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Application of novel cyclic peptide

A cyclic peptide and amino acid technology, applied in the field of diseases, can solve the problem that antimicrobial peptides are easily hydrolyzed by proteases

Active Publication Date: 2015-10-14
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most antimicrobial peptides enter the human body and are easily hydrolyzed by proteases. Currently, clinical trials are limited to local application.

Method used

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  • Application of novel cyclic peptide
  • Application of novel cyclic peptide
  • Application of novel cyclic peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Separation, Purification and Structure Identification of Natural Cyclic Peptides

[0066] Extraction of samples and separation of cyclic peptides Collect fresh Viola viola, crush it with a juice extractor, then extract it with 95% ethanol for 3 times, each time for 24 hours, combine the extracts, concentrate under reduced pressure and recover ethanol; the extracts are weighed with water Suspended and extracted with petroleum ether, ethyl acetate, and n-butanol in turn; the n-butanol part containing the cyclic peptide was concentrated under reduced pressure and then resuspended in water. The sample was subjected to Sephadex LH-20 column chromatography using 10%, 20%, 30% %, 40%, 50%, 60%, 80% methanol water gradient elution. The eluting fraction containing the cyclic peptide was determined to be the 30% fraction using mass spectrometry tracking. After concentrating again under reduced pressure, use Agilent1200 system and GRACE VYDAC protein polypeptide semi-preparative ...

Embodiment 2

[0077] SEQ ID NO:1 thermal stability test

[0078] (1). Peptide SEQ ID NO: 1 solution: 45 μL of 1 μg / μL polypeptide plus 180 μL of sterile water passed through the membrane, and mix well;

[0079] (2).0min heat resistance reaction test sample: take out 50μL of the mixed solution in step (1);

[0080] (3). Test samples with different heat-resistant reaction times: divide the mixture in step (1) into three samples of 50 μL, place them in a boiling water bath at 100°C, and take out a sample for about 10 minutes, 20 minutes, and 30 minutes respectively. , immediately placed on ice to cool for 10 minutes, and then centrifuged;

[0081] (4). The samples (2) and (3) were analyzed by HPLC using a chromatographic column (YMC-Pack, ODS-A, 250×4.6mm) ( Figure 9 ), the liquid chromatography elution conditions are as follows:

[0082]

[0083] Note: Phase A: 0.5%TFA Phase B: 90%AcN, 0.5%TFA; detection wavelength: 220nm

[0084] (5). Results: With the peak area of ​​0min SEQ ID NO:1 p...

Embodiment 3

[0087] SEQ ID NO:1 enzymatic stability test

[0088] 1. Solution preparation

[0089] (1). Peptide SEQ ID NO: 1: 1 μg / μL, prepared with sterile water through the membrane;

[0090] (2). Buffer for Trypsin test: 100mM Tris-HCl, pH8.0; Trypsin enzyme: 0.5μg / μL;

[0091] (3). Buffer for Chymotrypsin test: 100mM Tris-HCl, 10mM CaCl 2 ,pH7.8; Chymotrypsin enzyme: 0.5μg / μL;

[0092] (4). Enzyme hydrolysis termination solution: 10% TFA, prepared with corresponding reaction buffer.

[0093] 2. Trypsin enzymatic hydrolysis test of SEQ ID NO:1

[0094] (1). Peptide SEQ ID NO:1 solution: 25 μL 1 μg / μL polypeptide plus 95 μL buffer, mix well;

[0095] (2). Trypsin enzymatic hydrolysis reaction working solution: 0.35 μL 0.5 μg / μL peptide plus 3.15 μL buffer, mix well;

[0096] (3). Place solutions (1) and (2) in a 37°C incubator for 5 minutes;

[0097] (4).0min enzymatic hydrolysis test sample: take out 25 μL of the mixture in step (1), and add 3.75 μL of buffer;

[0098] (5). Test...

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Abstract

The invention relates to a cyclic peptide with antiviral, antibacterial and antifungal activity. The cyclic peptide has an amino acid residue sequence Cyclo (CGESCVFIPCITTVLGCSCSIKVCYKNGSIP) shown in SEQ ID NO:1, wherein three pairs of disulfide bonds are respectively formed by Cys in the first and the 17th sites, Cys in the fifth site and the 19th site and Cys in the 10th site and the 24th site, so that the cyclic peptide has thermotropy resistance and proteaseenzymolysis stability, and is low in hemolytic toxicity. Therefore, a pharmaceutical composition containing the cyclic peptide or salts thereof can be used for preventing or treating the diseases such as influenza viruses A, bacteria and fungi of people or livestock.

Description

technical field [0001] The present invention relates to a cyclic peptide with antiviral, bacterial and fungal activity, which contains 6 cysteine ​​residues and forms three pairs of disulfide bonds, and forms a unique cyclic cysteine ​​knot together with the cyclic backbone The pharmaceutical composition containing the cyclic peptide or its salt can be used to prevent or treat human or domestic animal infection with influenza A virus, bacteria, fungi and other diseases. Background technique [0002] Cyclotides (cyclotides) are a class of plant ring proteins with a novel structure, usually composed of 28-37 amino acids, three disulfide bonds in the molecule and a ring skeleton together form a unique ring cysteine ​​knot. Current studies suggest that such molecules mainly exist in Violaceae, Rubiaceae, Leguminosae and Cucurbitaceae plants, and the cyclic peptide molecule is encoded by the cyclic peptide precursor protein gene in plants, which is transcribed and expressed as a ...

Claims

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Application Information

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IPC IPC(8): C07K14/415A61K38/16A61K8/64A61P31/04A61P31/10A61P31/12A61P31/16A23L3/3544
CPCA61K38/00C07K14/415
Inventor 王伟刘忞之杨燕张书香唐亮王惠敏陈成娟程克棣孔建强
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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