A protein kinase polypeptide inhibitor

A peptide and injection technology, applied in the field of biomedicine, can solve the problem of lack of specificity of inhibitors

Active Publication Date: 2019-06-25
BEIJING PEPMAGIC BIOTECH CORPORATION LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the strong conservation of the kinase active site, a large number of kinase molecules in the body have similar active sites, so such inhibitors lack specificity; many studies have shown that these inhibitors are different from other kinases to varying degrees. cross-inhibition effect and drug off-target effect; in addition, in animal experiments, it also showed certain gene and liver toxicity
Therefore, there is currently no p38 inhibitor drug suitable for clinical treatment

Method used

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  • A protein kinase polypeptide inhibitor
  • A protein kinase polypeptide inhibitor
  • A protein kinase polypeptide inhibitor

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Experimental program
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Embodiment 1

[0025] Synthesis and purification of embodiment 1 polypeptide PI-38

[0026] 1. Synthesize the peptide PI-38 according to the Fmoc solid-phase peptide synthesis method, and the synthesis steps are as follows:

[0027] 1.1 To ensure that the C-terminus of the polypeptide is -CONH 2 Structure, choose Rink Amide resin as polymer solid phase carrier.

[0028] 1.2 Remove the Fmoc protecting group:

[0029] 1.2.1 Put the required polymer solid phase carrier into the synthesis column, add the organic solvent DMF (dimethylformamide) to soak for 10 minutes and then discharge; pass into 20% piperidine / DMF solution to cover the resin surface, pass through N 2 Discharge after reacting for 10 minutes; repeat to feed 20% piperidine / DMF, 2 Discharge after 10 minutes of reaction.

[0030] 1.2.2N 2 Under protection, pass through DMF solvent 5 times to thoroughly wash the above resin.

[0031] 1.3 Condensation reaction:

[0032] Amino Acid Activation: Weigh Fmoc-protected amino acid 3 t...

Embodiment 2PI-38

[0053] Example 2 Blocking effect of PI-38 on the interaction between p38MAPK protein and its effector protein.

[0054] Using SPOT technology, the MKK3b and MEF2A proteins that interact with the p38MAPK protein are prepared into a peptide array chip, and the peptides are used to block the interaction between the proteins on the array and the p38 recombinant protein.

[0055] Materials: Polypeptide array laboratory homemade, amino acids were purchased from Jill Biochemical, p38 recombinant protein was purchased from GeneTex, human p38 protein antibody was purchased from CST, and the secondary antibody of HRP-labeled anti-human p38 protein antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. The chemiluminescence reaction solution was purchased from Thermo Company

[0056] The peptide chip taken out from the -20 degree refrigerator was placed in a plate and washed 3 times with absolute ethanol, 5 minutes each time. Afterwards, the polypeptide chip membr...

Embodiment 3PI-38

[0058] Example 3 PI-38 inhibits p38MAPK protein kinase activity

[0059] p38 Kinase Assay / Inhibitor Screening Kit is a non-quantitative enzymatic detection method for detecting p38 protein activity. The ELISA plate used for detection in the kit is pre-coated with a substrate equivalent to ATF2. ATF2 contains Thr amino acid residues that can be effectively phosphorylated by p38. The detection antibody can specifically detect Thr phosphorylated by ATF2 by p38 kinase. . If the active polypeptide inhibitor is added, the active polypeptide with inhibitory effect can inhibit the kinase activity of p38, and the specific antibody cannot recognize the phosphorylated Thr on the ATF2 protein.

[0060] Materials: The p38 kinase assay / inhibitor screening kit was purchased from Abnova, the small molecule inhibitor SB202190 was purchased from MCE, and other reagents were provided with the kit.

[0061] Remove an appropriate number of microplate wells from the microplate provided in the kit...

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Abstract

The present invention provides a protein kinase polypeptide inhibitor PI-38, the amino acid sequence of which is: Ser-Gly-Arg-Val-Met-Arg-Gly-Pro-Arg-Ser-Ala. The polypeptide PI-38 can block the interaction between p38MAPK protein and its effector protein, and then specifically inhibit the activity of p38 kinase.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a protease polypeptide inhibitor. Background technique [0002] MAPKs are a class of protein kinases that widely exist from yeast to higher plants and animals. They are responsible for transducing specific extracellular stimulus signals to the nucleus and mediating specific responses in cells, so they play an important role in the process of cell signal transmission. . p38 MAPK was discovered in 1993 by Brewster et al. when studying the effect of hyperosmotic environment on fungi. Later, it was found that it also exists in mammalian cells, and it is also one of the subclasses of MAPKs. Its properties are similar to JNK, and it is also a stress-activated protein kinase. Studies have shown that the activators of the p38MAPK pathway are similar to the JNK pathway. Some pro-inflammatory factors (TNFα, IL-1) and stress stimuli (UV, H2O2, heat shock, hyperosmotic and protein s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06A61K38/08A61P29/00A61P35/00A61P35/02A61P19/02
CPCC07K7/06A61P29/00A61P35/00A61P35/02A61P19/02A61K38/00A61K38/08
Inventor 赵树民鲍勇刚石松传周方威廉姆·坎贝尔卡洛斯·阿德里尔
Owner BEIJING PEPMAGIC BIOTECH CORPORATION LTD
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