CN108409839B - Polypeptide inhibitor for interaction of MDM2 and p53 and application thereof - Google Patents
Polypeptide inhibitor for interaction of MDM2 and p53 and application thereof Download PDFInfo
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- CN108409839B CN108409839B CN201810149254.8A CN201810149254A CN108409839B CN 108409839 B CN108409839 B CN 108409839B CN 201810149254 A CN201810149254 A CN 201810149254A CN 108409839 B CN108409839 B CN 108409839B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a polypeptide inhibitor of the interaction of MDM2 and p53 and application thereof. The polypeptide protected by the invention is shown as a sequence 1 in a sequence table. The polypeptide is derived from Zika virus and has the function of a p53 protein-MDM 2 protein interaction inhibitor, namely the polypeptide can be effectively combined with MDM2 protein in an organism, so that the concentration of the cancer suppressor protein p53 protein in the organism is improved, and then the formation or growth of tumor cells can be effectively inhibited by accumulating more p53 protein in the organism, and finally the aim of inhibiting cancer is fulfilled. The invention has important significance for treating tumors.
Description
Technical Field
The invention relates to a polypeptide inhibitor of the interaction of MDM2 and p53 and application thereof.
Background
Among the various diseases of human beings, the death rate caused by malignant tumor is always high and is one of the most major diseases endangering human health. Therefore, the research on the mechanism of tumor formation, especially the therapeutic method of tumor, has become a very important field crossed with multiple disciplines such as biology, physics, chemistry and medicine.
In human cancers, either the tumor suppressor gene p53 is mutated or completely deleted and fails to synthesize a functional p53 protein, or the binding of MDM2 protein to p53 protein inhibits the activity of p 53. Therefore, designing a drug molecule capable of blocking the binding of MDM2 protein and p53 protein to achieve the goal of treating cancer is a preferred approach for cancer treatment at present. The drug molecule can specifically bind to MDM2 protein to treat tumor, and has no toxic and side effects.
The p53 gene was discovered in 1976, and it was considered that the p53 gene was a oncogene of SV virus. Later, intensive research shows that the p53 gene is a strong and effective cancer suppressor gene. The p53 gene is a tumor suppressor gene which is widely researched, and the p53 gene is ubiquitous in human tumors, such as p53 cancer suppressor gene in common human tumors including brain tumor, sarcoma, lung cancer, rectal cancer, breast cancer, microcephaly and the like. Expression of the p53 gene can drive the cell cycle into a arrest phase, after which the cell can further age or die. Inactivation or abnormality of the p53 gene can lead to tumor formation or accelerate the growth of tumor cells. The p53 protein is the primary "fort" of the cell's defense against DNA damage and is often referred to as the "gene guardian".
The MDM2 protein is an important down-regulation suppressor of the p53 protein and is often overexpressed in most tumor cells. The expression of the p53 protein can promote the expression of the MDM2 protein, but the over-expression of the MDM2 protein can inhibit the expression and the function of the p53 protein, and a negative feedback loop is formed between the two. When p53 protein interacts with MDM2 protein, it leads to the reduction of the activity of p53 protein or to the degradation of p53 protein, and thus the cancer-suppressing action is reduced or tumor formation is caused. Therefore, the p53 protein molecule can be released by a method of binding the p53 protein-MDM 2 protein interaction inhibitor to the binding cavity of MDM2 protein, thereby achieving the purpose of inhibiting tumor formation or weakening the growth of tumor cells.
Zika virus, dengue virus and West Nile virus belong to the genus flavivirus, Zika virus is transmitted by mosquito as a vector and threatens all places of the world. Most notably, Zika virus causes microcephaly and has serious influence on infants. Zika virus causes severe brain damage after birth in infants, but it is known or available to combat adult brain tumors.
Disclosure of Invention
The invention aims to provide a polypeptide inhibitor for interaction of MDM2 and p53 and application thereof.
The invention provides a polypeptide, which is derived from Zika virus and is shown as a sequence 1 in a sequence table.
The invention protects the application of the polypeptide in preparing products; the application of the product is (a1) or (a2) or (a3) or (a 4):
(a1) binding to MDM2 protein in cells;
(a2) reducing the level of free MDM2 protein in the cell;
(a3) increasing the expression level and/or activity of p53 protein in the cell;
(a4) inhibiting binding of MDM2 protein and p53 protein in cells.
The cells are in particular tumor cells.
The invention also protects the application of the polypeptide in preparing products; the application of the product is (b1) or (b 2):
(b1) inducing apoptosis of tumor cells;
(b2) reducing the activity of tumor cells.
The invention also protects the application of the polypeptide in preparing products; the application of the product is (c1) or (c 2):
(c1) inhibiting tumor growth;
(c2) treating tumor.
The invention also protects a product, the active ingredient of which is the polypeptide; the application of the product is (a1) or (a2) or (a3) or (a 4):
(a1) binding to MDM2 protein in cells;
(a2) reducing the level of free MDM2 protein in the cell;
(a3) increasing the expression level and/or activity of p53 protein in the cell;
(a4) inhibiting binding of MDM2 protein and p53 protein in cells.
The cells are in particular tumor cells.
The invention also protects a product, the active ingredient of which is the polypeptide; the application of the product is (b1) or (b 2):
(b1) inducing apoptosis of tumor cells;
(b2) reducing the activity of tumor cells.
The invention also protects a product, the active ingredient of which is the polypeptide; the application of the product is (c1) or (c 2):
(c1) inhibiting tumor growth;
(c2) treating tumor.
Any of the above products may specifically be a medicament.
The invention also protects the application of the polypeptide, which is any one of the following (1) to (8):
(1) binding to MDM2 protein in cells;
(2) reducing the level of free MDM2 protein in the cell;
(3) increasing the expression level and/or activity of p53 protein in the cell;
(4) inhibiting binding of MDM2 protein and p53 protein in cells.
(5) Inducing apoptosis of tumor cells;
(6) reducing the activity of tumor cells.
(7) Inhibiting tumor growth;
(8) treating tumor.
In the above (1) to (4), the cell is specifically a tumor cell.
Any of the above tumor cells may specifically be brain tumor cells, more specifically, brain glioma cells (e.g., U87 cells).
Any of the above tumors may be a solid tumor or a hematological tumor. The tumor may in particular be a brain tumor.
The inventor of the invention discovers a section of polypeptide which is derived from Zika virus and has the function of a p53 protein-MDM 2 protein interaction inhibitor, namely the section of polypeptide can be effectively combined with MDM2 protein in an organism, so that the concentration of the cancer suppressor protein p53 protein in the organism is improved, and then the formation or growth of tumor cells can be effectively inhibited by accumulating more p53 protein in the organism, and finally the aim of inhibiting cancer is fulfilled. The invention has important significance for treating tumors.
Drawings
FIG. 1 shows that the test polypeptide binds tightly to the MDM2 protein.
FIG. 2 shows that the control polypeptide is unable to bind tightly to the MDM2 protein.
FIG. 3 is a graph showing the change in concentration of free MDM2 protein in U87 cells after treatment with the experimental polypeptide.
FIG. 4 shows the concentration changes of p53 protein and apoptosis-related protein in U87 cells after experimental polypeptide treatment.
FIG. 5 shows the statistics of the cell viability after the treatment of the experimental polypeptide.
FIG. 6 is a graphical representation of the number of cells that changed in morphology after treatment with the experimental polypeptide.
FIG. 7 shows the change in apoptosis after treatment with experimental polypeptides.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
U87 cells: ATCC, No.:
example 1 discovery of the polypeptide
Through a large amount of sequence analysis and functional research, one polypeptide (derived from Zika virus) is screened out and is shown as a sequence 1 in a sequence table. Through molecular docking kinetic calculation, the polypeptide can be combined with the binding site of the p53 protein of the MDM2 protein, and is well combined with the MDM2 protein in a long time (figure 1), the interaction is strong, and other control polypeptides (such as LKRLPAGLLLGHGPIRMVLAILAF) cannot be well combined with the MDM2 protein (figure 2). This shows that the polypeptide has specific functions, i.e. it can strongly and perfectly bind with MDM2 protein, thus preventing the binding of p53 protein with MDM2 protein, further promoting the accumulation of intracellular p53 protein, and finally achieving the purpose of restoring p53 protein to inhibit the formation and growth of tumor cells.
Example 2 functional study of the Polypeptides
The experimental polypeptide sequences are as follows: KKEAMEIIGGGGGGLAAMLRIINA (SEQ ID NO: 1 of the sequence Listing).
The control polypeptide sequence is as follows: LKRLPAGLLLGHGPIRMVLAILAF are provided.
The polypeptide to be detected is an experimental polypeptide or a control polypeptide.
The polypeptide can effectively reduce the concentration of free MDM2 protein in U87 cells
1. U87 cells were seeded into 6-well plates (2 × 10 per well)6Individual cells) were cultured in DMEM medium containing 10% (by volume) fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin, 5% CO2And standing and culturing at 37 ℃ for 24 h.
2. After the step 1 is completed, the 6-hole plate is taken, the polypeptide to be detected is added, and the concentration of the polypeptide to be detected in the system is 80 mu M, 40 mu M, 20 mu M, 10 mu M, 5 mu M or 2.5 mu M (each concentration is provided with 3 duplicate wells; meanwhile, a blank control without the polypeptide to be detected is arranged, the blank control is provided with 3 duplicate wells), and 5% CO is added2Standing at 37 ℃ for 2 hours, and then using a commercial kit (human totalMDM2/HDM2 DuoSet IC ELISA kit (R)&D Systems)), the concentration of free MDM2 protein in U87 cells was determined according to the kit instructions.
The results after the care of the experimental polypeptides are shown in fig. 3. The results indicate that the test polypeptide can dose-dependently reduce the concentration of free MDM2 protein in U87 cells by binding to intracellular MDM 2. The control polypeptide did not achieve the same effect.
Secondly, the polypeptide can effectively improve the concentration of the p53 protein and apoptosis-related protein in U87 cells
1. U87 cells were seeded into 6-well plates (5 × 10 per well)5Individual cells) were cultured in DMEM medium containing 10% (by volume) fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin, 5% CO2And standing and culturing at 37 ℃ for 24 h.
2. After step 1 was completed, the 6-well plate was taken, the polypeptide to be tested was added to the plate to give a concentration of 0. mu.M, 5. mu.M or 10. mu.M in the system (3 duplicate wells were provided for each concentration; a blank control without the polypeptide to be tested was provided, and 3 duplicate wells were provided for the blank control), the cells were collected after 2 hours, total protein was extracted after lysis, and expression of p53 protein (Santa Cruze), Caspase-3 protein (Cell Signaling Technology) and Caspase-9 protein (Cell Signaling Technology) was detected by western blot using Tubulin protein (Sigma) as an internal reference protein.
The results after treatment with the experimental polypeptides are shown in FIG. 4. The results show that the levels of p53 protein and apoptosis-related protein in the cells are increased after the action of the experimental polypeptide. The control polypeptide did not achieve the same effect.
Thirdly, the polypeptide induces the apoptosis of U87 cells
1. U87 cells were seeded into 6-well plates (5 × 10 per well)5Individual cells) were cultured in DMEM medium containing 10% (by volume) fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin, 5% CO2And standing and culturing at 37 ℃ for 24 h.
2. After step 1, the 6-well plate is taken, the polypeptide to be detected is added, the concentration of the polypeptide to be detected in the system is 0 mu M, 5 mu M or 10 mu M (5 multiple wells are arranged for each concentration; meanwhile, a blank control without the polypeptide to be detected is arranged, and 3 multiple wells are arranged for the blank control), and the cell survival condition is detected by using a CCK8 kit (Dojindo Molecular Technologies, Inc., Kunamoto, Japan) after 6 h. The results after treatment with the experimental polypeptide are shown in FIG. 5.
3. After step 1 is completed, the 6-well plate is taken, the polypeptide to be detected is added, the concentration of the polypeptide to be detected in the system is 0 mu M, 5 mu M or 10 mu M (3 multiple wells are arranged for each concentration; meanwhile, a blank control without the polypeptide to be detected is arranged, 3 multiple wells are arranged for the blank control), the cell number morphology is observed after 6h, and the change of the Apoptosis condition of U87 cells is detected through an Apoptosis chip kit (Annexin V-FITC/propyiodiumiodine Apoptosis Detection kit; Dojindo Molecular Technologies, Inc., Kunamoto, Japan) according to the kit specification.
The results are shown in FIGS. 6 and 7. FIG. 6 morphological observations of cell morphology number after experimental polypeptide treatment. FIG. 7 is an observation of apoptosis following treatment with experimental polypeptides. In fig. 7, the number of apoptotic cells carrying green fluorescence increased with increasing polypeptide concentration. The result shows that the activity of the U87 cell treated by the experimental polypeptide is obviously reduced, and the polypeptide can induce apoptosis. The control polypeptide did not achieve the same effect.
<110> military medical research institute of military science institute of people's liberation force of China
<120> polypeptide inhibitor of MDM2 and p53 interaction and application thereof
<160>1
<210>1
<211>24
<212>PRT
<213> Artificial sequence
<220>
<223>
<400>1
Lys Lys Glu Ala Met Glu Ile Ile Gly Gly Gly Gly Gly Gly Leu Ala
1 5 10 15
Ala Met Leu Arg Ile Ile Asn Ala
20
Claims (6)
1. The polypeptide is shown as a sequence 1 in a sequence table.
2. Use of the polypeptide of claim 1 in the manufacture of a product; the use of the product is to inhibit the binding of MDM2 protein and p53 protein in a cell.
3. Use of the polypeptide of claim 1 in the manufacture of a product; the product is used for improving the expression level of p53 protein in cells.
4. Use of the polypeptide of claim 1 in the manufacture of a product; the product is used for inducing tumor cell apoptosis.
5. Use of the polypeptide of claim 1 in the manufacture of a product; the product can be used for treating tumor.
6. A medicament, the active ingredient of which is the polypeptide of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810149254.8A CN108409839B (en) | 2018-02-13 | 2018-02-13 | Polypeptide inhibitor for interaction of MDM2 and p53 and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810149254.8A CN108409839B (en) | 2018-02-13 | 2018-02-13 | Polypeptide inhibitor for interaction of MDM2 and p53 and application thereof |
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| Publication Number | Publication Date |
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| CN108409839A CN108409839A (en) | 2018-08-17 |
| CN108409839B true CN108409839B (en) | 2020-08-11 |
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| CN111574591B (en) * | 2020-04-29 | 2021-09-21 | 西安交通大学医学院第一附属医院 | Polypeptide and synthetic method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101023074A (en) * | 2004-09-22 | 2007-08-22 | 詹森药业有限公司 | Inhibitors of the interaction between MDM2 and P53 |
| EP0635068B2 (en) * | 1992-04-07 | 2009-04-22 | The Johns Hopkins University | Amplification of human mdm2 gene in human tumors |
| CN101775073A (en) * | 2009-01-08 | 2010-07-14 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Drug for inhibiting proliferation of tumor cells and application thereof |
| CN103269715A (en) * | 2010-11-05 | 2013-08-28 | 邓迪大学 | Method for improving the production of influenza viruses and vaccine seeds |
-
2018
- 2018-02-13 CN CN201810149254.8A patent/CN108409839B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0635068B2 (en) * | 1992-04-07 | 2009-04-22 | The Johns Hopkins University | Amplification of human mdm2 gene in human tumors |
| CN101023074A (en) * | 2004-09-22 | 2007-08-22 | 詹森药业有限公司 | Inhibitors of the interaction between MDM2 and P53 |
| CN101775073A (en) * | 2009-01-08 | 2010-07-14 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Drug for inhibiting proliferation of tumor cells and application thereof |
| CN103269715A (en) * | 2010-11-05 | 2013-08-28 | 邓迪大学 | Method for improving the production of influenza viruses and vaccine seeds |
Non-Patent Citations (3)
| Title |
|---|
| Mechanism of Competition between Nutlin3 and p53 for Binding with Mdm2;刘书霞等;《Chinese Physics Letters》;20171130;第34卷(第11期);第1-5页 * |
| Transcription factors that interact with p53 and Mdm2;Inoue K等;《HHS Public Access》;20160401;第138卷(第7期);第1-18页 * |
| 抑制p53-HDM2结合为靶点的候选药物研究进展;张国钧等;《中国药学杂志》;20090430;第44卷(第7期);第485-489页 * |
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