Crtc2/Creb compound blocking polypeptide and derivative medicinal polypeptide and application thereof

A compound and drug technology, which is applied to Crtc2/Creb complex blocking polypeptide and its derivative drug polypeptide and application fields, can solve the problems affecting the quality of life and longevity of patients, and achieve the goal of regulating blood sugar, regulating hepatic glucose and lipid metabolism, and achieving high-efficiency The effect of transmembrane efficiency

Active Publication Date: 2019-04-23
上海锐赛生物科技(集团)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These diseases not only seriously affect the quality of life and longevity of pa

Method used

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  • Crtc2/Creb compound blocking polypeptide and derivative medicinal polypeptide and application thereof
  • Crtc2/Creb compound blocking polypeptide and derivative medicinal polypeptide and application thereof
  • Crtc2/Creb compound blocking polypeptide and derivative medicinal polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Design and Synthesis of Polypeptide Molecules

[0023] Analyze the existing protein structure information of CRTC2 and CREB through bioinformatics, use the computer molecular docking software to analyze the binding domain and key amino acid residues of CRTC2-CREB, and then use the polypeptide-protein molecular docking software to analyze and design a high-affinity binding RBBp4 Protein and block the polypeptide molecule of SALL4-RBBp4 interaction, through screening to obtain the polypeptide that specifically targets and inhibits the formation of Crtc2 / Creb complex, the amino acid sequence is shown in SEQ ID NO: 1, hereinafter referred to as CCB-17.

[0024] Screening a membrane-penetrating peptide, the amino acid sequence of which is shown in SEQ ID NO:2, is used to guide the above-mentioned CCB-17 to efficiently enter the cytoplasm and nucleus. Design a drug polypeptide, hereinafter referred to as pCCB-17, including the penetrating peptide sequence and the C...

Embodiment 2

[0026] Example 2: Polypeptide membrane penetration and cell localization detection

[0027] 1. Resuscitate and culture LO2 cells.

[0028] 2. Inoculate the cells into a 96-well plate according to the appropriate number of cells, and inoculate three replicate wells for each cell strain.

[0029] 3. After 6 hours of cell inoculation, add the test polypeptide with a final concentration of 30uM.

[0030] 4. Record the FITC fluorescence of the cells after 24 hours of polypeptide treatment, and evaluate the membrane penetration efficiency and intracellular localization of the polypeptide.

[0031] The test results show that the drug polypeptide pCCB-17 can efficiently penetrate the cell membrane, and is located in the cytoplasm and nucleus in the cell. The test results are as follows: figure 1 shown.

Embodiment 3

[0032] Example 3: Co-immunoprecipitation detection of the blocking of the Crtc2 / Creb complex by the drug polypeptide pCCB-17

[0033] 1. Resuscitate and culture LO2 cells.

[0034] 2. Treat LO2 cells with a final concentration of 30uM membrane-penetrating peptide and drug polypeptide pCCB-17 for 24 hours, remove the medium, wash twice with PBS, centrifuge at 1000rpm for 5 minutes, and collect the cells.

[0035] 3. Suspend the cells in RIPA buffer, incubate on ice for 30 minutes, and lyse the cells with a sonicator (30s on, 30s off, 5 cycles).

[0036] 4. The lysate was centrifuged at 13,000 rpm for 10 min, and the supernatant was collected for protein quantification.

[0037] 5. Take the lysate of 1mg of total protein and incubate with 3ug CRTC2 antibody (abcam # ab109081) at 4°C and shake overnight.

[0038] 6. Add 50ul Protein A (Dynabeads®) to incubate for 1 hour, and wash the magnetic beads 6 times with IP buffer.

[0039] 7. Elute with 2X SDS buffer and incubate the p...

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Abstract

The invention relates to Crtc2/Creb compound blocking polypeptide and derivative medicinal polypeptide and application thereof. The amino acid sequence of the Crtc2/Creb compound blocking polypeptideis shown in SEQ ID NO:1, and the Crtc2/Creb compound blocking polypeptide can be used for preparing drugs for treating obesity, fatty liver, hyperlipemia and type-2 diabetes. The medicinal polypeptidecomprises a blocking function structure area and a membrane penetrating function structure area which are formed by a targeted-inhibition Crtc2/Creb compound, wherein the amino acid sequence of the blocking function structure area formed by targeted-inhibition Crtc2/Creb compound is shown in SEQ ID NO:1. The medicinal polypeptide has obvious effects on adjustment of blood sugar and blood fat andinhibition of liver lipid deposition and can be used as a treatment drug for treating metabolic diseases such as obesity, fatty liver and type-2 diabetes alone or in other auxiliary treatment modes.

Description

technical field [0001] The present invention relates to the field of disease treatment related to abnormal lipid metabolism, and more particularly relates to Crtc2 / Creb complex blocking polypeptide and its derivative drug polypeptide and application. Background technique [0002] With the rapid development of the national economy, the living standards of the Chinese people have been greatly improved. At the same time, the fast-paced lifestyle also greatly affects the diet structure of Chinese people. The high-sugar and high-fat fast food diet and the decrease in exercise frequency have brought about an increase in the incidence of metabolic diseases, mainly including obesity, fatty liver disease, hyperlipidemia, and diabetes. These diseases not only seriously affect the quality of life and longevity of patients, but also bring a huge economic burden to patients' families and society. Effective prevention and treatment of metabolic diseases is a problem that we need to solv...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K19/00A61K38/08A61P3/04A61P3/10A61P1/16
CPCA61P1/16A61P3/04A61P3/10C07K7/08A61K38/00C07K2319/10
Inventor 张腾罗卫峰谭圆圆丁越韩慧
Owner 上海锐赛生物科技(集团)有限公司
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