CN114349819B

CN114349819B - Gly-Pro-type peptide with DPP-IV inhibitory activity and application thereof

Info

Publication number: CN114349819B
Application number: CN202111587141.4A
Authority: CN
Inventors: 赵谋明; 徐琼瑶; 郑淋; 赵翊君
Original assignee: Guangdong Huatai Biological Technology Co ltd; South China University of Technology SCUT
Current assignee: Guangdong Huatai Biological Technology Co ltd; South China University of Technology SCUT
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2023-09-29
Anticipated expiration: 2041-12-23
Also published as: CN114349819A

Abstract

The invention discloses a Gly-Pro-type peptide with DPP-IV inhibitory activity and application thereof, wherein the sequence general formula of the Gly-Pro-Xaa1-Gly- [ Xaa2] _r ‑[Xaa3] _s ‑[Gly] _t ‑[Pro] _m ‑[Xaa4] _n The method comprises the steps of carrying out a first treatment on the surface of the Xaa1 is Ala (A), ser (S), arg (R), met (M), ile (I) or Gln (Q); xaa2 is Pro (P) or Ala (A). The Gly-Pro-type peptide has stronger DPP-IV inhibition effect than Gly-Pro-type peptides with other lengths, and the concentration for inhibiting half of DPP-IV enzyme activity is 114.09-445.86 mu M.

Description

Gly-Pro-type peptide with DPP-IV inhibitory activity and application thereof

Technical Field

The invention relates to bioactive peptides aiming at type 2 diabetes, in particular to Gly-Pro-type peptides with DPP-IV inhibitory activity and application thereof.

Background

Diabetes (Diabetes Mellitus) is one of the most rapidly growing diseases worldwide, and its symptoms and complications are the leading cause of death.

Type II diabetes is the most common type of diabetes. Currently, 9 drugs have been developed for controlling blood glucose in patients with type II diabetes, which are insulin and its analogues, sulfonylurea secretagogues, metformin, α -glucosidase inhibitors, thiazolidinedione derivative sensitizers, benzanilide derivative secretagogues, GLP-1 receptor agonists, DPP-IV enzyme inhibitors, and SGLT-2 inhibitors, respectively.

Among them, DPP-IV inhibitors are therapeutic drugs which have been developed and used for over twenty years, and which prolong the half-life of incretins (mainly GLP-1) by inhibiting the activity of DPP-IV enzyme, thereby achieving the purpose of regulating blood sugar by promoting the release of insulin. GLP-1 directly acts on endocrine systems such as pancreas, heart, stomach, brain and the like, has various physiological functions such as reducing gastric emptying, suppressing appetite and the like besides promoting insulin secretion, can assist in controlling blood glucose homeostasis, and has a regulating effect on cardiovascular functions.

Thus, regulation of blood glucose via the DPP-IV inhibition pathway is a pleiotropic strategy, and there is a need to develop more naturally safe DPP-IV inhibitors. Gly-Pro-Xaa-type peptides are reported to have DPP-IV inhibitory activity due to the Pro contained in the second position at the N-terminus and are present in large amounts in collagen sequences, however, it is not known how to elevate the DPP-IV inhibitory activity of Gly-Pro-Xaa-type peptides.

Disclosure of Invention

The invention aims to provide Gly-Pro-type peptide and application thereof in preparing medicines for treating type 2 diabetes.

The aim of the invention is achieved by the following technical scheme:

a Gly-Pro-type peptide consisting of 4-9 amino acid residues has the general sequence formula Gly-Pro-Xaa1-Gly- [ Xaa2] _r -[Xaa3] _s -[Gly] _t -[Pro] _m -[Xaa4] _n ；

Xaa1 is Ala (A), ser (S), arg (R), met (M), ile (I) or Gln (Q);

r is 0 or 1;

when r is 0, the s, t, m, n are all 0; when r is 1, s is 0 or 1;

xaa2 is Pro (P) or Ala (A);

s is 0 or 1;

when s is 0, t, m and n are all 0; when s is 1, t is 0 or 1;

xaa3 is Arg (R), ser (S) or Gln (Q);

t is 0 or 1;

when t is 0, m and n are 0; when t is 1, the m is 0 or 1;

m is 0 or 1;

when m is 0, n is 0; when m is 1, the n is 0 or 1;

xaa4 is Ala (A), ser (S) or Gln (Q);

the amino acid residues in the Gly-Pro-type peptide are L-shaped.

The Gly-Pro-type peptide consisting of 4-9 amino acid residues can be chemically synthesized, and a better synthetic route is selected based on a general chemical synthesis idea, so that a higher recovery rate can be obtained. For example, the synthesis is carried out on a solid phase synthesizer, 1g of dichloro resin is swelled, washed, fmoc protecting groups are removed, amino acid is added for condensation reaction, and the deprotection-condensation process is repeated until all amino acids are connected.

The Gly-Pro-type peptide has higher DPP-IV inhibitory activity, and can be used for preparing medicines for treating type 2 diabetes;

the medicine can also contain more than one pharmaceutically acceptable carrier or auxiliary materials.

The auxiliary materials are preferably sustained release agents, excipients, fillers, adhesives, wetting agents, disintegrating agents, absorption promoters, adsorption carriers, surfactants or lubricants and the like.

The carrier is at least one of microcapsule, microsphere, nanoparticle and liposome.

The medicine can be further prepared into various dosage forms, and the medicine in various dosage forms can be prepared according to a conventional method in the pharmaceutical field.

Compared with the prior art, the invention has the following advantages and effects:

the invention provides a Gly-Pro-type peptide consisting of 4-9 amino acids, which has stronger DPP-IV inhibition effect than Gly-Pro-type peptides with the same type (the same amino acid Xaa1 at the P3 position) and other lengths, and the concentration of inhibiting half of DPP-IV enzyme activity is 114.09-445.86 mu M.

Drawings

FIG. 1 shows DPP-IV inhibition by Gly-Pro-Xaa monomer (final concentration 1 mM).

FIG. 2 shows the concentration (IC) of GPA-series synthetic peptides inhibiting DPP-IV enzyme by 50% of the enzyme activity ₅₀ Values).

FIG. 3 shows the concentration (IC) of GPM-series synthetic peptides inhibiting DPP-IV enzyme by 50% of the enzyme activity ₅₀ Values).

FIG. 4 shows the concentration (IC) of GPR-series synthetic peptides inhibiting DPP-IV enzyme by 50% of the enzyme activity ₅₀ Values).

FIG. 5 shows the concentration (IC) of GPS-series synthetic peptides inhibiting DPP-IV enzyme by 50% of the enzyme activity ₅₀ Values).

FIG. 6 shows the concentration (IC) of GPI-series synthetic peptides inhibiting DPP-IV enzyme by 50% of the enzyme activity ₅₀ Values).

FIG. 7 shows the concentration (IC) of GPQ-series synthetic peptides inhibiting DPP-IV enzyme by 50% of the enzyme activity ₅₀ Values).

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.

Example 1

Synthesis of Gly-Pro-type peptide consisting of 4 amino acid residues by solid phase synthesis

Swelling and washing dichloro resin, removing Fmoc protecting group, adding amino acid for condensation reaction, and repeating the process of removing-protecting-condensing until all amino acids are connected. Cleavage of the resin gave a crude Gly-Pro-type peptide consisting of 4 amino acid residues (specific peptide sequences shown in Table 1) which was purified by reverse phase HPLC to give a purified polypeptide (> 95%).

Example 2

Synthesis of Gly-Pro-type peptide consisting of 5 amino acid residues by solid phase synthesis

The solid phase synthesis and purification method is the same as in example 1, and Gly-Pro-peptide pure product (> 95%) composed of 5 amino acid residues is finally obtained, and specific peptide sequences are shown in Table 1.

Example 3

Synthesis of Gly-Pro-type peptide consisting of 6 amino acid residues by solid phase synthesis

The solid phase synthesis and purification method is the same as in example 1, and Gly-Pro-peptide pure product (> 95%) composed of 6 amino acid residues is finally obtained, and specific peptide sequences are shown in Table 1.

Example 4

Synthesis of Gly-Pro-type peptide consisting of 7 amino acid residues by solid phase synthesis

The solid phase synthesis and purification method is the same as in example 1, and Gly-Pro-peptide pure product (> 95%) composed of 7 amino acid residues is finally obtained, and specific peptide sequences are shown in Table 1.

Example 5

Synthesis of Gly-Pro-type peptide consisting of 8 amino acid residues by solid phase synthesis

The solid phase synthesis and purification method is the same as in example 1, and Gly-Pro-peptide pure product (> 95%) composed of 8 amino acid residues is finally obtained, and specific peptide sequences are shown in Table 1.

Example 6

Synthesis of Gly-Pro-type peptide consisting of 9 amino acid residues by solid phase synthesis

The solid phase synthesis and purification method is the same as in example 1, and a Gly-Pro-peptide pure product (> 95%) composed of 9 amino acid residues is finally obtained, and the specific peptide sequences are shown in Table 1.

TABLE 1 peptides synthesized in examples

Comparative example 1

Synthesis of Gly-Pro-type peptide consisting of 3 amino acid residues by solid phase synthesis

The solid phase synthesis and purification method is the same as in example 1, and Gly-Pro-peptide pure product (> 95%) composed of 3 amino acid residues is finally obtained, and specific peptide sequences are shown in Table 2.

Comparative example 2

Synthesis of Gly-Pro-type peptide consisting of 12 amino acid residues by solid phase synthesis

The solid phase synthesis and purification method is the same as in example 1, and Gly-Pro-peptide pure product (> 95%) composed of 12 amino acid residues is finally obtained, and specific peptide sequences are shown in Table 2.

Comparative example 3

Synthesis of GPAG by solid phase Synthesis

The solid phase synthesis and purification method were the same as in example 1, finally obtaining pure GPAG (> 95%).

Table 2 peptides synthesized in comparative examples

Note that: amino acids are indicated in single letter abbreviations, wherein O represents hydroxyproline

The Gly-Pro-type peptides synthesized in examples 1-6 and comparative examples 1-3 were subjected to the following statistics and evaluation:

determination of DPP-IV inhibition: synthetic peptide powder was formulated into a sample solution of a certain concentration with Tris-HCl buffer (pH 8.0). 80uL of sample solution and 80uL of 0.5mM substrate (Gly-Pro-pNA) are added into a 96-well ELISA plate, the mixture is incubated for 10min at 37 ℃, 40uL of 12.5mU/mL DPP-IV reaction solution is then added, the mixture is precisely incubated for 120min at 37 ℃ after being uniformly mixed, and the absorbance at 405nm is measured every 2 min. The DPP-IV inhibition ratio of the sample to be tested is calculated according to the following formula.

DPP-IV inhibition rate calculation: two time points T1 and T2 are selected, the light absorption value of the two time points is changed in a linear range, and the slope delta A/min is calculated.

Slope= (A2-A1)/(T2-T1);

DPP-IV inhibition (%) = (Slope) _{Control group} -Slope _{Sample group} )*100/Slope _{Control group} 。

As shown in FIG. 1, 10 Gly-Pro-Xaa monomers showed different inhibition effects on DPP-IV enzyme at a final concentration of 1mM, wherein GPI inhibition rate on DPP-IV enzyme was the highest, 54.63+ -1.85%. The inhibition rates of GPP and GPO are the lowest, and little inhibition activity is exhibited. Whereas the inhibition rate of other 7 Gly-Pro-Xaa monomers to DPP-IV enzyme at the concentration of 1mM is between 9.38 and 52.37 percent.

Subsequently, gly-Pro-type tripeptides (GPI, GPM, GPQ, GPR, GPA and GPS) with the top six DPP-IV inhibition rates are selected as precursors, and amino acids are gradually increased at the C terminal of the tripeptides, so that Gly-Pro-type DPP-IV inhibition peptides with stronger activity are developed.

Concentration of synthetic peptide required for inhibiting half activity of DPP-IV enzyme (IC ₅₀ Value) calculation: determining DPP-IV inhibition rate of Gly-Pro-type peptide at different concentrations, and performing regression curve with logarithmic value of polypeptide concentration and inhibition rate to obtain regression equation, thereby calculating IC ₅₀ The value, i.e. the concentration of peptide at which 50% of DPP-IV enzyme activity is inhibited.

The amino acids were gradually increased at the C-terminus of GPI, GPM, GPQ, GPR, GPA and GPS, thereby forming 6 peptide series, GPI-, GPM-, GPQ-, GPR-, GPA-, and GPS-series, respectively, and their IC was measured ₅₀ Values.

The results of the synthetic peptide measurements are shown in FIGS. 2-7 and Table 3.

TABLE 3 IC of Gly-Pro-peptides of the invention against DPP-IV ₅₀ Value of

As shown in FIG. 2, the addition of amino acids at the C-terminus of GPA until the number of amino acids in the sequence is 12, the inhibitory activity of the formed novel peptide DPP-IV tends to increase rapidly and then decrease slowly with increasing peptide length, and the activity of all novel peptides is higher than that of tripeptide GPA, indicating that the appropriate increase of peptide length is one of the methods for improving the inhibitory activity of Gly-Pro-type peptide DPP-IV. GPAG is a DPP-IV inhibitory peptide which has been reported to date and IC thereof ₅₀ The value was 452.54.+ -. 5.66. Mu.M. The data show that other GPA-type peptides consisting of 5 to 9 amino acid residues, except 12 peptide GPAGPRGPSGAA, are higher in activity than GPAG and have IC50 values of 183.26 to 445.86 mu M for inhibiting DPP-IV enzyme.

The results in FIGS. 3-7 show that the DPP-IV inhibitory activity of GPM-, GPR-, GPS-, GPI-and GPQ-series peptides varies with the length of the peptide fragment similarly to the GPA-series. The DPP-IV inhibitory activities of peptides consisting of 4 to 9 amino acid residues in these series are higher than those of their corresponding tripeptides, and also higher than GPAG, IC for DPP-IV ₅₀ The value is 114.09-429.42 mu M.

In summary, the appropriate addition of amino acids at the C-terminus of Gly-Pro-Xaa monomers is an effective method for developing Gly-Pro-type peptides with enhanced DPP-IV inhibitory activity, wherein the increase in sequence length to 4-9 amino acids is desirable.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A Gly-Pro-type peptide is characterized in that the amino acid sequence is

GPQL。

The application of Gly-Pro-type peptide in preparing DPP-IV enzyme inhibitor is characterized in that the amino acid sequence of Gly-Pro-type peptide is at least one of the following:

GPIV、GPQL、GPRG、GPMG、GPSG、GPIG、GPMGP、GPRGP、GPAGA、GPSGP、GPIGPR、GPRGPS、GPMGPR、GPAGPS、GPSGPR、GPRGPSG、GPMGPRG、GPAGPRG、GPSGPRG、GPRGPSGP、GPMGPRGP、GPAGPRGP、GPSGPRGP、GPRGPSGPQ、GPIGPRGPA、GPMGPRGPA、GPAGPRGPS。

the application of Gly-Pro-type peptide in preparing medicine for treating type 2 diabetes, characterized in that the amino acid sequence of Gly-Pro-type peptide is at least one of the following:

4. a use according to claim 3, characterized in that: the medicine contains more than one pharmaceutically acceptable carrier or auxiliary materials.

5. The use according to claim 4, characterized in that: the auxiliary materials are sustained release agent, excipient, filler, adhesive, wetting agent, disintegrating agent, absorption promoter, adsorption carrier, surfactant or lubricant.

6. The use according to claim 4, characterized in that: the carrier is at least one of microcapsule, microsphere, nanoparticle or liposome.