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Polypeptide targeting to inhibit Wnt/beta-catenin signal activity and application thereof

A specific, peptide derivative technology, applied in the biological field, can solve the problems of complex Wnt/β-catenin signaling pathway regulation mechanism, slow drug development process, lack of inhibition of β-catenin transcriptional activation activity, etc. Safe, less toxic and side effects

Active Publication Date: 2019-09-03
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

[0004] The technical problem to be solved by the present invention is to provide an effective drug that inhibits the transcription activation activity of β-catenin in view of the complex regulatory mechanism of the Wnt / β-catenin signaling pathway, the slow progress of drug development, and the lack of effective drugs that inhibit the activation of β-catenin / TRIB3 interaction. Polypeptide for further weakening β-catenin / TCF4 transcriptional activity and its application in the preparation of drugs for treating and preventing tumors

Method used

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  • Polypeptide targeting to inhibit Wnt/beta-catenin signal activity and application thereof
  • Polypeptide targeting to inhibit Wnt/beta-catenin signal activity and application thereof
  • Polypeptide targeting to inhibit Wnt/beta-catenin signal activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Co-immunoprecipitation method confirms the domain that interacts with TRIB3 in β-catenin.

[0027] Co-immunoprecipitation reagents are as follows:

[0028] Lysis solution A: 0.6057g Tris base, 1.7532g NaCl, 0.1017g MgCl2 6H2O, 0.0742g EDTA, 10mL glycerin, 10mL 10% NP40, add deionized water to 150mL, adjust the pH value to 7.6 with HCl, and adjust the volume to 191mL , mixed thoroughly, filtered through a 0.45 μm membrane filter, and stored at 4°C.

[0029]Lysis Solution B: 200 μL 2M β-glycerol phosphate, 4 mL 2.5M NaF, 2 mL 8 mM NaVO3, 2 mL 100 mMPMSF, 200 μL 1M DTT, 200 μL each of 1 mg / mL Leu, Pep and Apr, total volume 9 mL. The mother liquor was stored at -20°C. Before use, thaw the mother liquor of each component in liquid B, add it to liquid A according to the above composition ratio and mix well. Protein A / GPlus-Agarose was purchased from Santa Cruz, USA. The specific operation steps are as follows:

[0030] First, the full-length gene of β-catenin ...

Embodiment 2

[0032] Example 2: Using surface plasmon resonance to screen peptides that bind to TRIB3 protein.

[0033] The M4 domain, which interacts with TRIB3, was analyzed using the I-TASSER website, and it was found that it contained 8 α-helical peptides. The peptide was synthesized with a peptide solid-phase synthesizer, and this process was carried out by Beijing Saibaisheng Gene Co., Ltd. EXAMPLES The entire screening process was carried out in a surface plasmon resonance instrument Biacore T200. The screening method is as follows: 1. The purified protein TRIB3 (purchased from Beijing Yiqiao Shenzhou Co., Ltd.) is coupled to the CM5 chip (purchased from GE Company) through amino groups, and the unbound protein is washed away at a flow rate of 10 μL / min, and the chip is equilibrated. Surface for 2 hours.

[0034] 200 μL of different concentrations of polypeptide fragments (200, 100, 50, 25, 12.5 nM) were automatically injected, and the whole process was carried out at 25°C. The buf...

Embodiment 3

[0037] Example 3 The ELISA method verifies the binding of the peptide ARM7 to the protein TRIB3.

[0038] The specific operation steps are as follows:

[0039] 1. Dilute human TRIB3 protein and bovine serum albumin (BSA) to 10 μg / ml with PBS, add 100 μl to each well, and coat 96-well ELISA plate overnight at 4°C.

[0040] 2. Wash three times with PBS containing 0.1% Tween-20. The plates were coated with 200 μl of blocking solution (10% bovine serum in PBS), and coated at 37° C. for 2 hours.

[0041] 3. Pour off the coating solution, add 200 μl of 1 μg / ml polypeptide ARM7 solution correspondingly, set up positive control wells at the same time, add 200 μl of 1 μg / ml β-catenin protein solution, and incubate at 37°C for 1 hour.

[0042] 4. Wash five times with PBS containing 0.1% Tween-20. Add 100 μl anti-TRIB3 monoclonal antibody diluted with blocking solution 1:4000 to each well, and incubate at room temperature for 1 h.

[0043] 5. Wash six times with PBS containing 0.1% T...

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Abstract

The invention discloses a polypeptide targeting to inhibit beta-catenin or a derivative thereof, and an application thereof in drugs for treating and preventing tumors. The polypeptide has the following amino acid sequence: ARM7:Leu-His-Tyr-Gly-Leu-Pro-Val-Val-Val-Lys-Leu-His-Pro-Pro. The polypeptide is screened by a surface plasmon resonance (Biacore) method, has the abilities to specifically bind to TRIB3 and block the binding of TRIB3 and beta-catenin protein, inhibits the activity of Wnt / beta-catenin signaling pathways, and inhibits the growth and metastasis of tumor cells. Therefore, thepolypeptide and the derivative thereof are applied to the preparation of drugs for treating and preventing tumors. The prepared drugs can be used for treating tumors, such as colon cancer, liver cancer, lung cancer, pancreatic cancer, glioma and breast cancer.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a polypeptide and its application in preparing medicines for treating and preventing 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 the β-catenin degradation complex disintegrate, and the β-catenin accumulated in the cytoplasm will translocate into the nucleus to form a transcription complex with the LEF / TCF family, and jointly promote the transcripti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K19/00A61K38/10A61K47/64A61P35/00
CPCC07K7/08A61K47/64A61P35/00A61K38/00C07K2319/10
Inventor 胡卓伟花芳尚爽杨雨薇余娇娇周丹丹张诚
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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