CN112194730A - Polypeptide TI-16 and application thereof - Google Patents

Polypeptide TI-16 and application thereof Download PDF

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CN112194730A
CN112194730A CN202011173540.1A CN202011173540A CN112194730A CN 112194730 A CN112194730 A CN 112194730A CN 202011173540 A CN202011173540 A CN 202011173540A CN 112194730 A CN112194730 A CN 112194730A
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polypeptide
seq
abeta
amino acids
amino acid
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CN112194730B (en
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郝丽英
杨琳林
孙旋旋
袁媛
魏郡
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China Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide TI-16 and application thereof. The polypeptide TI-16 has the amino acid sequence of SEQ ID No1, the N-terminal of the polypeptide TI-16 is connected with 11 amino acid leading cell penetrating peptides, the amino acid sequence of SEQ ID No.2 can carry the following 16 amino acids to penetrate cell membranes, and the following 16 amino acids SEQ ID No.3 are main bodies playing roles. The polypeptide is used for preparingThe application of the compound in medicaments for treating Alzheimer disease. The polypeptide can react with Abeta in vitro1‑42Binding and having a certain calcium concentration dependence; the polypeptide provided by the invention can penetrate cell membranes to enter nerve cells to inhibit A beta25‑35The resulting nerve cell damage; the polypeptide provided by the invention does not influence the nerve function, has a certain repairing effect on AD caused by Abeta, and lays a foundation for the treatment of AD.

Description

Polypeptide TI-16 and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide TI-16 and application thereof.
Background
Alzheimer's Disease (AD) is an senile neurodegenerative Disease that often results in impaired memory and other cognitive functions, and as the condition progresses, the patient gradually loses his ability to live, and in severe cases, even becomes life threatening. With the aging phenomenon of the social population becoming more and more serious, the incidence of AD increases year by year, and the AD becomes a great social public health problem. According to statistical data, the number of people suffering from AD in China is the first in the world, and the number of people dead due to AD is increased by 57.8% in 2005 to 2016. It is predicted that by 2050, 1 AD patient will occur every 33 seconds worldwide.
There are several hypotheses for the main pathological mechanism of AD, among which the theory of neurotoxic amyloid-beta toxicity dominates. A beta is prepared from Amyloid Precursor Protein (APP) by hydrolysis with beta-secretase and gamma-secretase, wherein the hydrolysis product comprises A beta1-40And Abeta1-42And A β1-42The brain is more prone to aggregate to form compact fiber plaques, and then neurotoxic cascade reaction is triggered, so that plaque formation, neurofibrillary tangle formation, tau protein abnormal phosphorylation, synapse loss and the like are caused, and finally, neuron death is caused. It is found that A beta25-35Is a region with strong amyloid toxicity, and has unique aggregation characteristics, so that short Abeta is often used for molding in experimental research.
Calmodulin (CaM) is a small molecule protein consisting of 148 amino acids and is highly conserved and ubiquitously expressed in all eukaryotes and cells. CaM is crucial in neuronal calcium signaling, is the major calcium binding protein involved in cytosolic calcium buffering, and is also involved in regulating other calcium signaling pathwaysA bond effector protein. The research finds that CaM is a beta peptide with neurotoxicity1-42One of the targets with the highest affinity to A β1-42The primary function of the interaction is to slow the formation of a β fibrils.
No specific therapeutic drugs for AD have been found, only five classes of drugs are approved for treatment, both acetylcholinesterase inhibitors and glutamate receptor antagonists, and only slow the symptoms and do not alter the disease process. Therefore, the search for a novel targeted drug capable of inhibiting the A beta cascade is very important in the treatment of AD, and the invention is based on the fact that CaM and A beta1-42Binding sites, and screening active peptide fragments as novel polypeptides, thereby providing a novel method for treating AD.
Research shows that the polypeptide can be used for inhibiting abnormal A beta deposition of neurons in brain, reducing apoptosis rate of neuron cells and the like, and can be widely applied in clinic. Therefore, the development of a targeted polypeptide medicament has an important effect on treating AD.
Disclosure of Invention
In view of the problems of the prior art, the present invention aims to provide a novel polypeptide TI-16 and the use thereof. The present invention establishes a polypeptide and attempts to treat AD due to abnormal deposition of a β, providing a polypeptide compound that can penetrate the cell membrane into neuronal cells and is stable in vivo. The invention designs a polypeptide compound TI-16 from CaM source, and the sequence and the length of a peptide fragment are determined by a computer-assisted molecular simulation technology; the cell experiment is utilized to observe the repairing effect of TI-16 on the neuronal cell damage caused by A beta; GST pull-down experiments are used for comparing the interaction between TI-16 and A beta under different calcium ion gradients, and the mechanism of the action of the polypeptide is discussed. The polypeptide can penetrate cell membrane to enter neuron cell, has effect in repairing neuron cell injury, and lays foundation for AD treatment.
In order to achieve the purpose, the invention adopts the following technical scheme.
A polypeptide TI-16, the amino acid sequence of which is SEQ ID No 1: YGRKKRRQRRRTMMARKMKDTDSEEEI, named TI-16.
The N-terminal of the polypeptide is connected with a leader membrane-penetrating peptide with 11 amino acids, and the amino acid sequence is SEQ ID No. 2: YGRKKRRQRRR, capable of carrying the next 16 amino acids to penetrate the cell membrane, the next 16 amino acids SEQ ID No. 3: TMMARKMKDTDSEEEI is the body that functions.
The polypeptide is prepared by a solid phase synthesis method.
The polypeptide is capable of reacting with A beta1-42Binding and having a certain calcium concentration dependence.
The polypeptide can penetrate cell membrane to enter nerve cell to inhibit A beta25-35Resulting in nerve cell damage.
A biologically active fragment or derivative comprising the polypeptide sequence of claim 1 as a core sequence, comprising a covalently linked compound and a multimeric mixture of core sequences.
A polynucleotide sequence encoding a polypeptide sequence comprising SEQ ID No.1 and SEQ ID No.3 and active fragments of the polypeptide of claim 4 and derivatives thereof.
The polypeptide, the bioactive fragment or derivative and/or the polynucleotide sequence are used for preparing a medicament for treating Alzheimer disease.
Further, the dosage form of the drug is any pharmaceutically therapeutically acceptable dosage form.
Further, the dosage of the drug is any therapeutically acceptable dosage of the drug.
Compared with the prior art, the invention has the following beneficial effects.
(1) Intracellular Ca2+Abnormal signaling, which can lead to a β accumulation and neurofibrillary tangle formation, is an important factor in early stage of neuronal degeneration. CaM is important calcium signal sensor and effector in brain, CaM and A beta1-42Has high affinity. The main function of their interaction is to slow the formation of a β fibrils. Simulation of Abeta by computer1-42The peptide segment can inhibit the damage of Abeta to nerve cells, regulate intracellular calcium signals to inhibit calcium overload, and treat ADAnd (4) acting.
(2) The polypeptide and the derivative thereof provided by the invention do not influence the nerve function, have a certain repairing effect on AD caused by Abeta, and lay a foundation for the treatment of AD.
Drawings
FIG. 1 is a MOE computer simulation of CaM and Abeta1-42The situation of binding.
FIG. 2 shows the purity of polypeptide TI-16 by HPLC.
FIG. 3 is an experimental demonstration of the polypeptides TI-16 and A.beta.with GST-Pull Down1-42The situation of binding.
FIG. 4 shows the polypeptide TI-16 vs. Abeta25-35Influence of cell survival when SH-SY5Y cells were induced to be injured. Data to
Figure BDA0002748055910000032
Represents P ≦ 0.05vs control group, ## P ≦ 0.001vs Abeta25-35And (4) grouping.
FIG. 5 is the polypeptide TI-16 vs. Abeta25-35Effect of SOD activity upon induction of SH-SY5Y cell damage. Data to
Figure BDA0002748055910000033
Figure BDA0002748055910000034
Represents a group of 0.01 vs. control, # P.ltoreq.0.05 vs. Abeta25-35And (4) grouping.
Detailed Description
The technical solutions and effects of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. The following examples are only preferred embodiments of the present invention and are not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art without departing from the spirit and the principle of the present invention, and any modifications, equivalents, improvements, etc. made within the scope of the present invention are intended to be covered by the present invention.
Example 1MOE computer simulation of CaM and Abeta1-42The situation of binding.
Simulation of in vivo CaM and Abeta by MOE software1-42Interaction of (1) with CaM as receptor, Abeta1-42And performing molecular docking as a ligand. Simulated binding was performed according to the virtual pocket of the receptor automatically screened by the software and the binding results were scored using the London G scoring function. Highest scoring CaM and Α β1-42The combination concept and combination parameters are shown in FIG. 1 and Table 1, and finally amino acids 70-85 of CaM and A beta can be determined1-42There are more binding sites.
TABLE 1 CaM and Abeta1-42And combining the parameters.
Figure BDA0002748055910000031
Example 2 solid phase Synthesis method to synthesize polypeptide TI-16.
The polypeptide TI-16: nanjing peptide Biotech Co., Ltd, Lot No: NJP 21915-200421.
The amino acid sequence of a polypeptide for treating AD is as follows: TI-16: YGRKKRRQRRRTMMARKMKDTDSEEEI MW: 3456.98. as shown in fig. 2 and table 2, the purity of the polypeptide TI-16 was (4382778/4529639) × 100% ═ 96.75%, as determined by HPLC analysis.
Table 2 TI-16HPLC assay related parameters.
Figure BDA0002748055910000041
EXAMPLE 3 Polypeptides TI-16 and A β1-42Protein interactions.
TI-16 and Abeta discussed by in vitro protein binding experiment1-42The combination of (1). As shown in FIGS. 3A and 3B, successful detection of TI-16 and A β1-42Combination of ([ Ca2 +)]Free, 2 mM). The final concentration of the CaM protein in the system is (in mu mol/L: 0.1, 0.3, 1.0, 3.0, 5.0, 10.0) and GST-Abeta1-42Fusion protein at 2mmol/L and Free [ Ca ]2+]Co-incubation under conditions pThe results of ul down assay are shown in FIGS. 3A and 3B, where A is 2mM calcium bound to SDS-PAGE under Free calcium. The CaM protein can be combined with GST-Abeta 1-42 protein, and the combination amount of the two is increased along with the increase of the concentration of the CaM protein. B: the binding curves of different concentrations of CaM protein to GST-Abeta 1-42 protein under the conditions of 2mM calcium ion and Free calcium ion are fitted. From the fitted curve, it can be seen that both binding is concentration dependent and binds more under 2mM calcium than under no calcium.
Example 4 polypeptide TI-16 vs. Abeta25-35Induce the protective effect of SH-SY5Y cell injury.
1. Effect of the polypeptide TI-16 on the cell survival rate.
The CCK8 method is adopted to detect the cell activity and observe the polypeptide TI-16 to A beta25-35SH-SY5Y cell activity after aging treatment. As shown in FIG. 4, A β25-35The cell viability of the group, the single TI-16 group and the TI-16 intervention treatment group is 83.40%, 105.44%, 90.79% and 114.39% of that of the control group respectively. Compared with the control group, the pure TI-16 group has no obvious change in cell viability. And with Abeta25-35Group comparison, Abeta25-35The cell viability of the + TI-16 (8. mu.M) group was significantly increased (P)<0.001). Data to
Figure BDA0002748055910000042
Represents P ≦ 0.05vs control group, ## P ≦ 0.001vs Abeta25-35And (4) grouping.
2. Effect of SOD Activity upon injury of the polypeptide TI-16 cells.
After cells were sonicated, A.beta.was measured using SOD kit25-35Induce SH-SY5Y cell damage SOD value. As shown in FIG. 5, A β25-35The SOD activity of the cells of the group and the TI-16 intervention treatment group is respectively 80.74 percent, 98.37 percent and 93.85 percent of that of the control group. Abeta compared with control group25-35The SOD activity of the group cells is obviously reduced, and the statistical difference (P) is obvious<0.01). And Abeta25-35Compared with the group, the SOD activities of the cells of the TI-16 intervention treatment group are obviously improved (P)<0.05). Data to
Figure BDA0002748055910000043
Represents a group of 0.01 vs. control, # P.ltoreq.0.05 vs. Abeta25-35And (4) grouping.
Sequence listing
<110> university of Chinese medical science
<120> a polypeptide TI-16 and uses thereof
<130> 3
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 27
<212> PRT
<213> Artificial sequence
<400> 1
TYR GLY ARG LYS LYS ARG ARG GLN ARG ARG ARG THR MET MET ALA ARG LYS MET LYS ASP THR ASP SER GLU GLU GLU ILE
<210> 2
<211> 11
<212> PRT
<213> Artificial sequence
<400> 2
TYR GLY ARG LYS LYS ARG ARG GLN ARG ARG ARG
<210> 3
<211> 16
<212> PRT
<213> Artificial sequence
<400> 3
THR MET MET ALA ARG LYS MET LYS ASP THR ASP SER GLU GLU GLU ILE

Claims (10)

1. A polypeptide TI-16, wherein the amino acid sequence of said polypeptide is SEQ ID No 1: YGRKKRRQRRRTMMARKMKDTDSEEEI, named TI-16.
2. The polypeptide TI-16 as claimed in claim 1, wherein the N-terminus of said polypeptide is linked to a leader membrane-penetrating peptide of 11 amino acids having the amino acid sequence of SEQ ID No. 2: YGRKKRRQRRR, capable of carrying the next 16 amino acids to penetrate the cell membrane, the next 16 amino acids SEQ ID No. 3: TMMARKMKDTDSEEEI is the body that functions.
3. The polypeptide TI-16 of claim 1, wherein said polypeptide is produced by solid phase synthesis.
4. The polypeptide TI-16 of claim 1, wherein the polypeptide is capable of binding to A β1-42Binding and having a certain calcium concentration dependence.
5. The polypeptide TI-16 of claim 1, wherein said polypeptide permeates cell membranes into nerve cells and inhibits a β25-35Resulting in nerve cell damage.
6. A biologically active fragment or derivative comprising the polypeptide sequence of claim 1 as a core sequence, comprising a covalently linked compound and a multimeric mixture of core sequences.
7. A polynucleotide sequence encoding a polypeptide sequence comprising SEQ ID No.1 and SEQ ID No.3 and active fragments of the polypeptide of claim 4 and derivatives thereof.
8. Use of the polypeptide TI-16 according to claim 1, the biologically active fragment or derivative according to claim 6 and/or the polynucleotide sequence according to claim 7 for the preparation of a medicament for the treatment of alzheimer's disease.
9. The use of claim 8, wherein the medicament is in a dosage form that is any pharmaceutically therapeutically acceptable dosage form.
10. The use of claim 8, wherein the dose of the medicament is any pharmaceutically therapeutically acceptable dose.
CN202011173540.1A 2020-10-28 2020-10-28 Polypeptide TI-16 and application thereof Active CN112194730B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118001370A (en) * 2024-04-10 2024-05-10 北京汉氏联合生物技术股份有限公司 Compositions and methods for treating neurodegenerative diseases

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105175494A (en) * 2015-09-12 2015-12-23 复旦大学 Polypeptide capable of inhibiting AD (Alzheimer's disease) protein Abeta (beta-amyloid) accumulation activity and application thereof
CN108715601A (en) * 2018-05-24 2018-10-30 华南理工大学 It is a kind of while there is polypeptides that are anti-oxidant and inhibiting 42 aggregation properties of A β and its application and the gene that encodes the polypeptide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105175494A (en) * 2015-09-12 2015-12-23 复旦大学 Polypeptide capable of inhibiting AD (Alzheimer's disease) protein Abeta (beta-amyloid) accumulation activity and application thereof
CN108715601A (en) * 2018-05-24 2018-10-30 华南理工大学 It is a kind of while there is polypeptides that are anti-oxidant and inhibiting 42 aggregation properties of A β and its application and the gene that encodes the polypeptide

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118001370A (en) * 2024-04-10 2024-05-10 北京汉氏联合生物技术股份有限公司 Compositions and methods for treating neurodegenerative diseases

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