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A kind of small molecule polypeptide and its application in preparing medicine for preventing and treating ischemic stroke

A technology for ischemic stroke and small molecule peptides, applied in the field of medicine, can solve problems such as inappropriateness, achieve the effect of improving nervous system symptoms, high synthesis purity, reducing nerve cell death and brain damage

Active Publication Date: 2021-07-27
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since glutamate receptors play an important physiological role in normal neuronal activity, direct blockade of glutamate receptors is not appropriate for the treatment of ischemic stroke

Method used

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  • A kind of small molecule polypeptide and its application in preparing medicine for preventing and treating ischemic stroke
  • A kind of small molecule polypeptide and its application in preparing medicine for preventing and treating ischemic stroke
  • A kind of small molecule polypeptide and its application in preparing medicine for preventing and treating ischemic stroke

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Synthesis of TAT-CDK5-CTM

[0029] The sequence of TAT-CDK5-CTM is shown as SEQ ID NO.1, which was artificially synthesized by Jiangsu Qiangyao Biotechnology Co., Ltd. The synthesis report is as follows, and the chromatogram is as follows figure 1 shown.

[0030] TAT-CDK5-CTM synthetic HPLC report

[0031]

[0032] Table 1

[0033] wavelength time Peak area Peak Area Percentage (%) 1 220nm 7.310 17438 0.2265 2 220nm 9.067 55117 0.716 3 220nm 9.360 7404161 96.18 4 220nm 9.933 118775 1.543 5 220nm 11.207 38910 0.5055 6 220nm 12.239 63670 0.8271

[0034] The purity of the synthesized TAT-CDK5-CTM polypeptide is 96.18%, 1 mg per tube, in the form of white powder, completely soluble in water, sealed and protected from light, and stored at -20°C. Before use, dilute it with normal saline for injection according to the specified concentration, and prepare it for immediate use.

[0035] The contro...

Embodiment 2

[0038] TAT-CDK5-CTM blocks the combination of neuronal NR2B receptor and CDK5 under the condition of ischemia and hypoxia, and inhibits the neuronal apoptosis and necrosis caused by the downstream of CDK5.

[0039] The primary neurons on day 10 were cultured in vitro and treated with oxygen glucose deprivation (OGD) to mimic the cell model of ischemic stroke. After 90 minutes of OGD treatment, normal culture was performed for 2 hours, and 5uM TAT-CDK5-CTM polypeptide or control TAT-s-CDK5 polypeptide or vehicle was incubated. Cellular proteins were extracted after 2 hours. Anti-NR2B antibody was used to precipitate cellular protein, and then anti-CDK5 antibody was used to detect the precipitated protein. The black blot on the NC membrane was detected to indicate the interaction between NR2B and CDK5. The results showed that after administration of TAT-CDK5-CTM, the anti-CDK5 antibody could hardly detect the CDK5 protein on the NC membrane, suggesting that TAT-CDK5-CTM blocked...

Embodiment 3

[0041] Application of TAT-CDK5-CTM in cell model of ischemic stroke

[0042] (1) Construction of primary neuron culture and glucose and oxygen deprivation simulated ischemic stroke cell model

[0043] Fetal mice on the 18.5th day of the embryonic period were taken from the uterus of pregnant mice, and after decapitation, the prefrontal cortex of the brain was taken out in the dissecting solution (Hank's Balance Solution), and the cells were digested with 0.125% trypsin. Cells were planted on 20ng / ul poly-lysine and laminin-coated coverslips, and the coverslips were placed in 12-well plates with a cell planting density of 100-150 cells per square millimeter and medium without Serum Neurobasal medium (21103, gibco) was added with 2% B27, and the same medium was used for half replacement every three days, that is, half of the old medium was discarded and half of the fresh medium was added. Immunofluorescent labeling of the cultured cells with the neuron marker β-tubulin III (Tuj...

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Abstract

The invention discloses a small molecule polypeptide TAT‑CDK5‑CTM and its application in the preparation of drugs for preventing or treating ischemic stroke. By synthesizing the fusion protein TAT-CDK5-CTM of the TAT protein transduction domain, the protein polypeptide that can bind and inhibit CDK5, and the autophagy element CTM, TAT is used to carry the CDK5 protein polypeptide through the blood to transport through the blood-brain barrier, and taken up by neurons. Applying the peptide to ischemic stroke models in vitro and in vivo, it can effectively block the NR2B subunit of N-methylaspartate receptor (NMDAR) and cyclin-dependent protein kinase 5 (CDK5) binding, and the polypeptide molecule bound to CDK5 protein is mediated by CTM, enters the lysosome for degradation, inhibits neuronal apoptosis and necrosis caused by the downstream of CDK5, and reduces brain damage after ischemic stroke. Drug development for the treatment of ischemic stroke provides molecular targets.

Description

technical field [0001] The invention belongs to the field of medicine, relates to small molecular polypeptides and their medical applications, in particular to an artificially synthesized small molecular polypeptide TAT-CDK5-CTM, and the use of small molecular polypeptide TAT-CDK5-CTM in the preparation of prevention or treatment of ischemic stroke application in medicine. Background technique [0002] Ischemic stroke is a severe neurological disease in which cerebral blood flow is interrupted, causing thrombosis or embolism, leading to cerebral ischemia. Patients will suffer from paralysis, language barriers, vision loss and even death. Because of its high morbidity, high mortality and high disability rate, it is considered as a common refractory disease that seriously endangers human health and life safety. According to the 2016 Stroke Epidemiology Report, there are 70 million stroke patients in my country, 2 million new strokes each year, and 1.65 million stroke deaths e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00A61K38/10A61P9/10A61P25/00
CPCA61K38/00C07K7/08C07K2319/10
Inventor 朱铃强周雅帆刘丹汪晶邓曼菲
Owner HUAZHONG UNIV OF SCI & TECH
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