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Polypeptide with PINK1 kinase agonist activity and application thereof

An active, dl-pt-1 technology, applied in the field of biochemistry, can solve the problems of clinical application limitations and achieve non-cytotoxic effects

Pending Publication Date: 2022-04-01
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these drugs cannot fundamentally improve the degenerative process of dopaminergic neurons, and their clinical applications are limited

Method used

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  • Polypeptide with PINK1 kinase agonist activity and application thereof
  • Polypeptide with PINK1 kinase agonist activity and application thereof
  • Polypeptide with PINK1 kinase agonist activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Separation and purification of polypeptide components

[0026] 1. Guided by the determination of PINK1 enzyme activity, five polypeptide components with anti-Parkinson potential activity were isolated and purified from the extract of Earthworm. The specific separation method is as follows: the dried earthworm is crushed and soaked in 80% methanol for extraction, and the extract is concentrated under reduced pressure to obtain the earthworm extract. Sephadex LH-20 gel column was used to separate the earthworm extract by column chromatography to obtain four fractions: DL-A, DL-B, DL-C and DL-D; the DL-B fraction has PINK1 agonistic activity, Therefore, it is further separated by C-18 reverse phase column chromatography to obtain DL-B-1, DL-B-2, DL-B-3, DL-B-4, DL-B-5, DL-B-6 , DL-B-7, DL-B-8, DL-B-9 and DL-B-10 are ten parts in total; the DL-B-8 part has PINK1 agonistic activity, so preparative HPLC (C- 18 column) to separate and purify it, and finally obtain...

Embodiment 2

[0037] Example 2: Structural Identification of Polypeptide Components

[0038] 1. Analyze the amino acid sequence of DL-B-8-c obtained in Example 1 by using a high-resolution liquid-mass spectrometry system (Q-Exactive-MS / MS), and analyze the mass spectrometry data with Proteomo Discoverver 1.4 and Peaks Pnovo , and using the polypeptide and protein sequence library of Dilong as the reference file for searching, 6 polypeptides were identified from DL-B-8-c, and the sequences are shown in Table 2. According to these six amino acid sequences, corresponding polypeptides were synthesized for subsequent testing of PINK1 agonistic activity and anti-PD activity.

[0039] Table 2 Comparison table of sequencing results of each polypeptide

[0040] polypeptide amino acid sequence Molecular weight (Da) DL-Pt-1 SEQ ID NO.1 610.4166 DL-Pt-2 SEQ ID NO.2 639.4319 DL-Pt-3 SEQ ID NO.3 1528.7271 DL-Pt-4 SEQ ID NO.4 1501.7512 DL-Pt-5 SEQ ID N...

Embodiment 3

[0045] Example 3: Cytotoxic activity test of polypeptide DL-Pt-6

[0046] Dilute the SH-SY5Y cell suspension at 1×10 4 The density of each well was inoculated in a 96-well plate, and 100 μL of complete medium (DMEM high-glucose medium containing 1% streptomycin / penicillin double antibody, 10% fetal bovine serum FBS) was added to each well, and the cell culture incubator ( 37°C, 5% CO 2 ) for 24 hours. After the cells enter the logarithmic growth phase (adhesive growth to 70%-80%), replace the medium with 100 μL of complete culture containing different concentrations of drugs (0.01, 0.05, 0.1, 0.5 μM DL-Pt-6) base, and continue to culture for 24 h. After the cultivation, the CCK8 kit was used to detect the absorbance at 450 nm, and the cell survival rate was calculated.

[0047] The formula for calculating the cell survival rate is: cell survival rate (%)=[(A-B) / (C-D)]×100%. Wherein A is the absorbance of the drug group (containing cells and the complete medium containing ...

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Abstract

The invention belongs to the field of biochemistry, and particularly relates to a polypeptide with PINK1 kinase agonist activity and application thereof. According to the technical scheme, the polypeptide or the polypeptide mixture comprises polypeptide DL-Pt-6, and the amino acid sequence of the DL-Pt-6 is as shown in SEQ ID NO. 6. A series of polypeptide compounds are separated and identified from the earthworm extract, DL-Pt-1, DL-Pt-3, DL-Pt-5 and DL-Pt-6 all have PINK1 agonist activity, the PINK1 agonist rate of DL-Pt-6 is the highest, cytotoxicity is avoided, and the polypeptide compounds have the remarkable effect of saving MPP + damaged nerve cells and have the prospect of preparing the anti-Parkinson's disease medicine.

Description

technical field [0001] The invention belongs to the field of biochemistry, and specifically relates to a polypeptide with PINK1 kinase activating activity and application thereof. Background technique [0002] Parkinson's disease (PD), also known as essential parkinsonism, is the second most common chronic degenerative disease of the central nervous system in the world. The pathological feature of PD is the progressive degeneration of dopaminergic neurons in the substantia nigra compacta, and a significant decrease in dopamine levels in the striatum. The pathogenesis of PD is complex and has not yet been elucidated; the existing anti-PD drugs are mainly divided into two categories: drugs affecting dopaminergic neurons and anticholinergic drugs. Among them, the representative drug affecting dopaminergic neurons is levodopa, which can be catalyzed by dopa decarboxylase in the brain and converted into dopamine, so as to realize the supplement of dopamine and produce therapeuti...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K7/06C12N9/12A61K38/08A61K38/10A61P25/16
CPCY02A50/30
Inventor 廖循袁韫聪白小琳何柯杭
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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