence of IA at the beginning complies with the rules for DPP-4 to hydrolyze both amino acids Xaa-Ala at the N-terminus of the peptide chain. This is also consistent with the result of selecting the active site of DPP-4 as the docking site in molecular docking.

TABLE 7 fitting of DPP-4 inhibited oligopeptide inhibition types

Example 5: oral hypoglycemic experiments of oligopeptides

The experiments were performed using 8 week old male ICR mice, 5 mice per group. The oligopeptides used in the experiments were all dissolved in physiological saline. After all mice were fasted without water for 8 hours, fasting blood glucose of each mouse was measured with a blood glucose meter, and the body weight of the mice was weighed. Subsequently, each group of mice was administered with IAVP, IAVPGE or IAVPGEVA solution by gavage at 8.5mg/kg, and the control group of mice was gavaged with an equal dose of physiological saline. After 1h, each mouse was again perfused with 2g/kg of a gastric glucose solution and blood glucose levels were measured at subsequent 30, 60, 90 and 120 minutes. The results show (fig. 6) that after each oligopeptide was administered by gastric lavage, blood glucose was significantly reduced at 30min compared to the control group, and the glucose tolerance curves of the oligopeptide group were more gentle compared to the control group, demonstrating that IAVP and its functional variant IAVPGE, IAVPGEVA both improved oral glucose tolerance in mice.

IAVPGE was selected for subsequent experiments because of its similar inhibition and oral hypoglycemic activity, and better water solubility.

Example 6: therapeutic effect of oligopeptide IAVPGE on high-fat diet induced type 2 diabetes C57BL/6J mice

Clean male C57BL/6J mice (SCXK 2022-0009, provided by the university of Yangzhou comparative medical center for laboratory animal production license number), 6 weeks old, 18-22g weight, and 8 animals selected randomly as normal control groups after 3 days of adaptive feeding, and were always fed with common feed. The rest is fed with high-fat feed (product number: H10060, license number: SCXK 2019-0008, kyowa biological technology Co., ltd.) with fat energy ratio of 60%, after 12 weeks of feeding with high-fat feed, fasted mice are fasted for 8 hours, body weight is weighed, blood is taken from the tail tip, fasting blood glucose of the mice is measured by blood glucose test strips, and average fasting blood glucose value of more than 7.0mmol/L is regarded as successful molding. Mice were randomly and equally divided into model groups, IAVPGE low dose group (3 mg/kg), IAVPGE medium dose group (9 mg/kg), IAVPGE high dose group (15 mg/kg) and positive drug group (sitagliptin, 15 mg/kg) according to fasting blood glucose and body weight. The IAVPGE and sitagliptin solution was prepared using physiological saline, and the treated mice were perfused with corresponding doses of IAVPGE or sitagliptin solution daily for 4 consecutive weeks in groups, with normal control and model groups perfused with equal doses of physiological saline.

Experimental animals were kept in the university of chinese pharmacopoeia experimental animal center, license number: SYXK 2021-0011, raising conditions are 24+ -2deg.C, 50% -60% humidity, 12 hours of light and night circulation. The experimental dosing period was 28 days, the mice in the experiment were free to eat and drink, the body weight, food intake and water intake of the mice were measured 2 times a week from the start of dosing, and fasting blood glucose was measured 1 time a half month after 8 hours of fasting of the mice. At the end of the experiment, oral glucose tolerance and insulin tolerance assays were performed. Wherein, oral glucose tolerance test, after all mice are fasted without water for 8 hours, their fasting blood glucose is measured, and blood glucose values are measured at the following 30, 60, 90 and 120 minutes according to 2g/kg of a gastric lavage glucose solution. Insulin resistance test fasting blood glucose was measured after all mice had fasted for 4 hours without water deprivation, insulin solution was injected intraperitoneally at 0.5U/kg, and blood glucose values were measured at the following 30, 60, and 120 minutes. After all experiments are finished, the mice are fasted without water inhibition for 8 hours, the mice are killed by removing cervical vertebrae after blood taking, blood is collected, part of the blood is collected in a common centrifuge tube to separate serum, the other part of the blood is collected in an anticoagulation tube containing EDTA-2K to separate plasma and blood cells, and the blood cells are lysed by double distilled water to prepare blood-dissolving liquid. Determining insulin, GLP-1, AST and ALT in mouse serum, total cholesterol and triglycerides in plasma, and glycosylated hemoglobin levels in blood using a kit; separating pancreatic tissues, simultaneously taking the tail parts of pancreas with rich islet content, placing the tail parts of pancreas into tissue fixing liquid for fixing for 48 hours, and carrying out paraffin embedding slicing and H & E staining; liver was separated, and the surface water was sucked by a piece of absorbent paper and weighed.

The results show that: the model mice showed a significant increase in body weight compared to the normal group before administration (fig. 7A), combined with a fasting blood glucose value of greater than 7.0mmol/L (fig. 7D), and it was confirmed that model was successful in early stage type 2 diabetes. After oligopeptide IAVPGE dry prognosis, mice all exhibited significant reductions in body weight, average food intake, average water intake, and fasting blood glucose values compared to the model group (fig. 7A-D), and the reductions in average water intake and fasting blood glucose values exhibited dose-dependency. Sitagliptin is a DPP-4 inhibitor hypoglycemic agent which is marketed, and is used for clinical first-line administration of type 2 diabetes. The weight, average food intake and fasting blood glucose values of the sitagliptin mice also exhibited a significant decrease compared to the model group, but there was no significant difference in average water intake. The results show that the oligopeptide IAVPGE has obvious improvement on the physiological index of the type 2 diabetes mice, and the improvement on the empty stomach blood sugar value of the high-dose group is equivalent to that of the sitagliptin group, and the empty stomach blood sugar value of the high-dose group is in the normal empty stomach blood sugar range after the administration is finished.

Glycosylated hemoglobin level (DCCT-HbAlc) effectively reflects the average blood glucose level over the past 8-12 weeks. Based on the results of the mice glycosylated hemoglobin (fig. 7E), the mice of the model group had significantly increased glycosylated hemoglobin levels, which indicated that the mice of the model group had significantly increased blood glucose. The low, medium and high doses of oligopeptide and the positive drug sitagliptin have different degrees of reducing effects on glycosylated hemoglobin of diabetic mice. The results show that the oligopeptide IAVPGE has similar effects to the sitagliptin, and can effectively reduce the content of glycosylated hemoglobin.

Serum insulin results showed (fig. 7F) that the serum insulin content of the model mice was not significantly reduced, as it was in the compensatory high expression phase of insulin resistance. The elevated serum insulin levels in mice exhibited a dose-dependent response following oligopeptide stem prognosis, and the high dose group had a significant difference compared to the model group, comparable to the sitagliptin group. Calculating insulin resistance index (HOMA-IR, HOMA-ir=fasting blood glucose (mmol/L) ×fasting insulin (mu U/mL)/22.5) from the fasting blood glucose level and insulin level of the mice, the results show (fig. 7G) that the HOMA-IR index of the mice in the model group is significantly elevated compared to the normal group, indicating that the mice in the model group have developed severe insulin resistance; the mice with the oligopeptide stem prognosis have significantly reduced HOMA-IR index, and are dose-dependent, and the medium and high dose groups are equivalent to the sitagliptin group. The results show that the oligopeptides IAVPGE and sitagliptin can promote insulin secretion and better improve insulin resistance.

Inhibition of DPP-4 is directly related to GLP-1 levels, and serum GLP-1 results show (FIG. 7H) that GLP-1 levels are significantly impaired in the model group compared to the normal group; serum GLP-1 levels exhibited a dose-dependent restoration after oligopeptide stem prognosis, with the high dose group levels comparable to the sitagliptin group, all returning to normal levels. The results show that the oligopeptides IAVPGE and sitagliptin can both improve GLP-1 level, thereby playing a beneficial role in promoting insulin secretion and regulating blood glucose balance.

Obesity also causes an increase in liver weight in the high-fat diet-induced type 2 diabetes model, which in turn leads to the formation of fatty liver. Liver index is the percentage of liver weight to body weight, and the results show (fig. 7I) that the model group significantly increased liver index compared to the normal group, whereas the middle and high dose groups of oligopeptides significantly decreased liver index compared to the model group, and that there was no significant difference in the sitagliptin group. Likewise, obesity can also lead to elevated total cholesterol and triglycerides in mice. Based on the mouse plasma total cholesterol (fig. 7N) and triglyceride results (fig. 7O), the plasma total cholesterol and triglyceride of the mice of the model group were significantly elevated; the plasma total cholesterol and triglyceride of the mice in the low, medium and high dose groups are obviously reduced after oligopeptide dry prognosis. The plasma triglycerides were significantly reduced in the sitagliptin group mice, and total cholesterol was also reduced, but with no statistical differences. The results show that the oligopeptide IAVPGE can reduce the liver index, total cholesterol and triglyceride rise caused by obesity, and has better lipid-lowering effect compared with sitagliptin.

The results of the oral glucose tolerance test in mice showed that the mice were fed with 2g/kg glucose (0.4 g/mL) after 8 hours of fasting, the blood glucose fluctuation amplitude was small in normal group mice and recovered to the initial level after 2 hours, while the diabetic mice in model group had a marked impaired glucose tolerance, and blood glucose was sharply increased. The impaired glucose tolerance of the mice in the low, medium and high dose groups and the positive drug group of the oligopeptide is significantly improved, and the area under the curve (figure 7K) is significantly reduced compared with the model group. The results of the mice intraperitoneal insulin resistance test showed (FIG. 7L), that after 4 hours of fasting, i.e., 30 minutes of intraperitoneal insulin injection into mice at 0.5U/kg, the blood glucose reduction rate was greater in normal mice than in model mice and recovered within two hours, while model mice recovered more slowly, indicating impaired insulin resistance in model mice, reduced sensitivity to insulin action, and poor control of blood glucose homeostasis; the high dose group of oligopeptide and the sitagliptin group showed an improvement in the condition of mice compared with the model group, the blood glucose reduction rate in the initial time was higher, and the area under the curve of the administration group showed a significant reduction compared with the model group (fig. 7M). The results show that the mice of the model group have serious impaired glucose tolerance, have disorder on the regulation of insulin, have poor regulation capability on blood sugar, can not regulate the blood sugar steady state in vivo, and have good improvement effect on the regulation of the glucose tolerance and the blood sugar steady state of the mice.

Glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) are very high in liver, and they escape into blood after liver cells are damaged, resulting in a significant increase in serum content. Based on the mouse serum AST and ALT results (fig. 7P-Q), AST and ALT levels were significantly increased in the serum of the model group compared to the normal group. The oligopeptide dry prognosis, the decrease of AST and ALT in serum of mice in low, medium and high dose groups is dose dependent, and the medium and high dose groups have significant differences compared with the model group. The sitagliptin group was not significantly different from the model group. The results show that the oligopeptide IAVPGE has no hepatotoxicity after long-term administration, can effectively reduce the damage of liver cells and prevent liver injury.

According to the results of the rat islet HE staining (fig. 7R), the islet structure of normal group of rats was approximately circular, well defined and normal in size. The model group mice show compensatory increase of islets, blurring of boundaries, damage of islets, abnormal increase of islet alpha cells at the edges, and reduction and rarefaction of islet beta cells in the interior. The boundaries of the islets of the mice in the oligopeptide low-dose group are clear, and the morphology is improved; the islets of mice in the medium-high dose group and the sitagliptin group are better improved in size and shape, the boundaries are clear, the shape is approximate to a circle, and the density of islet beta cells in the mice is increased. The above results demonstrate that the oligopeptides IAVPGE and sitagliptin have repair and protection effects on damaged islets.

In conclusion, the oligopeptide IAVPGE has a therapeutic effect on high-fat diet induced type 2 diabetes C57BL/6J mice, a high-dose group and an equal-dose positive drug sitagliptin group have similar therapeutic effects on weight, fasting blood glucose, glycosylated hemoglobin, insulin and GLP-1 level, oral glucose tolerance test and the like, and the oligopeptide IAVPGE has a therapeutic effect superior to that of sitagliptin in liver index, total cholesterol, AST and ALT, and shows that the oligopeptide IAVPGE has advantages in lipid reduction and liver injury prevention.

Example 7: safety of oligopeptides IAVPGE

1. Cytotoxicity assays for oligopeptides IAVPGE

L02 and Rin-m5f in logarithmic growth phase were each set at 5×l0 ³ Individual cells/wells were plated in 96-well plates at 37 ℃ in complete medium, 5% co ₂ Culturing for 24h under the condition, sucking and removing the complete culture medium, adding serum-free 1640 culture medium containing oligopeptides IAVPGE (0, 10, 25, 50, 100, 250 and 500 mu M) with different concentrations to act on L02 and Rin-M5f cells respectively, sucking and removing the culture medium in a 96-well plate after culturing for 24h, adding 100 mu L of serum-free 1640 culture medium into each well, adding 10 mu L of 5mg/mL MTT into each well, and standing and culturing the treated 96-well plate in a cell culture box for 4h. The supernatant was aspirated, 150. Mu.L of DMSO was added to each well, the mixture was placed on a plate shaker and shaken for 10min, absorbance was measured at 490nm in an ELISA reader, and cell viability was calculated according to the following formula:

cell viability (%) = [ (OD) _{Experimental hole} -OD _{Blank hole} )/(OD _{Negative control well} -OD _{Blank hole} )]×100％

The results show that when the concentration of the oligopeptide IAVPGE is less than or equal to 500 mu M, the method has no obvious influence on the survival rate of L02 cells (figure 8A) and Rin-M5f cells (figure 8B), and the extremely high concentration has no obvious toxicity to cells and high safety.

2. Hemolytic activity detection of oligopeptide IAVPGE

Blood of a healthy c57BL/6J mouse is collected in an anticoagulant tube by an eyeball picking and blood taking method, and then transferred into a triangular flask containing glass beads to be gently shaken for 10min, so that the blood becomes defibrinated blood. Then adding proper volume of physiological saline, gently mixing, centrifuging at 4deg.C for 15min, removing supernatant, and washing the precipitated red blood cells with physiological saline until the supernatant does not appear red. The obtained erythrocytes were resuspended in physiological saline and prepared into a 2% (v/v) erythrocyte suspension tube for further use. After centrifugation of the erythrocyte suspension to remove the supernatant, oligopeptides IAVPGE (1, 10, 100. Mu.M) of different concentrations prepared using physiological saline were added to the packed erythrocytes while using physiological saline and 0.1% (v/v) Triton X-100 as a negative control group and a positive control group, respectively, and after mixing, they were incubated in an incubator at 37℃to observe and record the hemolysis of each tube. The haemolysis at 0.25, 0.5, 1, 2, 4 and 8 hours was observed, the supernatant was centrifuged, the absorbance of each sample at 540nm was read using an enzyme-labeled instrument to determine the amount of haemoglobin released by red blood cell disruption or lysis, and the haemolytic activity of the oligopeptide IAVPGE was calculated.

The results showed (FIG. 8C) that when the concentration of oligopeptide IAVPGE was 1. Mu.M, there was almost no hemolytic activity after the action for a different period of time as compared with the negative control group; at a concentration of 10 μm, there was almost no hemolytic activity in the 0-4h relative to the negative control group, and a slight increase of 2% occurred at 8h, still within the safe range; at a very high concentration of 100. Mu.M, there was almost no hemolytic activity in 0-2h compared to the negative control, and slight increases of 1% and 2% occurred at 4h and 8h, respectively, while still being within safe range. The experiment shows that the oligopeptide IAVPGE has almost no hemolytic activity and high safety.

3. Major organ tissue safety of oligopeptides IAVPGE on high fat diet induced type 2 diabetes C57BL/6J mice

After 6 weeks of C57BL/6J mice are fed with high-fat feed with the fat energy ratio of 60%, the fasting blood glucose value is measured, and the average fasting blood glucose value is more than 7.0mmol/L, so that the model of type 2 diabetes is considered to be successful; the type 2 diabetes model mice are randomly divided into a control group and an oligopeptide IAVPGE group, and are continuously fed with high-fat feed, wherein the IAVPGE group is filled with 15mg/kg IAVPGE solution every day for 4 weeks, and the control group is filled with normal saline with the same dosage. After the end of the administration, the mice were sacrificed by cervical vertebrae removal, and the heart, liver, spleen, lung and kidney of the mice were isolated and fixed in tissue fixative for 48H, paraffin embedded sections and H & E staining were performed.

The results of H & E staining showed no apparent lesions in heart, spleen, lung and kidney tissues of the control and IAVPGE groups (fig. 8D). Because of the high-fat feed feeding, fat accumulation phenomenon and partial hepatocyte dissolution necrosis appear in liver tissues of two groups, inflammatory cell infiltration also appears in a control group, and the IAVPGE group is improved, so that a certain anti-inflammatory treatment effect is shown. The result shows that after the oligopeptide IAVPGE is used for intervening the type 2 diabetes mouse for 4 weeks, the main organ tissues of the mouse do not generate lesions on the original basis, and the in vivo safety is high.

In summary, in vivo and in vitro experiments prove that the oligopeptide IAVPGE has high safety and almost no toxic or side effect.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A DPP-4 inhibitory oligopeptide, comprising the amino acid sequence IAVP and functional variants thereof, wherein the functional variants are variants of IAVP resulting from the C-terminal addition of any one of the following sequences: G. GD, GE, GDV, GEV, GDL, GEL, GDI, GEI, GDVA, GEVA, GDLA, GELA, GDIA or GEIA.

2. The DPP-4 inhibitory oligopeptide according to claim 1, wherein the inhibitory oligopeptide is IAVP, IAVPGE or IAVPGEVA.

3. The use of a DPP-4 inhibiting oligopeptide according to claim 1 or 2 for the treatment or prevention of DPP-4 mediated diseases.

4. The use according to claim 4, wherein the DPP-4 mediated condition is diabetes, diabetic complications, obesity, atherosclerosis or nonalcoholic fatty liver disease.

5. A pharmaceutical composition comprising the inhibitory oligopeptide of claim 1 or 2 and a pharmaceutically acceptable adjuvant.

6. The pharmaceutical composition according to claim 6, wherein the dosage form comprises a tablet, granule, capsule, injection, pill, oral liquid, inhalant, ointment, suppository or patch.