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Polypeptide, its derivative and its application in the preparation of drugs for preventing and treating tumors

A technology of derivatives and peptides, applied in the direction of antineoplastic drugs, drug combinations, peptides containing positioning/targeting motifs, etc., can solve the problems of complex signaling pathway regulation mechanism, slow and lack of drug development process, etc.

Active Publication Date: 2021-04-09
胡卓伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the defects of complex regulatory mechanism of Wnt / β-catenin signaling pathway in the prior art, slow drug development process, and lack of a drug that can effectively inhibit the transcription activation activity of β-catenin, the present invention provides a polypeptide, its derivatives and Application in the preparation of drugs for preventing and treating tumors

Method used

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  • Polypeptide, its derivative and its application in the preparation of drugs for preventing and treating tumors
  • Polypeptide, its derivative and its application in the preparation of drugs for preventing and treating tumors
  • Polypeptide, its derivative and its application in the preparation of drugs for preventing and treating tumors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 The surface plasmon resonance method is used to screen peptides that bind to β-catenin protein.

[0034]Firstly, the TRIB3 segment is truncated into different peptide fragments, and the peptides are synthesized with a peptide solid-phase synthesizer. This process is carried out by Beijing Saibaisheng Gene Co., Ltd. The entire screening process was carried out in a surface plasmon resonance instrument Biacore T200. The screening method is as follows:

[0035] 1) The purified protein β-catenin (purchased from Beijing Yiqiao Shenzhou Co., Ltd.) was coupled to the CM5 chip (purchased from GE Company) through amino groups, and the unbound protein was washed away at a flow rate of 10 μL / min, and the surface of the chip was equilibrated 2 Hours, the buffer for washing and equilibration was glycine buffer at pH 2.5.

[0036] 2) 200 μL of different concentrations of polypeptide fragments (200, 100, 50, 25, 12.5 nM) were automatically injected, and the whole process w...

Embodiment 2

[0040] Example 2 ELISA method to verify the binding of peptide T3A6 to protein β-catenin

[0041] The specific operation steps are as follows:

[0042] 1. Dilute human β-catenin protein and bovine serum albumin (BSA) with PBS to 10 μg / ml respectively, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.

[0043] 2. Wash three times with PBS containing 0.1% Tween-20, coat the plate with 200 μl blocking solution (10% bovine serum PBS), and coat at 37° C. for 2 hours.

[0044] 3. Pour off the blocking solution used for coating, and add 200 μl of 1 μg / ml polypeptide T3A6 solution; at the same time, set up a positive control, add 200 μl of 1 μg / ml TRIB3 protein solution (purchased from RD Company), and incubate at 37°C for 1 hour.

[0045] 4. Wash five times with PBS containing 0.1% Tween-20, add 100 μl anti-β-catenin monoclonal antibody diluted with blocking solution 1:4000 (v / v) to each well, and incubate at room temperature for 1 hour.

[0046] 5. Wash six t...

Embodiment 3

[0051] Example 3 The method of competition ELISA verifies that the peptide T3A6 can compete for the binding of TRIB3 and β-catenin protein

[0052] The specific operation steps are as follows:

[0053] 1. Dilute human β-catenin protein and bovine serum albumin (BSA) with PBS to 10 μl / ml, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.

[0054] 2. Wash three times with PBS containing 0.1% Tween-20, coat the plate with 200 μl blocking solution (10% bovine serum PBS), and coat at 37° C. for 2 hours.

[0055] 3. Pour off the coated blocking solution, add 200 μl of 1 μg / ml TRIB3 protein solution, and incubate at 37°C for 1 hour.

[0056] 4. Wash five times with PBS containing 0.1% Tween-20; add 100 μl of blocking solution to each well as a negative control and 100 μl of horseradish catalase-labeled polypeptide T3A6 diluted with blocking solution, and incubate at room temperature for 1 h.

[0057] 5. Wash six times with PBS containing 0.1% Tween-20; prepare...

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Abstract

The invention discloses a polypeptide specifically binding to β-catenin, which is a TRIB3 mutant whose amino acid sequence is substituted, deleted or added with one or several amino acids and has TRIB3 activity. Also disclosed is a derivative of the polypeptide, which is a chimeric peptide formed by linking the polypeptide with a membrane-penetrating peptide. The invention discloses an anti-tumor pharmaceutical composition containing the polypeptide or polypeptide derivative as an active ingredient. Also disclosed are applications of the polypeptides, polypeptide derivatives and pharmaceutical compositions in the preparation of drugs for preventing and / or treating diseases related to TRIB3 and β-catenin, especially tumors. The pharmaceutical composition has the ability to specifically bind to β-catenin, block the binding of TRIB3 and β-catenin protein, and reduce the protein level of TRIB3. The prepared medicine has the advantages of remarkable curative effect, less toxic and side effects and safe use.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to polypeptides, their derivatives and their application in the preparation of drugs for preventing and treating tumors. Background technique [0002] The Wnt / β-catenin signaling pathway is abnormally activated in a variety of tumors (colon cancer, liver cancer, lung cancer, pancreatic cancer, glioma and breast cancer). , tumor drug resistance and maintenance of tumor stemness and other aspects play a role in promoting. As the effector molecule of this signaling pathway, β-catenin is usually phosphorylated by its degradation complex members Axin-2, APC, and GSK-3β, and then enters the ubiquitination pathway for degradation. When Wnt signaling is abnormally activated, the members of its degradation complex disintegrate, and the β-catenin accumulated in the cytoplasm will translocate into the nucleus and form a transcription complex with the LEF / TCF family, which jointly promo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/705C07K19/00A61K38/17A61P35/00
CPCA61K38/00A61P35/00C07K14/705C07K2319/10
Inventor 胡卓伟花芳尚爽
Owner 胡卓伟
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