Stable polypeptide protein covalent inhibitor targeting anti-apoptotic protein BFL-1

A BFL-1, anti-apoptotic protein technology, applied in the direction of peptide/protein components, medical preparations containing active ingredients, peptides, etc., can solve problems such as poor efficacy, broaden the scope of application, and solve drug resistance of tumor cells Effect

Inactive Publication Date: 2021-01-19
PEKING UNIV SHENZHEN GRADUATE SCHOOL +1
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Problems solved by technology

[0006] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a stable polypeptide protein covalent inhibitor targeting anti-apoptotic protein BFL-1, said targeting anti-apoptotic protein BFL-1 Stable polypeptide protein covalent inhibitors need to solve the technical problem that the drugs in the prior art are not effective in treating melanoma or pancreatic cancer

Method used

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  • Stable polypeptide protein covalent inhibitor targeting anti-apoptotic protein BFL-1
  • Stable polypeptide protein covalent inhibitor targeting anti-apoptotic protein BFL-1
  • Stable polypeptide protein covalent inhibitor targeting anti-apoptotic protein BFL-1

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Embodiment 1

[0029] The present invention adopts the sulfonium salt stabilized polypeptide technology reported in the previous literature (D.Wang, M.Yu, et al.Chem.Sci.10, 4966-4972), through the methionine, cysteine ​​and dialkylene on the polypeptide The sulfonium salt cyclic peptide can be reacted with an alkylation reagent, which can not only stabilize the polypeptide, but also covalently modify the cysteine ​​on the interaction site of the target protein, block the combination of anti-apoptotic proteins and pro-apoptotic proteins, and promote The release of apoptotic proteins promotes the apoptosis of tumor cells.

[0030] The inventors synthesized a number of different polypeptides, and focused on seven of them, as shown in Table 1. Then through the covalent reaction to BFL-1 to screen the polypeptide molecule with good covalent effect. Figure 11It is a schematic diagram of anti-tumor covalent polypeptide inhibitor promoting cell apoptosis through covalent reaction with BFL-1.

[...

Embodiment 2

[0035] Preparation and separation and purification steps of the polypeptide of embodiment 2:

[0036] According to the amino acid sequence of solid-phase synthesis of polypeptides, the core steps of preparing the above-mentioned stable polypeptides are as follows (taking B4-MC as an example):

[0037] Specific operation steps ( figure 1 )for:

[0038] (1) Polypeptide solid-phase synthesis: Weigh Rink amide MBHA resin into a peptide tube, add dichloromethane (DCM), and swell with nitrogen gas for 30 minutes. 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 alternately with DMF and DCM, the prepared Fmoc-AA-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea Fluorophosphate ester (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. Take out th...

Embodiment 3

[0041] Example 3 Polypeptide Molecule and Protein BFL-1 Covalent in Vitro

[0042] Methionine and cysteine ​​on different polypeptides react with dialkylating reagents to form sulfonium salt cyclic peptides and co-incubate with proteins respectively, and the protein and sulfonium salt polypeptides undergo a covalent reaction, and obvious upward shifting bands can be seen . The covalent reaction between protein and peptide is dose-dependent. The polypeptide has good specificity, and it mainly reacts covalently with the cysteine ​​at position C55 of BFL-1. The cysteine ​​at position 55 of BFL-1 is mutated to serine, and the polypeptide basically does not covalently interact with BFL-1. valence reaction. In addition, some proteins containing cysteine ​​were selected, and the sulfonium salt polypeptide did not covalently react with it ( figure 2 ).

[0043] The polypeptide B4-MC was selected to be co-incubated with the protein BFL-1, and the primary mass spectrometry verified...

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Abstract

The invention provides a stable polypeptide protein covalent inhibitor targeting an anti-apoptosis protein BFL-1. The amino acid sequence of the stable polypeptide protein covalent inhibitor is Ac-cyclic(MIAQC)LR(Aib)IGD(Aib)FNAYYARR, also provides application of the stable polypeptide protein covalent inhibitor in preparation of a drug for targeting the anti-apoptotic protein BFL-1, and further provides application in preparation of a drug for treating melanoma or pancreatic cancer with BFL-1 high-expression. According to the invention, the sulfonium salt cyclic peptide targeting BFL-1 is stabilized by adopting a method of reacting methionine-cysteine on a polypeptide with a dialkylating reagent to form a single sulfonium salt. Cell proliferation and apoptosis experiments also prove thatthe polypeptide kills tumor cells with high expression of BFL-1, such as melanoma cells A375 and pancreatic cancer cells Miapaca2, through an apoptosis pathway.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a polypeptide, specifically a stable polypeptide protein covalent inhibitor targeting anti-apoptosis protein BFL-1. Background technique [0002] China is a big cancer country in the world. In 2017, the number of new cases of malignant cancer in China was 3.8 million, and the number of cancer deaths was 260,000. According to the National Bureau of Statistics, malignant tumors have become one of the main causes of death in China. Currently, lymphoma is one of the fastest-growing malignant tumors in the world, and its incidence rate increases by an average of 4% every year. In recent years, the incidence of malignant lymphoma in my country has increased rapidly, and it has become one of the top ten malignant tumors in my country. The drug resistance and recurrence of malignant lymphoma have brought huge economic and psychological burdens to society and families, and also brought great ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08A61K38/10A61P35/00
CPCA61K38/00A61P35/00C07K7/08
Inventor 李子刚尹丰刘娜王冬园廉晨珊
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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