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Tubulin polymerization agent polypeptide and application thereof

A tubulin and polymerizing agent technology, applied in the fields of peptide/protein components, peptides, drug combinations, etc., can solve the problem of lack of specificity of small molecular polymerizing agents, so as to increase the survival rate, improve the survival rate, and promote the polymerization activity. Effect

Active Publication Date: 2013-09-11
南京利康医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many successful small-molecule polymerizers can promote the activity of tubulin at the nanomolar level, but small-molecule polymerizers lack specificity. If the lack of specificity of tubulin polymerizers is used for a long time in chronic diseases, large side effect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Effect of the tubulin polymerizing agent polypeptide 8 on tubulin polymerization and depolymerization in vitro.

[0018] Take fresh bovine brain tissue, peel off the meninges and large blood vessels, cut into pieces, wash 1-2 times with cold MES buffer, add MES buffer at a ratio of 0.5-1ml per gram of brain tissue, at 4°C, use electric Homogenize with a homogenizer; centrifuge at 105,000 g for 1 hour at 4°C, take the supernatant, add an equal volume of microtube polymerization buffer, and incubate in a water bath at 37°C for 30 minutes. Centrifuge at 105,000g at 26°C for 1 hour, take the precipitate, add about 1 / 10 of the homogenate volume of cold MES buffer, stir gently or crush the precipitate with a homogenizer; place the suspension in an ice bath for 30 minutes to dissolve the precipitate completely. Protein content was determined by Lowry's method (SDS polyamide gel electrophoresis). Tubulin was diluted to 4-5mg / ml with MES buffer and stored in liquid nitrogen. T...

Embodiment 2

[0022] IC50 of tubulin polymerizing agent polypeptide 8 on the growth and survival of tumor cells cultured in vitro.

[0023] Using the MTT colorimetric method. The logarithmic growth of U937 cells was added to a 96-well culture plate at 1.0×105, and cultured for 24 hours. Different concentrations of the experimental drug tubulin polymerizer polypeptide 8 and the positive control drug paclitaxel were added to the experimental wells and positive drug control wells; blank The same volume of solvent was added to each group. Set up five duplicate wells per well, culture for 48h, add MTT to each well at 0h, 2h, 8h, 14h, 20h, 24h, 36h, and 48h respectively, add DMSO after 4h, incubate for 30min, and incubate at 620nm in a microplate reader. Measure the absorbance A value, according to the formula tumor cell growth inhibition rate=(1-absorbance value of the experimental group / absorbance value of the control group)×100%. The calculated IC50 of the test drug was 4.66 μM.

Embodiment 3

[0025] In vivo activity of tubulin polymerizing agent polypeptide 8 was tested in a tumor model.

[0026] The U937 tumor model was established, and the positive control drug paclitaxel was added; the same volume of solvent was added to the blank group, and 3 doses were set to the experimental group: 0.75, 1.5 μM, and 3 μM mg / Kg. After 21 days, the number of surviving mice was observed, and the survival rate was calculated. The results showed that the tubulin polymerizing agent polypeptide 8 could effectively protect mice and improve the survival rate of tumor-bearing mice, with the survival rate reaching 81.4%.

[0027]

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PUM

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Abstract

The invention relates to the field of medicaments, and in particular relates to a polypeptide which can promote the polymerization of tubulin and treat acute lymphocytic leukemia. The sequence of the polypeptide is EVVPFQEVWGRSYCRA and is a new sequence; the polymerization of the tubulin can be promoted by 1 micromolar level of the polypeptide in vitro, and the survival rate of tumor-bearing mice can be increased in vivo experiments; and therefore, the polypeptide has a potential new medicament development value.

Description

technical field [0001] The present invention relates to tubulin polymerizing agent polypeptide 8 and its application, in particular to a polypeptide capable of promoting tubulin polymerization and treating acute lymphocytic leukemia. Background technique [0002] Acute lymphoblastic leukemia (ALL) is a progressive malignant disease characterized by large numbers of immature white blood cells that resemble lymphoblasts. These cells can be found in the blood, bone marrow, lymph nodes, spleen and other organs. Acute lymphoblastic leukemia accounts for 80% of acute leukemia in children, and the peak incidence is between 3 and 7 years old. ALL can also occur in adults and accounts for 20% of all adult leukemias. In recent years, with the deepening of medical research, great progress has been made in the understanding and treatment of acute lymphoblastic leukemia. Among them, tubulin plays an important role in acute lymphoblastic leukemia. [0003] Microtubules are the main co...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61P35/02
Inventor 罗瑞雪
Owner 南京利康医药科技有限公司
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