CN111592583B

CN111592583B - Bioactive polypeptide and application thereof in preparation of dipeptidyl peptidase 4 inhibitor

Info

Publication number: CN111592583B
Application number: CN202010530946.4A
Authority: CN
Inventors: 朱奕; 刘长军; 孟尔; 王珊; 项王菲; 李佳豪; 王凤
Original assignee: Hunan University of Science and Technology
Current assignee: Hunan University of Science and Technology
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2022-09-06
Anticipated expiration: 2040-06-11
Also published as: CN111592583A

Abstract

The invention discloses a bioactive polypeptide, and the sequence of the bioactive polypeptide is NH 2-Val-Thr-Tyr-Glu-Trp-Pro-Asn-Ser-Gly-Phe-Ser-Glu-OH. The relative molecular weight of the bioactive polypeptide is 1415.82 Da. The biologically active polypeptide has an IC50 value of 1.48 mM. The bioactive polypeptide can be used for preparing dipeptidyl peptidase 4 inhibitors.

Description

Bioactive polypeptide and application thereof in preparation of dipeptidyl peptidase 4 inhibitor

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a bioactive polypeptide and application of the polypeptide in preparation of a dipeptidyl peptidase 4 (DPP-4) inhibitor.

Background

Diabetes is a metabolic disease characterized by hyperglycemia. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. Hyperglycemia, which is present in long-term diabetes, leads to chronic damage to, dysfunction of, various tissues, particularly, the eyes, kidneys, heart, blood vessels, nerves. About 4.63 hundred million 20-79 year old adults with diabetes in 2019 worldwide (1 out of 11 people is diabetic); it is predicted that by 2030, diabetics will reach 5.784 billion; it is expected that by 2045 years, diabetics will reach 7.002 billion.

In recent years, DPP-4 inhibitors, as newly developed hypoglycemic drugs, have become one of the important research directions for type II diabetes drugs. DPP-4 is an in vivo enzyme, i.e., an enzyme. Its main function is to break down proteins in the body. In humans, DPP-4 can break down glucagon-like peptide-1 (GLP-1) which has the effect of stimulating insulin secretion, and GLP-1 can lower blood sugar by stimulating insulin, inhibiting glucagon, inhibiting gastric emptying and allowing islet cells to regenerate. The DPP-4 inhibitor can effectively antagonize the action, control the blood sugar of a patient, particularly the postprandial blood sugar by inhibiting the degradation of GLP-1, and improve the symptoms of the patient such as the sugar tolerance, the insulin resistance and the like. The DPP-4 inhibitor may play a role in other various diseases of the body, and is also found to improve cardiovascular system complications such as atherosclerosis, left diastolic dysfunction and the like of a diabetic patient while treating type 2 diabetes. DPP-4 inhibitors that result in the inactivation of DPP-4 and thus do not break down GLP-1 have been one of the leading targets for the treatment of diabetes. The DPP-4 inhibitors which are currently studied most intensively and have been applied clinically are Sitagliptin (Sitagliptin), Vildagliptin (Vildagliptin), Saxagliptin (Saxagliptin), Alogliptin (Alogliptin) and linagliptin (linagliptin). However, the FDA in the united states has warned that saxagliptin and alogliptin may cause side effects such as heart failure in patients, especially in patients who have cardiovascular disease or renal failure themselves.

Long-acting DPP-4 is widely distributed in human body, and inhibitors thereof may have influence on other substrate functions. The small molecule drugs have relatively poor specificity due to their small spatial structure, and are prone to side effects.

Therefore, research and development of a DPP-4 inhibitor with few side effects is a focus in the field of diabetes research.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a bioactive polypeptide and application of the polypeptide in preparing a dipeptidyl peptidase 4 (DPP-4) inhibitor.

The invention obtains a random polypeptide which can interact with an extracellular domain in human intestinal dipeptidyl peptidase 4 (DPP-4) by a yeast two-hybrid method, the sequence of the polypeptide is shown as SEQ ID NO.1, and the random polypeptide specifically comprises the following components: NH2-Val-Thr-Tyr-Glu-Trp-Pro-Asn-Ser-Gly-Phe-Ser-Glu-OH (VTYEWPNSGFSE), which is a cell surface serine protease inhibitor. The theoretical relative molecular weight of the molecular weight is 1415.82 Da through MALDI-TOF mass spectrum detection; the polypeptide can inhibit the activity of dipeptidyl peptidase 4 (DPP-4) through detection, and the IC50 value of the polypeptide is 1.48mM, namely, the polypeptide can be used as a dipeptidyl peptidase 4 (DPP-4) inhibitor and is named DPP 4P-1.

Compared with small molecule compounds, the polypeptide has a better spatial structure, so that the polypeptide has better specificity on the action of a target, is easy to degrade in vivo and cannot cause damage due to accumulation in vivo. The polypeptide is extracted from nature to be artificially synthesized, is used as a new medicine, and shows unique superiority in clinical application and production and preparation modes. In clinic, the polypeptide medicament is similar to recombinant protein medicaments and monoclonal antibody medicaments, and has the advantages of strong specificity, good curative effect and the like; in the production and preparation mode, the polypeptide medicament is close to a micromolecular medicament, has the characteristics of high purity, controllable quality, easily determined structure and the like, and is a potential therapy with high selectivity, effectiveness and relative safety in polypeptide treatment.

The other various experimental procedures involved in the present invention are conventional procedures in the field of biotechnology, and are not specifically described herein, and those skilled in the art can refer to various common tool books, scientific documents, or relevant specifications, manuals, etc. before the filing date of the present application.

Drawings

FIG. 1 is an RP-HPLC separation and purification profile of DPP4P-1, wherein the arrow indicates the peak of interest;

FIG. 2 is a MALDI-TOF mass spectrometry identification map of DPP4P-1, wherein the arrow indicates the peak of interest;

FIG. 3 is a concentration-inhibition curve of DPP4P-1 vs. DPP-4 enzyme inhibitory activity.

Detailed Description

EXAMPLE 1 Yeast two-hybrid approach Positive clones were obtained and verified by self-activation

The invention adopts MATCHMAKER GAL Two-Hybrid System 3 System of Clontech company to carry out yeast Two-Hybrid experiment:

1) bait plasmid (BD) containing the extracellular domain of human intestinal dipeptidyl peptidase 4 (DPP-4) was transferred into yeast strain AH109 and made competent by the PEG/LiAc method.

2) 10. mu.g of plasmid DNA from a random library of random dodecapeptide polypeptides was transferred to the competence of the previous step.

3) Finally, the competent cells are smeared on a growth-defective culture medium and cultured at 30 ℃ until positive clones grow out.

4) And performing small-scale amplification culture on the positive clone, extracting the plasmid, transforming the plasmid into AH109, and coating the AH109 on a proper defective culture medium to perform repeated experiments and self-activation verification.

5) Positive plasmids without self-activation were sent to sequencing company for sequencing.

Through the steps, 1 library plasmid which can interact with the BD plasmid is obtained through screening, and the amino acid sequence of the library plasmid is as follows: NH 2-Val-Thr-Tyr-Glu-Trp-Pro-Asn-Ser-Gly-Phe-Ser-Glu-OH.

EXAMPLE 2 chemical polypeptide Synthesis of candidate polypeptide sequences

Rink Amide NovaPEG resin is used, 20% piperidine/DMF solution is used as a deprotection agent, peptide bond formation is carried out by HBTU coupling, and the reactant feeding is 4 times of the molar number of the resin loading. Polypeptide is synthesized on a CEM Liberty microwave-assisted full-automatic polypeptide synthesizer by adopting an Fmoc protection strategy, the microwave power is 35W, and the highest reaction temperature is 75 ℃. After completion of the polypeptide chain construction, 2g of peptide resin was obtained. The peptide resin was cleaved and TFA removed under reduced pressure to precipitate the crude peptide. And then purifying the crude peptide by reverse phase high performance liquid chromatography (RP-HPLC) (wherein figure 1 is RP-HPLC purification map, and acetonitrile concentration is eluted from 5% -30%), detecting a target product by a MALDI-TOF mass spectrometer (see figure 2), and finally obtaining the high-purity polypeptide freeze-dried powder by steps of freeze drying and the like.

Example 3 in vitro enzyme activity assay for detecting inhibitory Activity of polypeptide DPP-4 enzyme

Under alkaline conditions, DPP-4 can catalyze the substrate Gly-Pro-pNA to hydrolyze to generate glycylproline and yellow p-nitroaniline (pNA), the latter has a characteristic absorption peak at the wavelength of 405 nm, and the absorbance of the yellow p-nitroaniline (pNA) is measured at the wavelength of 405 nm by an enzyme-labeling instrument to reflect the activity of the enzyme. Adding 110 mu L of 10 mmol/LTris-HCl (pH 7.8), 20 mu L of 0.2U/mL DPP-4 enzyme and 20 mu L of polypeptide solution (10 mmol/L Tris-HCl (pH 7.8) solution is used for preparing polypeptide solution with different concentrations) in sequence into a 96-hole enzyme-labeled plate hole, incubating the mixture in an ice bath for 1 h after mixing, adding 50 mu L of 300 mmol/L Gly-Pro-pNA, reacting the mixture at the constant temperature of 37 ℃ for 2 h after mixing, detecting the absorbance value of the mixture at the wavelength of 405 nm by an enzyme-labeling instrument, repeating the experiment for 3 times, and averaging.

Formula of DPP-4 enzyme activity inhibition ratio:

I％＝[1－(A1－A2)/(A3－A4)]×100%；

in the formula: a1 is the absorbance value of the sample group, a2 is the background absorbance value of the sample group measured by replacing the enzyme solution with 10 mmol/L Tris-HCl (pH 7.8), A3 is the background absorbance value of the blank group measured by replacing the sample solution with 10 mmol/L Tris-HCl (pH 7.8), and a4 is the background absorbance value of the blank group measured by replacing the sample solution with 10 mmol/L Tris-HCl (pH 7.8) and the enzyme solution with 10 mmol/L Tris-HCl (pH 7.8).

The polypeptide DPP4P-1 was detected to have inhibitory activity against DPP-4, with an IC50 value of 1.48mM (as shown in FIG. 3).

Sequence listing

<110> Hunan university of science and technology

<120> bioactive polypeptide and application thereof in preparation of dipeptidyl peptidase 4 inhibitor

<141> 2020-06-11

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> null

<400> 1

Val Thr Tyr Glu Trp Pro Asn Ser Gly Phe Ser Glu

1 5 10

Claims

1. A bioactive polypeptide, characterized in that the sequence of the bioactive polypeptide is NH 2-Val-Thr-Tyr-Glu-Trp-Pro-Asn-Ser-Gly-Phe-Ser-Glu-OH.

2. The biologically active polypeptide of claim 1, having a relative molecular weight of 1415.82 Da.

3. The biologically active polypeptide of claim 1, having an IC50 value of 1.48 mM.

4. Use of a biologically active polypeptide according to any one of claims 1 or 2 for the preparation of a dipeptidyl peptidase 4 inhibitor.