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Stable polypeptide protein-targeted chimeric molecule, preparation method and application thereof

A polypeptide and protein target technology, applied in the field of bioengineering, can solve the problems of poor breast cancer effect and easy drug resistance, and achieve the effect of significant technological progress, good degradation ability and good proliferation

Active Publication Date: 2018-05-18
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] (1) Aiming at the above-mentioned technical problems in the prior art, the present invention provides a stable polypeptide protein-targeting chimera molecule and its preparation method and application. The stable polypeptide protein-target chimera molecule and Its preparation method and use should solve the technical problems that the drugs in the prior art have poor effect in treating breast cancer and are prone to drug resistance

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  • Stable polypeptide protein-targeted chimeric molecule, preparation method and application thereof
  • Stable polypeptide protein-targeted chimeric molecule, preparation method and application thereof
  • Stable polypeptide protein-targeted chimeric molecule, preparation method and application thereof

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Embodiment 1

[0038] The present invention adopts protein targeting chimera technology, and a pentapeptide (LAP(OH)YI) that can bind to VonHippel-Lindau (VHL) E3 ubiquitin ligase and a polypeptide (TD) that can target ERα are combined through a linking group. -PERM) to design peptides. Then recruit VHL ubiquitin ligase, and then ubiquitinate ERα, degrade ERα by starting proteasome, and then affect the downstream pathway of ERα, and inhibit the proliferation ability of breast cancer cell lines with high expression of ERα.

[0039] Such as Figure 21 As shown, a stable helical polypeptide (TD-PERM) targeting ERa was obtained by connecting chiral diacid-modified polypeptides at the end side chain-tail end, and then combined with a Von Hippel-Lindau (VHL) E3 ubiquitin ligase A stable polypeptide protein targeting chimera molecule (TD-PROTAC) targeting estrogen receptor alpha obtained by linking the pentapeptide (LAP(OH)YI) to degrade ERα protein through the ubiquitin-proteasome pathway.

[00...

Embodiment 2

[0045] In order to better carry out the following research, the design of the control peptide is necessary. As shown in Table 2, we designed a series of control polypeptides. Our research group previously developed an estrogen receptor regulatory polypeptide stabilized by the terminal aspartic acid strategy and named it TD-PERM, and the pentapeptide that binds to VHL E3 ubiquitin ligase was named HIF. In addition, we scrambled the TD-PERM sequence to obtain the control polypeptide TD-PROTACsc, and mutated the key amino acid residues in the HIF polypeptide that bind to VHL E3 ligase into alanine to obtain the control polypeptide TD-PROTACmut. In addition, we also synthesized a peptide PROTAClinear that did not use the ring-closing strategy.

[0046] Table 2: TD-PROTAC and its control polypeptide sequence (peptides without Tyr residues in the sequence are introduced into Try at the nitrogen end to determine the concentration of the polypeptide. For fluorescence polarization exp...

Embodiment 3

[0048] Preparation and separation and purification steps of the polypeptide of embodiment 3:

[0049] According to the amino acid sequence of solid-phase synthesis of polypeptides, the core steps of preparing the above stable polypeptides are as follows (TD-PROTAC as an example):

[0050]

[0051] The specific operation steps are:

[0052] (1) Polypeptide solid-phase synthesis: Weigh 100 mg Rink amide MBHA resin into a 10 ml peptide tube, add dichloromethane (DCM), and swell with nitrogen gas for 30 min. Add 50% (v / v) morpholine in N,N-dimethylformamide (DMF) solution, blow nitrogen gas for 30 minutes, and remove the Fmoc protecting group. After washing the resin 10 times alternately with DMF and DCM, the prepared (1) Fmoc-Ile-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1,1,3,3-tetramethyl Urea hexafluorophosphate (HCTU) (5eq, 0.38M, DMF) solution and N,N-diisopropylethylamine (DIPEA) (10eq) were mixed well, then added to the resin and blown with nitrogen for 1h. ...

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Abstract

The invention provides a stable polypeptide protein-targeted chimeric molecule with an amino-acid sequence structure being described in the specification. The invention also provides an application ofthe stable polypeptide protein-targeted chimeric molecule in degrading ERalpha protein. The invention also provides an application of the stable polypeptide protein-targeted chimeric molecule in preparing a medicine for treating breast cancer. The invention also provides a preparation method of the stable polypeptide protein-targeted chimeric molecule. Proved by a fluorescent quantitative PCR (Polymerase Chain Reaction) experiment, the stable polypeptide protein-targeted chimeric molecule provided by the invention has the advantage that the expression of downstream genes of the ERalpha can beinhibited. Also proved by a cell proliferation experiment and cell apoptosis experiment, the polypeptide can kill breast cancer cells by a apoptosis pathway.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a polypeptide, in particular to a stable polypeptide-like protein-targeted chimera molecule and its preparation method and application. Background technique [0002] Breast cancer has become a major public health problem in the current society. 99% of breast cancers occur in women and only 1% in men. The global incidence of breast cancer has been on the rise since the late 1970s. In recent years, the growth rate of the incidence of breast cancer in my country is 1-2 percentage points higher than that of high-incidence countries, and the incidence of breast cancer in the national tumor registration area ranks first among female malignant tumors. Estrogen receptor (ERα) is highly expressed in 70% of breast cancers. The growth of estrogen receptor-positive cancer cells depends on estrogen. They can be treated with estrogen antagonist drugs such as tamoxifen, and usually have a bette...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K38/10A61P35/00
CPCA61K38/00C07K7/08
Inventor 李子刚蒋艳红尹丰
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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