CN112375150A - Application of binary polypeptide compound - Google Patents
Application of binary polypeptide compound Download PDFInfo
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- CN112375150A CN112375150A CN202011333520.6A CN202011333520A CN112375150A CN 112375150 A CN112375150 A CN 112375150A CN 202011333520 A CN202011333520 A CN 202011333520A CN 112375150 A CN112375150 A CN 112375150A
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- egfr
- polypeptide
- growth factor
- epidermal growth
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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
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
Abstract
The invention relates to a binary polypeptide of a target Epidermal Growth Factor Receptor (EGFR) specific binding polypeptide and application thereof, belonging to the technical field of biology. Wherein the polypeptide consists of an EFGR-targeting affibody and a transmembrane normal-induced protein (TMEPAI) amino acid truncation at position 148-242, and a linker (four glycines-serine, GGGGS). The polypeptide can specifically bind to EGFR and E3 ubiquitin ligase (NEDD 4). The polypeptide prepared by the invention has the functions of targeted combination and degradation of high expression EGFR in lung cancer cells so as to inhibit tumor growth, and the method has the characteristics of reversibility, timing and quantitative targeted degradation of high expression EGFR in tumor cells.
Description
Technical Field
The invention relates to an application of a binary polypeptide compound, in particular to an application of a targeting degradation tumor cell high-expression epidermal growth factor receptor.
Background
Cancer has become one of the major diseases that endanger human life and health. The world health organization reports show that 1810 thousands of cancer patients are newly added in 2018 all over the world, and the number of deaths reaches 960 thousands. Epidermal Growth Factor Receptor (EGFR) is involved in regulating cell proliferation, survival, growth and differentiation, but if EGFR is overexpressed, it will be directly associated with human cancer. Therefore, the reduction of EGFR is a strategy for treating certain cancers. There are two main methods of interfering with protein expression in the past: DNA knockout and RNA interference. However, the above methods are all methods for permanently reducing the protein. This means that they are limited to studying functions that are temporarily or partially deficient in a certain protein. In recent years, with the progress of intensive research on malignant tumors and research on antitumor drugs, various methods for temporarily consuming a target protein using a cellular protein degradation system have been developed. One approach is to attach a small hydrophobic moiety to the target protein, thereby mimicking a misfolded protein. These proteins can be further degraded by the proteasome. Another approach is targeted proteolytic-targeting chimera (PROTAC) technology. The PROTAC technique degrades the target protein through the ubiquitin-proteasome system by recruiting E3 ubiquitin ligase.
In the last 20 years, a variety of EGFR targeting drugs have been designed. One type is monoclonal antibodies, such as cetuximab and panitumumab. Monoclonal antibodies bind to the extracellular domain of EGFR and compete with endogenous ligands by blocking ligand binding regions to block ligand-induced activation of EGFR tyrosine kinase. The second is a Tyrosine Kinase Inhibitor (TKI), such as gefitinib, erlotinib, lapatinib and canertinib. TKIs reversibly compete with 5' adenosine triphosphate, bind to the intracellular catalytic domain of EGFR tyrosine kinase and inhibit EGFR autophosphorylation and downstream signaling. Another type is antibody-based immunoconjugates, such as trastuzumab-entamoenom and EQ 75-ADR. The mechanism is to change its distribution in vivo by changing its binding to a monoclonal antibody targeting the tumor, thereby improving the therapeutic range of the chemotherapeutic agent or rendering the drug inactive (as a prodrug). In addition, antisense oligodeoxynucleotide (GEM 231) is another drug. They can reduce the expression of EGFR and modulate cell proliferation for potential anti-cancer treatment. The patent aims to develop a multifunctional polypeptide designed and synthesized based on the PROTAC technology to target-bind and degrade EGFR, so that the multifunctional polypeptide is applied to the targeted therapy of malignant tumors.
Disclosure of Invention
The polypeptide capable of specifically degrading the epidermal growth factor receptor is synthesized based on a targeted protein hydrolysis chimera (PROTAC) technology, and the binding capacity and the binding specificity of the polypeptide and the epidermal growth factor receptor are verified through a Western blot experiment. The result shows that the obtained polypeptide can be specifically combined with the epidermal growth factor receptor and promote the degradation of the epidermal growth factor receptor.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the polypeptide sequence specifically bound aiming at the epidermal growth factor receptor consists of an affibody targeting EFGR, a 148-position 242-amino acid truncation of a transmembrane prostate protein (TMEPAI), and a linker (four glycines, one serine, GGGGS), and has high affinity activity of binding the epidermal growth factor receptor and a function of specifically degrading the epidermal growth factor receptor.
The polypeptide for targeted degradation of the epidermal growth factor receptor can be combined with the epidermal growth factor receptor and E3 ubiquitin ligase NEDD4, and degradation of the epidermal growth factor to different degrees can be realized by concentration gradient and time gradient. The polypeptide which can degrade the epidermal growth factor receptor in a targeted way also has influence on the cell viability, the proliferation capacity and the migration capacity of the NCI-H460 lung cancer cells. Such polypeptides are considered to be included within the scope of the present invention.
The polypeptide sequence is used for targeting the degradation of the epidermal growth factor receptor. The application of the polypeptide in timed and quantitative reversible targeted degradation of epidermal growth factor receptors.
The application of the polypeptide His-HA-ALT in tumor cells is disclosed, wherein the tumor is lung cancer.
The invention has the advantages and positive effects that:
1. the invention aims at degrading the EGFR with high expression in tumor cells by synthesizing a binary polypeptide compound His-HA-ALT based on a targeted protein hydrolysis chimera (PROTAC) technology. Compared with the monoclonal antibody, the monoclonal antibody has the remarkable advantages of low preparation cost, good stability and the like. Compared with a gene knockout strategy PROTAC technology, the method has the advantages of reversibility and timed and quantitative targeted degradation of EGFR.
2. The binary polypeptide compound obtained by the invention can achieve the effect of treating lung cancer by specifically and rapidly targeting and degrading EGFR.
Drawings
FIG. 1 shows His-HA-ALT interaction with EGFR;
FIG. 2 is a graph of His-HA-ALT interaction with NEDD 4;
FIG. 3 is EGFR degradation following treatment of NCI-H460 cells with different concentrations of the binary polypeptide complex His-HA-ALT;
FIG. 4 EGFR degradation after treatment of NCI-H460 cells with the binary polypeptide complex His-HA-ALT at a concentration of 50. mu.g/mL for various periods of time;
FIG. 5 shows the results of the MTT method of the present invention for detecting the activity of His-HA-ALT of the binary polypeptide complex of the present invention on NCI-H460 lung cancer cells;
FIG. 6 shows the result of detecting the proliferation of the binary polypeptide complex His-HA-ALT of the present invention on NCI-H460 lung cancer cells by the EdU method of the present invention;
FIG. 7 shows the inhibition of the binary polypeptide complex His-HA-ALT on the NCI-H460 lung cancer cell migration by the scarification method.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description will be given with reference to specific embodiments.
Example 1
His-HA-ALT interaction with EGFR
Taking 30 mu L of Ni-beads to put in a 1.5mL EP tube, centrifuging for 1min at 10000rpm, and removing the supernatant; wash 1 time with 500. mu.L lysis buffer. 5-10. mu.g of His-HA-ALT fusion protein was added to the above EP tube, and lysis buffer was added to make up to 600. mu.L. The above samples were incubated at 4 ℃ for 2 hours on a roller, centrifuged at 10000rpm for 1min, the supernatant was discarded, washed 1 time with 500. mu.L lysis buffer and the supernatant discarded.
Pancreatin digestion and collection of 6cm plates of NCI-H460 cells
Add 300. mu.L of 1% NP-40 lysate (containing PIC and PMSF) to lyse the cells, lyse on ice for 30min, and blow-suck every 10 min. The mixture was centrifuged at 14000rpm for 10min at 4 ℃ and the supernatant was collected. mu.L of the solution was taken and 20. mu.L of 2 XSDS loading buffer was added, and the sample was boiled at 95 ℃ for 5min and labeled input. The supernatant was added to beads and roller incubated at 4 ℃ for 4 hours or overnight. Centrifuging at 10000rpm for 1min, collecting supernatant 20 μ L, adding 20 μ L2 xSDS loading buffer, and boiling at 95 deg.C for 5min to obtain UB. The beads were washed 3 times with 500. mu.L lysis buffer (containing 1% NP-40 or Triton X-100) and finally 1 time with PBS at 10000rpm for 30s, and the supernatant was discarded. Add 40. mu.L of 2 xSDS loading buffer to the beads, and cook the sample at 95 ℃ for 5min, which is recorded as Bound.
As shown in FIG. 1, the results of the experiment show that His-HA-ALT interacts with EGFR, and in FIG. 2, the results of the experiment show that His-HA-ALT interacts with NEDD 4.
Example 2
SDS-PAGE detection of EGFR degradation by His-HA-ALT
Treating NCI-H460 cells with His-HA-ALT at different concentrations for 24H; NCI-H460 cells were treated with 50. mu.g/mL His-HA-ALT for various periods of time. Collecting the above cell sample, extracting protein, adding 5 xSDS, mixing, and decocting at 95 deg.C for 5min to fully denature protein. After boiling, the mixture was homogenized in a suspension shaker and centrifuged instantaneously, and the sample was stored at-20 ℃. And finally, carrying out immunoblot detection.
As shown in FIG. 3 and FIG. 4, the results of the experiments indicate that the binary polypeptide complex His-HA-ALT promotes EGFR degradation in NCI-H460 cells.
MTT assay
Logarithmic NCI-H460 lung cancer cells were collected, the concentration of the cell suspension was adjusted, 200. mu.L of medium was added to each well, and the cells to be tested were plated at a density of 8000 cells/well. 5% CO2Incubate at 37 ℃ for 24h, and add His-HA-ALT. 4 concentration gradients (0. mu.g/mL, 2)5. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL), and 3 multiple wells. 5% CO2Incubate at 37 ℃ for 24h, add 20. mu.L MTT per well (ready-to-use, 5mg/mL MTT solution in PBS) and continue incubation for 4 h. The liquid in the wells is carefully washed away and the precipitate must not be sucked away. Adding 200 μ L dimethyl sulfoxide into each well to dissolve precipitate, and shaking for 10min to dissolve crystal completely. The absorbance of each well was measured at 490nm in an enzyme linked immunosorbent assay.
As shown in FIG. 5, the results of the experiment show that the binary polypeptide complex His-HA-ALT inhibits the activity of NCI-H460 lung cancer cells.
EdU test
Taking cells in logarithmic growth phase at a rate of 7X 10 cells per well3Individual cells were seeded in 96-well plates and cultured to normal growth stage. His-HA-ALT with concentration gradient of 0, 25, 50, 100. mu.g/mL was added for treatment. The EdU solution was diluted 1000: 2 with cell complete medium to prepare an appropriate amount of 100. mu.M EdU medium. Add 100. mu.L of 100. mu.M EdU medium to each well and incubate for 2h, discard the medium. The cells were washed 2 times with PBS for 5min each. mu.L of cell fixative (i.e., 4% paraformaldehyde in PBS) was added to each well and incubated at room temperature for 30min, and the fixative was discarded. Add 50. mu.L 2mg/mL glycine to each well, decolorize and shake incubate for 5min, discard the glycine solution. Add 100. mu.L PBS to each well, wash on a decolorization shaker for 5min, discard PBS. Add 100. mu.L of penetrant (0.5% TritonX-100 PBS) to each well, decolour and shake the wells for 10min, wash the wells with PBS 1 time for 5 min. Add 100. mu.L of 1 XApollo staining reaction solution into each well, incubate for 30min in dark, room temperature, decolorizing and shaking table, discard the staining reaction solution. Adding 100 μ L of penetrant, washing with a decolorizing shaker for 3 times (10 min each time), and discarding the penetrant. Add 100. mu.L of methanol to wash each well 2 times for 5min each time, and wash with PBS 1 time for 5 min. Diluting with deionized water at a ratio of 100: 1, preparing a proper amount of 1 × Hoechst33342 reaction solution, and storing in dark place. Add 100. mu.L of 1 × Hoechst33342 reaction solution into each well, incubate for 30min in dark at room temperature in a decolorization shaker, and then discard the staining reaction solution. Wash 3 times with 100 μ LPBS per well. Photographic analysis was performed with a fluorescence microscope.
As shown in FIG. 6, the results of the experiment show that the binary polypeptide complex His-HA-ALT inhibits the proliferation of the NCI-H460 lung cancer cells.
4. Scratch test
NCI-H460 cells were prepared into cell suspension, diluted to an appropriate concentration, and counted using a hemocytometer at 6X 105Concentration of individual cells per well, cells were seeded into 6-well plates with a total final culture medium of 2mL per well, placed in a cell incubator at 37 ℃ with 5% CO2Culturing under the conditions of (1). When the cells grow to 90-100% of confluence, a well is drawn at the bottom of the dish. The culture medium in the dish was discarded, washed 3 times with DPBS, and the scraped cells were rinsed clean. DMEM medium containing 0.5% serum was added to reduce the effect of cell proliferation on the scratch test. Photographs were taken at 0h, 24h, 48h, 72h post-scratching, respectively, using a Nikon inverted fluorescence microscope. The light source uses white light and the objective lens uses a 10-fold mirror. And selecting the position near the intersection position of the scratch as much as possible to take a picture so as to ensure that the visual field is consistent every time. Each well takes a picture of not less than 3 fields of view.
As shown in FIG. 7, the results of the experiment show that the binary polypeptide complex His-HA-ALT inhibits NCI-H460 lung cancer cell migration.
Claims (3)
1. A polypeptide that specifically binds to Epidermal Growth Factor Receptor (EGFR) consisting of an EGFR-targeting affibody and a 148-242 amino acid truncation of the prostate transmembrane protein (TMEPAI), and a linker (four glycines-one serine, GGGGS).
2. The polypeptide of claim 1, wherein: can bind to Epidermal Growth Factor Receptor (EGFR) and E3 ubiquitin ligase (NEDD 4).
3. The use of the polypeptide of claim 1, specifically targeting and degrading tumor cells to highly express Epidermal Growth Factor Receptor (EGFR).
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011333520.6A CN112375150A (en) | 2020-11-25 | 2020-11-25 | Application of binary polypeptide compound |
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| CN202011333520.6A CN112375150A (en) | 2020-11-25 | 2020-11-25 | Application of binary polypeptide compound |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109152843A (en) * | 2016-05-20 | 2019-01-04 | 豪夫迈·罗氏有限公司 | PROTAC antibody conjugates and its application method |
| CN109928956A (en) * | 2019-02-27 | 2019-06-25 | 杭州偶联医药科技有限公司 | A kind of compound of targeting ubiquitination degradation EGFR albumen and its pharmaceutical composition and application |
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- 2020-11-25 CN CN202011333520.6A patent/CN112375150A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109152843A (en) * | 2016-05-20 | 2019-01-04 | 豪夫迈·罗氏有限公司 | PROTAC antibody conjugates and its application method |
| CN109928956A (en) * | 2019-02-27 | 2019-06-25 | 杭州偶联医药科技有限公司 | A kind of compound of targeting ubiquitination degradation EGFR albumen and its pharmaceutical composition and application |
Non-Patent Citations (3)
| Title |
|---|
| UNIPROT: "Accession NO.：Q969W9", 《UNIPROT数据库》 * |
| YANYUN DU ET AL: "The transmembrane protein TMEPAI induces myeloma cell apoptosis by promoting degradation of the c-Maf transcription factor", 《J. BIOL. CHEM.》 * |
| YUXUAN DAI ET AL: "Development of cell-permeable peptide-based PROTACs targeting estrogen receptor α", 《EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY》 * |
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