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Targeted tumor stem cell marker CD133 stapled peptide and application thereof

A technology of tumor stem cells and stapled peptides, applied in the fields of biomedical detection and medicinal chemistry, can solve the problems of low affinity of small molecule peptides, difficulty in maintaining natural conformation, and difficulty in exerting synergistic effects, etc., to achieve increased α-helices and stability Enhanced, increased stability effects

Inactive Publication Date: 2021-08-27
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there have been many reports on the use of targeted peptides in tumor diagnosis and treatment, including small molecular peptides and peptide nano-self-assembly materials, etc., but there are still many problems, such as low affinity and insufficient stability of small molecular peptides; Most of the assemblies are in the form of "building blocks" (recognition units, signal units, and hydrophilic or hydrophobic units spliced ​​together), and orderly assemblies can be formed under strict control of various conditions (solution purity, concentration, acidity, etc.) in vitro However, it is difficult to play a good synergistic effect in vivo; moreover, linear peptides are prone to proteolytic degradation, and it is difficult to maintain their natural conformation, resulting in inefficient cell penetration

Method used

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  • Targeted tumor stem cell marker CD133 stapled peptide and application thereof
  • Targeted tumor stem cell marker CD133 stapled peptide and application thereof
  • Targeted tumor stem cell marker CD133 stapled peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0039] Experimental example 1 Construction and screening of CD133 targeting polypeptide screening system of the present invention

[0040] 1) Experimental instruments and materials

[0041] N-methylmorpholine (NMM), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), ninhydrin, vitamin C, phenol, tetramethyluronium hexafluorophosphate (HBTU), hexahydro Pyridine, triisopropylsilane (TIS), ethanedithiol (EDT), N,N dimethylformamide (DMF), anhydrous ether, resin, methanol, various Fmoc protected amino acids, 4,7-bis (5-bromo-2-thienyl)-2,1,3-benzothiadiazole (DBT), dichloroethane (EDC), streptavidin magnetic beads (MB-Streptavidin), peptide synthesis tube , shaker, vacuum water pump, rotary evaporator, laser confocal microscope (Olympus FV1000-IX81), the above reagents and materials were obtained from commercial sources.

[0042] 2) Synthesis of CD133 "one bead one object" polypeptide library

[0043] Use the Fmoc solid-phase peptide synthesis method to synthesize t...

experiment example 2

[0064] Experimental Example 2 The i and i+7 sites of CCP were stapled to generate and synthesize stapled peptides by introducing two unnatural amino acids containing α-alkenyl groups to form a ring.

[0065] The two unnatural amino acids introduced into the linear peptide chain by the present invention are Fmoc-2-(7'-octenyl)alanine and Fmoc-2-(4'-pentenyl)alanine. Preferably, the synthetic staple peptide sequence is denoted NH 2 - SKDEEW R8 HIWIKR S5HSYNLEGR-Ac (SCCP). Wherein, R8 and S5 are Fmoc-2-(7'-octenyl)alanine and Fmoc-2-(4'-pentenyl)alanine respectively. The synthesis of the linear peptide is consistent with the method steps of Experimental Example 1. After the synthesis of the linear peptide was completed, Grubbs-catalyzed ring-closing olefin metathesis (RCM) was performed to connect the side chains of R8 and S5 for cyclization. Afterwards, use a cleavage reagent (87.5% TFA+5% thioanisole+2.5% phenol+2.5%EDT+2.5%H 2 O) Peptides were cleaved from Wang resin with ...

experiment example 3

[0066] Experimental Example 3 The affinity between CD133 positive polypeptides CCP and SCCP and CD133 protein was detected by surface plasmon resonance (SPRi) method.

[0067] Spot 1 mg / mL CCP, SCCP and 1×PBS on the chip, incubate overnight at 4°C under humid conditions, then wash with 10×PBS for 10 minutes, then with 1×PBS for 10 minutes, and finally with deionized water for 2 minutes. Once, 10min each time, immersed in 1×PBS containing 5% milk, incubated overnight at 4°C, then washed with 10×PBS for 10min, 1×PBS for 10min, and finally washed twice with deionized water, 10min each time , dried with nitrogen, and mounted on a chip (Plexera HT surface plasmon resonance imaging system).

[0068] The mobile phase was sequentially passed through 1×PBS, 2×PBS, 0.78μg / mL, 1.56μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL and 25μg / mL human CD133 purified protein, and the SPRi signal was recorded and analyzed .

[0069] Depend on figure 2 It can be seen that CCP ( figure 2 a) with SCC...

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Abstract

The invention relates to a stapled peptide of a targeted tumor stem cell marker CD133 and an application of the stapled peptide, and belongs to the field of biomedical detection and medicinal chemistry. The polypeptide amino acid sequence SKDEEX1X2X3X4X5X6X7X8X9X10X11X12X13X14X15 is stapled, the resulting stapled peptide comprising two non-natural amino acids X which are crosslinked at i and i+n (n=3, 4, 5, 6, 7, 8, 9) positions by a linker forming a macrocycle. The stapled peptide disclosed by the invention can be used for generating in-situ induced self-assembly of the CD133 tumor stem cell marker. The stapled peptide provided by the invention is more stable in alpha-helical conformation and stronger in self-assembly capability to a certain extent, and the plasma stability is greatly enhanced. The stapled peptide disclosed by the invention can achieve a targeting effect on CD133 positive cells, and can be used as a targeting material for specifically targeting cancer cells.

Description

technical field [0001] The invention relates to a staple peptide targeting tumor stem cell marker CD133 and its application, in particular to the screening of tumor stem cell marker CD133-specific targeting polypeptides and full-carbon chain stapling, and its application as a drug carrier, belonging to biological Fields of medical testing and medicinal chemistry. Background technique [0002] In recent years, malignant tumors have become an important disease that endangers human health, and its morbidity and mortality are increasing year by year. For most malignant tumors, surgery, radiotherapy and chemotherapy, targeted and immunotherapy can kill most tumor cells, but the vast majority of tumors still cannot be cured, and are prone to recurrence and metastasis. In 2001, the concept of cancer stem cells (Cancer stem cells, CSCs) was formally proposed. Cancer stem cells are a very small amount (1%) of cells with self-renewal, unlimited proliferation and multi-directional di...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K19/00G01N33/68A61K47/64A61K47/42
CPCC07K14/001G01N33/6845A61K47/64A61K47/42C07K2319/00
Inventor 王蔚芝郭明媚
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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