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Alpha-helical polypeptide and application thereof

A helical and multipurpose technology, applied in the field of polypeptides and α-helical polypeptides, can solve the problems of poor anti-tumor effect and achieve the effect of inhibiting tumor growth

Active Publication Date: 2017-02-22
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the above-mentioned technical problems in the prior art, the present invention provides an α-helical polypeptide and its use. The α-helical polypeptide and its use are intended to solve the poor anti-tumor effect of the drugs in the prior art. technical issues

Method used

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  • Alpha-helical polypeptide and application thereof
  • Alpha-helical polypeptide and application thereof
  • Alpha-helical polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] MeR, PhR are dual inhibitors of MDM2 and MDMX and exhibit nuclear accumulation

[0047]According to the phage display technology, the MDM2 / MDMX dual inhibitor polypeptide PDI sequence LTFEHYWAQLTS was discovered, and based on the consideration of the membrane penetration of the polypeptide, the 4th E in the sequence was mutated to Q (PDI-1), thus simulating a series of template design The stable α-helical peptide sequence of p53. Considering that the side chain structure of the polypeptide often affects the hydrophobic pocket of MDM2 / MDMX binding, and keeping the amino acid of the action site unchanged, a series of i, i+4 stable conformation α-helical polypeptides (PDI 2-12) were designed and synthesized. Such as side chain construction at different positions, substitution of different side chain substituent structures (Me or Ph) at chiral positions, and amino acid mutations at different specific positions.

[0048] Preparation and separation and purification steps of ...

Embodiment 2

[0065] MeR, PhR activate p53 signaling in cancer cells

[0066] Inhibiting the combination of p53 protein and its negative regulatory proteins MDM2 and MDMX is the way to stabilize and activate p53 protein. Activation of the p53 pathway occurs only in cells expressing wild-type p53 protein, but not in cells with mutant forms of p53. In order to evaluate the activity of MeR and PhR synthesized in the above Example 1, two cancer cell lines overexpressing MDM2 (PA-1 ovarian teratoma) or MDMX (MCF-7 breast cancer) were selected as representatives. Meanwhile, two mutant p53 cell lines (SKOV3 and MDA-231) were selected to exclude the non-specific toxicity of MeR and PhR. And two normal cell lines (HEK293 and QSG7701), and take the same peptide treatment to exclude cytotoxicity. IC was detected by cell survival assay 50 , nutlin-3a was selected as a positive control.

[0067] Cell viability was determined by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylt-etrazolium bromide...

Embodiment 3

[0073] Reactivation of partial p53 cellular functions in tumor cells overexpressing MDM2 / MDMX

[0074] 1. The effect of CIH polypeptide on apoptosis

[0075]The tumor suppressor gene p53 has many cellular functions, the most important of which is the induction of apoptosis and cell cycle arrest. PA-1 and MCF-7 cells were treated with CIH polypeptide (40 μM) or nutlin-3a (5 μM) for 48 h, and Annexin-V / PI assay (BDPharmingenTM) was used to quantify the apoptotic effects of MeR and PhR. Cells were harvested, washed twice with cold PBS, and suspended in buffer. During the process of cell apoptosis, FITC was marked on the combination of Annexin-V and phosphatidylserine (PS), and the nuclei were stained with propidium iodide (PI). Stained cells were analyzed by flow cytometry to differentiate apoptotic cells. Cells with both FITC and PI fluorescence signal intensities represent apoptotic cell counts. Whereas the degree of apoptosis is measured by exposing cells to caspase-3 spec...

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Abstract

The invention discloses an alpha-helical polypeptide and application thereof. The amino acid sequence structure of the alpha-helical polypeptide is shown as the specifications. The invention further discloses the application of the alpha-helical polypeptide in preparing active medicine capable of inhibiting the growth of tumor stem cells. MDM2 / MDMX dual inhibitor polypeptide PDI sequences is adopted, with a formation method of alpha-helix by chiral induction and by experiments of fluorescence polarization detection, an immunoblotting analysis, a gene chip analysis, human-oriented tumor heterograft model and the like, it is proved that PhR can simulate the interaction of p53-MDM2 / MDMX through cell membrane penetration property and good binding affinity of the PhR and can reactivate an apoptosis pathway of the p53 and remove malignant CSC cells. The PhR is an efficient dual inhibitor active against both the MDM2 and the MDMX, can conduct chiral adjustment and control on molecular properties through polypeptide side chains, and inhibit the growth of tumor stem cells.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a polypeptide, specifically an α-helical polypeptide and its application. Background technique [0002] Cancer is one of the most incurable diseases in the world. Cancer stem cells (CSC) play a key role in the occurrence and development of tumors, and are considered to be closely related to tumor metastasis, drug resistance and recurrence after treatment. For most tumors, surgery, chemotherapy, radiotherapy and other means can only kill part of the tumor cells, but they cannot kill the tumor stem cells and cure the tumor fundamentally. More and more research is investing in the development of anti-CSC drugs, but due to the high expression of drug-resistant proteins on the surface of CSC membranes, CSC forms self-protection and high tolerance to existing tumor drugs, which is the main reason for the failure of current tumor treatment. one of the reasons. Therefore, its drug resistanc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C12N15/11A61K38/10A61P35/00
CPCA61K38/00C07K7/08
Inventor 李子刚胡宽尹丰孙程洁李文君
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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