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Active polypeptide with function of antagonizing chemokine receptor CXCR4 as well as design preparation and biomedical application of active polypeptide

A technology of peptide derivatives and activity, which is applied in the field of design and preparation of active peptides with antagonistic chemokine receptor CXCR4 and biomedical applications, which can solve the problems of low survival rate and poor prognosis of patients, and achieve biological stability Good, strong anti-HIV-1 virus invasion, strong anti-tumor cell metastasis effect

Active Publication Date: 2015-09-09
徐岩 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In breast cancer patients, the positive expression of CXCR4 is associated with lymph nodes and distant metastasis, and the prognosis of breast cancer patients with high expression of CXCR4 is poor
Studies on colorectal cancer have shown that high expression of CXCR4 is associated with tumor recurrence and liver metastasis, and the corresponding patient survival rate is also low

Method used

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  • Active polypeptide with function of antagonizing chemokine receptor CXCR4 as well as design preparation and biomedical application of active polypeptide
  • Active polypeptide with function of antagonizing chemokine receptor CXCR4 as well as design preparation and biomedical application of active polypeptide
  • Active polypeptide with function of antagonizing chemokine receptor CXCR4 as well as design preparation and biomedical application of active polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Design of polypeptides.

[0032] Peptide design was performed using molecular dynamics simulations. Specifically, the Biopolymer module in the SYBYL-x software package is used to perform simulation calculations to predict the binding conformation of the designed polypeptide to the CXCR4 protein. The CXCR4 protein model was constructed from the published crystal structure (PDB: 3ODU). Use software to organize the crystal structure of CXCR4, remove redundant ligand molecules, water molecules and other small molecules, add hydrogen and charge to the protein file, and obtain the mol2 file. The structure of the polypeptide molecule undergoes simulated annealing and energy minimization to obtain a relatively low-energy conformation. By manual adjustment, the peptide molecule is moved to the surface of the protein molecule, and the dihedral angle is adjusted to avoid molecular collision. In the process of molecular dynamics simulation, the structures of polypeptid...

Embodiment 2

[0034] Example 2 Synthesis and purification of the polypeptide.

[0035] Polypeptides were synthesized using solid-phase synthesis methods. The solid-phase resin adopts TentaGel Amide resin, and the polypeptide obtained by separation is a C-terminally aminated polypeptide. All amino acids were amino protected with 9-fluorenylmethoxycarbonyl (9-Fmoc). During the synthesis process, the carboxyl group of the first amino acid forms an amide bond with the activated amino group of the solid phase resin and connects to the solid phase. Through the deprotection of 20% piperidine 80% dimethylformamide (DMF), the Fmoc group is removed to expose the amino group, and under the action of the condensing agent DIC (5 equivalents) and HOBt (5 equivalents) and The carboxyl group of the next amino acid (5 equivalents) was reacted. Excess reactants and condensing agents were removed after the reaction by washing the solid phase resin with DMF. In this way, the sequence of the polypeptide is ...

Embodiment 3

[0038] Example 3 Affinity activity test of the polypeptide.

[0039] HEK293 cells are exogenously inserted with a CXCR4 plasmid, which can stably express CXCR4 protein. The cells were cultured in RPMI1640 (10% calf serum, 100 IU penicillin, 0.1 mg / mL streptomycin, 2 mM glutamine), and added 0.4 mg / mL G418 for selection. Before the experiment, HEK293 cells were digested with trypsin and collected, washed twice with FACS buffer (0.5% BSA, 0.05% sodium azide in PBS) at 4°C, and finally diluted with buffer to 1×10 7 cells / mL. Cells were mixed evenly and placed in a 96-well plate with conical wells, 5×10 per well 5 cell. The primary antibody (12G5, mouse anti-human CXCR4 antibody, 1:3000) and different concentrations of polypeptides were incubated with the cells on ice for 40 min, and then the cells were washed twice with FACS buffer. After completion, add secondary antibody (anti-mouse antibody-FITC, 1:250) and incubate on ice in the dark for 30 min. After the reaction, the c...

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Abstract

The invention discloses an active polypeptide designed to have a natural chemotactic factor N-end region. The active polypeptide inhibits HIV-1 from invading and infecting cells by antagonizing the activity of the chemokine receptor. The invention further discloses a design method for the active polypeptide. According to molecular dynamic simulation, an appropriate connecting bridge is designed for connecting two polypeptide fragments, so that the polypeptide synthesis sequence is determined, the polypeptide is synthesized in a solid-phase mode, and finally the biologic activity of the active polypeptide is tested. The active polypeptide disclosed by the invention can be used as a CXCR4 receptor antagonist, a precursor medicine for treating AIDS and a stem cell mobilizer, and is used for treating acute myeloid leukemia and various CXCR4-associated diseases.

Description

technical field [0001] The invention relates to an active polypeptide with antagonism chemokine receptor CXCR4, its preparation method and application. Background technique [0002] CXCR4 is a member of the CXC chemokine receptor family and has a total of 356 amino acid residues. CXCR4 is the specific receptor of the chemokine stromal cell-derived factor-1 (SDF-1, CXCL12), which can induce chemotaxis of cells after binding with SDF-1. SDF-1a and CXCR4 are widely expressed in a variety of cells and tissues, including immune cells, brain, heart, kidney, liver, lung, and spleen, and play critical roles in the development of the immune system, circulatory system, and central nervous system role. [0003] CXCR4 protein has been found to be closely related to various diseases. In the 1990s, researchers discovered the co-receptors of HIV virus infection, namely CXCR4 and CCR5. Both of these two transmembrane proteins belong to the family of chemokine receptors, and are closely ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/04A61K38/17A61P35/00A61P35/02A61P31/18A61P37/00
Inventor 不公告发明人
Owner 徐岩
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