CN114349819A

CN114349819A - Gly-Pro-type peptide with DPP-IV inhibitory activity and application thereof

Info

Publication number: CN114349819A
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: 2022-04-15
Anticipated expiration: 2041-12-23
Also published as: CN114349819B

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-type peptide is Gly-Pro-Xaa1-Gly- [ Xaa2]_r‑[Xaa3]_s‑[Gly]_t‑[Pro]_m‑[Xaa4]_n(ii) a 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 a bioactive peptide aiming at type 2 diabetes, in particular to a Gly-Pro-type peptide with DPP-IV inhibitory activity and application thereof.

Background

Diabetes Mellitus (Diabetes mellitis) is one of the fastest growing diseases worldwide, and its symptoms and complications are the leading causes of death.

Type two diabetes is the most common type of diabetes. At present, 9 drugs aiming at different therapeutic targets have been developed aiming at the control of blood sugar of the patients with type II diabetes, and the drugs are insulin and analogues thereof, sulfonylurea secretagogues, metformin, alpha-glucosidase inhibitors, thiazolidinedione derivative sensitizers, anthranilic acid derivative secretagogues, GLP-1 receptor agonists, DPP-IV enzyme inhibitors and SGLT-2 inhibitors.

The DPP-IV inhibitor is a therapeutic drug which is developed and mature in the last two decades and achieves the aim of regulating blood sugar by inhibiting the activity of DPP-IV enzyme and prolonging the half-life of secretin (mainly GLP-1) so as to promote the release of insulin. GLP-1 directly acts on endocrine systems such as pancreas, heart, stomach, brain, etc., has effects of promoting insulin secretion, reducing gastric emptying, suppressing appetite, etc., can assist in controlling blood sugar homeostasis, and has regulating cardiovascular function.

Therefore, regulating blood glucose by the DPP-IV inhibitory pathway is a pleiotropic strategy, and there is a need to develop more natural and safe DPP-IV inhibitors. The Gly-Pro-Xaa-type peptide has been reported to have DPP-IV inhibitory activity because it contains Pro at the second position on the N-terminal, and is present in a large amount in a collagen sequence, but how to enhance the DPP-IV inhibitory activity of the Gly-Pro-Xaa-type peptide is not known.

Disclosure of Invention

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

The purpose of the invention is realized by the following technical scheme:

a Gly-Pro-type peptide consisting of 4-9 amino acid residues, the general formula of the sequence is 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, s, t, m and 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, the 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, both m and n are 0; when t is 1, m is 0 or 1;

m is 0 or 1;

when m is 0, n is 0; when m is 1, 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 all in L configuration.

The Gly-Pro-type peptide consisting of 4 to 9 amino acid residues can be prepared by a chemical synthesis method, and a better synthesis route is selected based on a general chemical synthesis thought, so that higher recovery rate can be obtained. For example, the synthesis is carried out on a solid phase synthesizer, 1g of dichloro resin is swelled and 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 medicaments for treating type 2 diabetes;

the medicament can also contain more than one pharmaceutically acceptable carrier or auxiliary material.

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

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

The medicine can be further prepared into various dosage forms, and the medicines of various dosage forms can be prepared according to the 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 of the same type (the amino acid Xaa1 at the P3 position is the same) with other lengths, and the concentration for inhibiting the activity of half DPP-IV enzyme is 114.09-445.86 mu M.

Drawings

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

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

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

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

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

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

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

Detailed Description

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

Example 1

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

And swelling and washing the dichloro resin, removing the Fmoc protecting group, adding amino acid for condensation reaction, and repeating the processes of removing, protecting and condensing until all the amino acid is connected. The resin was cleaved to give a crude Gly-Pro-type peptide consisting of 4 amino acid residues (the specific peptide sequence is shown in Table 1), which was purified by reversed-phase high performance liquid chromatography to give a pure polypeptide (> 95%).

Example 2

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

Solid phase synthesis and purification method as in example 1, finally obtained pure product (> 95%) of Gly-Pro-type peptide consisting of 5 amino acid residues, and the specific peptide sequence is shown in table 1.

Example 3

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

Solid phase synthesis and purification method as in example 1, finally yielded pure (95%) Gly-Pro-type peptide consisting of 6 amino acid residues, the specific peptide sequence is shown in table 1.

Example 4

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

Solid phase synthesis and purification method as in example 1, finally yielded a pure (95%) Gly-Pro-type peptide consisting of 7 amino acid residues, the specific peptide sequence is shown in table 1.

Example 5

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

Solid phase synthesis and purification method as in example 1, finally yielded pure (95%) Gly-Pro-type peptide consisting of 8 amino acid residues, the specific peptide sequence is shown in table 1.

Example 6

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

Solid phase synthesis and purification method as in example 1, finally yielded pure (95%) Gly-Pro-type peptide consisting of 9 amino acid residues, the specific peptide sequence is shown in table 1.

Table 1 peptides synthesized in the examples

Comparative example 1

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

Solid phase synthesis and purification method as in example 1, finally yielded pure (95%) Gly-Pro-type peptide consisting of 3 amino acid residues, the specific peptide sequence is shown in table 2.

Comparative example 2

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

Solid phase synthesis and purification method as in example 1, finally obtained pure product (> 95%) of Gly-Pro-type peptide consisting of 12 amino acid residues, and the specific peptide sequence is shown in table 2.

Comparative example 3

Solid phase synthesis method for synthesizing GPAG

Solid phase synthesis and purification were performed as in example 1 to obtain pure GPAG (> 95%).

TABLE 2 peptides synthesized in comparative examples

Note: amino acids are indicated in the form of their single-letter abbreviations, in which 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 evaluations:

determination of DPP-IV inhibition: the synthetic peptide powder was prepared into a sample solution of a certain concentration using Tris-HCl buffer (pH 8.0). Adding 80uL of sample solution and 80uL of 0.5mM substrate (Gly-Pro-pNA) into a 96-well enzyme label plate, mixing, incubating at 37 ℃ for 10min, adding 40uL of 12.5mU/mL DPP-IV reaction solution, mixing uniformly, incubating accurately at 37 ℃ for 120min, and measuring the light absorption value at 405nm every 2 min. The DPP-IV inhibition rate of the sample to be detected is calculated according to the following formula.

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

Slope ═ (a2-a 1)/(T2-T1);

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

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

And then Gly-Pro-type tripeptides (GPI, GPM, GPQ, GPR, GPA and GPS) with the highest DPP-IV inhibition rate of six are selected as precursors, and amino acids are gradually added at the C terminal of the tripeptides, so that the Gly-Pro-type DPP-IV inhibitory peptide with stronger activity is developed.

Concentration of synthetic peptide (IC) required for inhibiting half the activity of DPP-IV enzyme₅₀Value) calculation: determining DPP-IV inhibition rate of Gly-Pro-type peptide under different concentrations, making regression curve with logarithm value of polypeptide concentration and inhibition rate to obtain regression equation, and calculating IC₅₀The value, i.e. the concentration of peptide at which 50% of the DPP-IV enzyme activity is inhibited.

The C-terminal of GPI, GPM, GPQ, GPR, GPA and GPS was gradually added with amino acids to form 6 peptide series, GPI-, GPM-, GPQ-, GPR-, GPA-, and GPS-series, and their IC was determined₅₀The value is obtained.

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

TABLE 3 IC of Gly-Pro-type peptides of the invention on DPP-IV₅₀Value of

As shown in FIG. 2, the number of amino acids is increased from the C-terminal of GPA to 12, and the DPP-IV inhibitory activity of the formed new peptide is dependent on the length of the peptide segmentThe increase of (b) shows a tendency of increasing sharply and then decreasing slowly, and the activity of all new peptides is higher than that of the tripeptide GPA, indicating that a suitable increase of the length of the peptide fragment is one of the methods for improving the DPP-IV inhibitory activity of the Gly-Pro-type peptide. GPAG is a currently reported DPP-IV inhibitory peptide, the IC of which₅₀The value was 452.54. + -. 5.66. mu.M. The data show that the activity of GPA-type peptides consisting of 5-9 amino acid residues is higher than that of GPAG except for 12 peptides GPAGPRGPSGAA, and the IC50 value of the GPA-type peptides inhibiting DPP-IV enzyme is 183.26-445.86 mu M.

The results in FIGS. 3-7 show that the DPP-IV inhibitory activity of the GPM-, GPR-, GPS-, GPI-and GPQ-series peptides varies with the length of the peptide fragment in a manner similar to the GPA-series. The DPP-IV inhibitory activity of peptides consisting of 4-9 amino acid residues in the series is higher than that of corresponding tripeptides, simultaneously higher than that of GPAG, and the IC of the peptides on DPP-IV₅₀The value is between 114.09 and 429.42 mu M.

In summary, adding an amino acid to the C-terminus of the Gly-Pro-Xaa monomer is an effective method for developing Gly-Pro-type peptides with stronger DPP-IV inhibitory activity, wherein the sequence length is increased to 4-9 amino acids.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A Gly-Pro-type peptide consisting of 4-9 amino acid residues, which is characterized in that the sequence general formula is 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).

2. The Gly-Pro-type peptide of claim 1, wherein: xaa2 is Pro (P) or Ala (A).

3. The Gly-Pro-type peptide of claim 1, wherein: xaa3 is Arg (R), Ser (S) or Gln (Q).

4. The Gly-Pro-type peptide of claim 1, wherein: xaa4 is Ala (A), Ser (S) or Gln (Q).

5. The Gly-Pro-type peptide of claim 1, wherein: the amino acid residues in the Gly-Pro-type peptide are all in L configuration.

6. Use of a Gly-Pro-type peptide according to any of claims 1 to 5 for the preparation of a medicament for the treatment of type 2 diabetes.

7. Use according to claim 6, characterized in that: the medicine contains more than one pharmaceutically acceptable carrier or auxiliary material.

8. Use according to claim 7, characterized in that: the auxiliary materials are sustained release agents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption enhancers, adsorption carriers, surfactants or lubricants.

9. Use according to claim 7, characterized in that: the carrier is at least one of a microcapsule, a microsphere, a nanoparticle or a liposome.