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Protein kinase polypeptide inhibitor

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

Active Publication Date: 2018-01-09
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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0027] 1.1 To ensure that the C-terminal of the polypeptide is -CONH 2 Structure, Rink Amide resin is selected as the 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) and soak it for 10 minutes before discharging; pass 20% piperidine / DMF solution to cover the resin surface, pass through N 2 Discharge after 10 minutes of reaction; repeat to feed 20% piperidine / DMF, pass N 2 It was discharged after 10 minutes of reaction.

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

[0031] 1.3 Condensation reaction:

[0032] Amino acid activation: Weigh 3 times ...

Embodiment 2PI-38

[0053] Example 2 The blocking effect of PI-38 on the interaction of p38 MAPK protein with its effector proteins.

[0054] Using SPOT technology, MKK3b and MEF2A proteins that interact with p38MAPK protein were prepared into a peptide array chip, and the interaction between the proteins on the array and the p38 recombinant protein was blocked by peptides.

[0055] Materials: The peptide array was self-made in the laboratory, the amino acids were purchased from Jill Biochemical, the p38 recombinant protein was purchased from GeneTex, the human p38 protein antibody was purchased from CST, and the HRP-labeled anti-human p38 protein antibody secondary antibody was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Chemiluminescence reaction solution was purchased from Thermo Company

[0056] The peptide chip taken out from the -20 degree refrigerator was placed in a dish and washed three times with absolute ethanol for 5 minutes each time. After that, the pept...

Embodiment 3PI-38

[0058] Example 3 Inhibitory effect of PI-38 on p38MAPK protein kinase activity

[0059] The p38 Kinase Assay / Inhibitor Screening Kit is a non-quantitative enzymatic assay for the detection of 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 efficiently phosphorylated by p38. The detection antibody can specifically detect the Thr phosphorylated by p38 kinase of ATF2. . If an 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 Company, the small molecule inhibitor SB202190 was purchased from MCE Company, and other reagents were provided with the kit.

[0061] Pipette the appropriate number of microtiter wells fro...

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Abstract

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

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a protease polypeptide inhibitor. Background technique [0002] MAPKs are a class of protein kinases that are widely found in yeast to higher plants and animals. They are responsible for transducing specific extracellular stimuli to the nucleus and mediating specific responses in cells, so they play an important role in cell signaling. . p38 MAPK was discovered in 1993 by Brewster et al. 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 they are both stress-activated protein kinases. Studies have shown that the activator of the p38MAPK pathway is similar to the JNK pathway. Some pro-inflammatory factors (TNFα, IL-1), stress stimuli (UV, H2O2, heat shock, hypertonicity and protein synthesis inhibitors) that can activate JNK can also activate p38. In addition, p38 can also be activated by ...

Claims

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Application Information

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Patent Type & Authority Applications(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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