A polypeptide mse responding targeted degradation of egfr protein and its anti-tumor use

Abstract

The application belongs to the technical field of biological materials, and particularly relates to a polypeptide MSE for enzyme-responsive targeted degradation of EGFR protein and anti-tumor use thereof. The amino acid sequence of the polypeptide MSE is shown as SEQ ID NO. 1. The polypeptide MSE can be co-localized and combined with EGFR protein on the surface of a cell membrane, degrades the EGFR protein on the cell membrane, promotes tumor cell apoptosis through an active oxygen signal pathway, has anti-tumor effects in vivo and in vitro, has good biological safety, and is expected to provide a new idea for the treatment of cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and specifically relates to a polypeptide MSE for targeted degradation of EGFR protein in response to enzymes and anti-tumor use thereof. BACKGROUND

[0002] Epidermal growth factor receptor (EGFR) is a transmembrane protein, which belongs to the tyrosine kinase receptor and has the activity of tyrosine kinase. It is one of the members of the epidermal growth factor receptor (HER) family. In addition to EGFR, the family also includes HER2, HER3 and HER4. When EGFR binds to its ligand to form a dimer, it is then phosphorylated and activated. The activated EGFR can be recognized by various downstream signal molecules such as JAK, PI3K and Ras, and the related signals are transmitted to the nucleus through various signal transduction pathways such as PI3K-AKT-mTOR and Ras-Raf-MEK, thereby regulating various cell functions such as proliferation, apoptosis and angiogenesis. EGFR is widely present in various types of tumor cells such as colorectal cancer, lung cancer and breast cancer, and the development of cancer is closely related to EGFR mutation. Therefore, EGFR has become a classic potential therapeutic target for cancer. So far, relevant personnel have developed three generations of specific tyrosine kinase inhibitors including gefitinib, afatinib and osimertinib. In addition, monoclonal antibody drugs represented by pertuzumab are widely used in clinical treatment because they can bind to the extracellular domain of EGFR, thereby blocking the binding of the receptor and the ligand. However, some cancer patients often have EGFR mutations, and EGFR mutants often exhibit ligand-independent activity, so antagonizing the ligand-receptor interaction cannot completely prevent intracellular signal transduction. Moreover, the above-mentioned drugs are generally used for the treatment of lung cancer or breast cancer in clinical practice, and are less used for the treatment of other types of cancer. Therefore, although the survival status of cancer patients has been improved with the emergence of the above-mentioned targeted drugs, the treatment prospects still face great challenges.

[0003] The polypeptide sequence is reasonably modified and designed to have the ability to bind to a specific target, and has a larger binding surface than small molecules, which is not prone to drug resistance. In order to achieve precise drug release and improve its biological stability, researchers have designed polypeptide in-situ self-assembly. This technology relies on hydrogen bonds and other interactions caused by aromatic amino acid residues to induce the spontaneous conversion of polypeptides into more complex multimers. Enzymes, pH, reactive oxygen species (ROS), reducing glutathione (GSH) and other physical and chemical biological signals can be used as stimulus response signals to induce the spontaneous conversion of self-assembly precursors to achieve high-level enrichment of drugs, which is a feasible method that can solve the above-mentioned problems. SUMMARY

[0004] The application synthesizes an enzyme-responsive self-assembly polypeptide, which has a module for precisely targeting EGFR, self-assembles nanofibers by utilizing MMPs and through specific interactions, and then targets the overexpressed membrane protein EGFR in tumor cells. Matrix metalloproteinases (MMPs) are overexpressed in various tumor tissues, but not expressed or expressed at very low levels in corresponding normal tissues. The nanofibers generated in situ on the cell surface self-assemble and, after binding to EGFR, are endocytosed to lysosomes by the action of lysosomal sorting sequences, degrading EGFR. We have verified that it can effectively induce EGFR degradation, leading to tumor cell death, and play an anti-tumor role in mice. This method of enzyme-responsive self-assembly targeted degradation of membrane proteins is expected to provide a new idea for the treatment of cancer.

[0005] The application adopts the following technical solutions:

[0006] The application provides an enzyme-responsive polypeptide MSE for targeted degradation of EGFR protein, and the amino acid sequence of the polypeptide MSE is YHWYGYTPQNVINPGY-KLVFFPLGYLGEEE (SEQ ID NO. 1).

[0007] In the above technical solution, further, the polypeptide MSE is applied to preparation of an anti-tumor drug.

[0008] In the above technical solution, further, the tumor is lung cancer, colorectal cancer or ovarian cancer.

[0009] In the above technical solution, further, the application is that the polypeptide MSE is co-localized and combined with EGFR protein on the surface of a cell membrane, and EGFR is degraded.

[0010] In the above technical solution, further, the application is that the polypeptide MSE promotes tumor cell apoptosis through an active oxygen signaling pathway.

[0011] The application provides application of a pharmaceutical composition in preparation of an anti-tumor drug, and the pharmaceutical composition comprises the aforementioned polypeptide MSE.

[0012] In the above technical solution, further, the tumor is lung cancer, colorectal cancer or ovarian cancer.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The polypeptide MSE of the present application utilizes the overexpressed membrane protein EGFR and MMPs in tumor cells and self-assembles nanofibers through specific interaction, precisely targets and degrades EGFR of tumor cells. The tumor overexpressed trend matrix metalloproteinase (MMPs) is cut, the self-assembled nanofibers are proved by electron microscopy, which can promote it and under the action of lysosomal sorting sequence, endocytosis to lysosome, and degrade EGFR. The strategy of in situ self-assembly can effectively improve the tissue selectivity and accumulation ability of the disease site of MSE, and improve the safety and effectiveness of MSE in degrading EGFR. We have verified that it can effectively induce EGFR degradation in vivo and in vitro, thereby causing tumor cell death. This method of targeted degradation of membrane proteins through enzyme-responsive self-assembly is expected to provide a new idea for the treatment of cancer. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The structure of MSE is shown.

[0016] Figure 2 The reverse high performance liquid chromatography analysis (A) and mass spectrometry analysis (B) of MSE are shown.

[0017] Figure 3 MSE in aqueous solution is shown, which is converted into a unique nanofilament structure after co-incubation with MMP-2.

[0018] Figure 4 The fluorescence map of MSE successfully binding and co-localization with EGFR receptor on the surface of cells after being taken up by cells for 6 hours is shown.

[0019] Figure 5 The killing effect of MSE and GE11 on tumor cells; A. The killing effect of MSE on H226, HCT-116, SKOV3, 293T; B. The killing effect of GE11 on H226, HCT-116, SKOV3.

[0020] Figure 6 The results of cell live and dead staining experiment are shown; A. H226 cells, B. HCT-116 cells.

[0021] Figure 7 The results of intracellular reactive oxygen species level fluorescence detection are shown; A. H226 cells, B. HCT-116 cells.

[0022] Figure 8 EGFR degradation in HCT-116 cells induced by MSE at different times (A) and at different concentrations (B).

[0023] Figure 9In vivo anti-tumor effect of MSE in nude mice model; A. nude mice images and tumor growth curve; B. nude mice tumor pictures and their weights.

[0024] Figure 10 Results of biological safety evaluation of MSE in nude mice model; A. AST, ALT, UREA level detection, B. body weight change chart of nude mice in each group. DETAILED DESCRIPTION

[0025] The application is further described below in conjunction with specific embodiments, but in no way limits the application.

[0026] First, the enzyme-responsive self-assembly polypeptide MSE was successfully synthesized, and the self-assembly change was verified by electron microscopy. Next, the fluorescence co-localization of MSE was verified by microscopy. The results prove that H226 has good uptake ability for MSE and can be co-localized and combined with EGFR protein on the surface of the cell membrane. Then, the in vitro anti-tumor ability of MSE was verified by different experiments. The influence of different concentrations of MSE on the growth and proliferation of various cancer cells and normal cells was detected by CCK-8 method, and the results show that after a certain concentration of MSE acts on cancer cells, corresponding proliferation inhibition effect can be produced, and this effect presents drug concentration dependence, that is, the higher the concentration, the lower the relative activity of the cells. However, this effect is not reflected on the normal cells 293T, which may be related to the relatively low expression of EGFR in 293T cells. The EGFR-binding peptide GE11 reported in the prior art does not have obvious proliferation inhibition effect on cancer cells or normal cells at the same concentration. Cell live and dead staining and plate cloning experiments also verify the in vitro anti-tumor ability of MSE, and in the active oxygen experiment, the cancer cells have a significant change of green fluorescence enhancement, and we have reason to infer that MSE is very likely to promote cell apoptosis through the active oxygen signaling pathway, thereby playing a drug role.

[0027] To study the effect of MSE on EGFR, the selective degradation of EGFR protein level by MSE was detected. The Western blot results show that MSE degrades EGFR in a time- and concentration-dependent manner, verifying the degradation effect of MSE on EGFR. In the following tumor formation experiment of nude mice, according to the phenomenon that the tumor growth is obviously inhibited, we also have reason to prove that MSE has in vivo anti-tumor effect, and according to the body weight change of nude mice and the detection of biochemical indicators, it can be seen that this drug has good biological safety.

[0028] The application proposes to use the new strategy of enzyme-responsive self-assembly to selectively degrade EGFR protein on the cell membrane, verifies its anti-tumor effect, and analyzes the possible mechanism of action, and expects that the new strategy of the application can provide a new perspective for the treatment of cancer.

[0029] Design, synthesis and characterization of example 1MSE:

[0030] The enzyme-responsive precursor MSE (YHWYGYTPQNVI-NPGY-KLVFF-PLGYLG-EEE) is composed of five modules, EGFR binding peptide module, lysosome sorting module, self-assembly module, enzyme-responsive module, and hydrophilic module. Figure 1 The polypeptide was synthesized by solid-phase FMOC method, and amino acids were added one by one from the carboxyl end to the amino end. After synthesis, the side chain protecting group was removed, and the polypeptide was cut from the resin. Purified by high performance liquid chromatography, and identified by mass spectrometry. The results show that Figure 2 MSE was successfully synthesized, purified by high performance liquid chromatography, and identified by mass spectrometry, with a molecular weight of 3595.03 Da.

[0031] The morphology of the enzyme reaction MSE before and after the addition of MMP-2 was detected by transmission electron microscopy (TEM), and the results show that Figure 3 MSE in aqueous solution is not aggregated, and is converted into a unique nanofilament structure after incubation with MMP-2 at a final concentration of 1 μg / mL at 37°C for 2 hours.

[0032] Example 2: Cell uptake and fluorescence co-localization of MSE:

[0033] In order to evaluate the uptake of MSE in vitro, cell uptake experiments were performed.

[0034] 60 μM of MSE was labeled with Cy3 dye which can emit red fluorescence. In order to verify whether the drug taken up by H226 cells is combined with the EGFR receptor, we observed fluorescence co-localization. The results show that Figure 4 There is obvious co-localization, which indicates that MSE can successfully combine with the cell surface EGFR receptor after being taken up by cells for 6 hours.

[0035] Example 3: Toxic effect of MSE on tumor cells:

[0036] In order to more intuitively reflect the pharmacodynamic advantage of MSE, GE11 was compared with it. GE11 (YHWYGYTPQNVI) is an EGFR ligand that has been widely reported and verified to be effective, and can specifically bind to EGFR.

[0037] The inhibitory effect of the two drugs on tumor cells was verified by treating cells with drug concentration gradient for 24 hours and cck8 experiment cell activity detection experiment. The results show that Figure 5), MSE had good killing effect on H226, HCT-116, SKOV3 cells in a dose-dependent manner, and had no significant killing effect on 293T. Further analysis of the toxic effect of GE11 on tumor cells found that the activity of the above tumor cells did not change significantly.

[0038] In order to further more intuitively verify the killing effect of the drug on tumor cells, a cell live and dead staining experiment was performed. Figure 6 ) Cells were seeded in 48-well plates, and after the cells were fully adherent, 60 μM drug was added for treatment, and PBS was used as a control group, and staining was performed after incubation in the incubator for 24 h. Calcein is a calcium ion fluorescent indicator. Calcein-AM has good membrane penetration ability. After entering the cell, strong green fluorescence occurs. It is only used to label live cells. PI cannot penetrate the cell membrane of live cells and is excluded outside the live cells, but can penetrate the damaged cell membrane and embed in the DNA structure of the nucleus to emit red fluorescence. Based on this principle, the above two dyes can be used in combination to distinguish and identify live and dead cells. The results show that in H226 and HCT-116 cell lines, the amount of live cells in the MSE treatment group is significantly reduced, and the amount of dead cells is increased, compared with the control group.

[0039] Next, in order to explore whether MSE causes damage to tumor cells through the active oxygen signaling pathway, ROS level detection was performed. Active oxygen is a substance with oxidative activity in cells, and when it is produced too much, it will cause oxidative stress and damage to cells. DCFH-DA is an indicator probe that does not emit fluorescence itself and can freely penetrate the cell membrane. After entering the cell, it is hydrolyzed to DCFH under the catalysis of intracellular esterase and remains in the cell. ROS can oxidize non-fluorescent DCFH to generate DCF that can emit green fluorescence. Based on this principle, the active oxygen level in the cell can be determined by observing the fluorescence intensity, and then it can be judged whether the drug causes damage to tumor cells through the active oxygen signaling pathway. Cells were seeded in 48-well plates, and after the cells were fully adherent, 60 μM drug was added for treatment, and PBS was used as a control group, and staining was performed after incubation in the incubator for 24 h. The results show that Figure 7 ), in H226 and HCT-116 cell lines, the active oxygen content of the MSE treatment group was significantly increased, compared with the control group.

[0040] Example 4: Effect of selective degradation on EGFR expression:

[0041] The differential EGFR degradation induced by MSE in HCT-116 cells was investigated. CCK-8 results showed that the IC50 of MSE in H226 and HCT-116 cells was approximately 60 μM. Therefore, a final concentration of 60 μM MSE was added to H226 cells, and cells were collected at different time points. Total protein was extracted and Western blot was performed. The results showed... Figure 8 A) The effects of drug action at different time periods will have different effects on the degree of EGFR degradation; the longer the time, the higher the degree of degradation.

[0042] Furthermore, the effect of different concentrations of MSE on EGFR protein degradation was investigated. Different concentrations of MSE diluted in DMEM medium were added to H226 cells, and total protein was collected after 24 hours for Western blot analysis. The results demonstrated that (… Figure 8 B) The degradation effect of MSE on EGFR is concentration-dependent, and the expression level of EGFR gradually decreases with increasing MSE concentration.

[0043] Example 5: In vivo antitumor effect of MSE:

[0044] Nine nude mice were used to establish a xenograft tumor model by subcutaneous injection of HCT-116 cells, and then randomly divided into three groups. Each mouse in the experimental group was injected with 50 μl of either 1.5 μM / kg MSE or GE11 solution, while the control group received an equal volume of physiological saline. Injections were administered continuously for 8 days. During this period, the weight and volume of the mice were measured every two days. The results showed that both MSE and GE11 had varying degrees of inhibitory effects on tumor growth, but the inhibitory effect was significantly stronger in the MSE group. Figure 9 A). After photographing and weighing the tumors from these three groups, it was found that the tumor volume and size in the MSE experimental group were significantly smaller than those in the control group ( Figure 9 B).

[0045] The extracted serum was used to detect three biochemical indicators: ALT, AST, and UREA. The results showed that ( Figure 10 There was no significant difference between the experimental group and the control group. Figure 10 A). The weight changes of nude mice were also analyzed, and it was found that the weight of the mice remained stable in both the experimental and control groups. Figure 10 B).

Claims

1. A polypeptide MSE that targets and degrades EGFR protein in response to enzyme activity, characterized in that, The amino acid sequence of the polypeptide MSE is YHWYGYTPQNVINPGY-KLVFFPLGYLGEEE.

2. The use of the polypeptide MSE according to claim 1 in the preparation of drugs for treating lung cancer, colorectal cancer, or ovarian cancer.

3. The application according to claim 2, characterized in that, The application involves the peptide MSE co-localizing and binding to the EGFR protein on the cell membrane surface, thereby degrading EGFR.

4. The application according to claim 2, characterized in that, The application involves the peptide MSE promoting tumor cell apoptosis through the reactive oxygen species signaling pathway.

5. Use of a pharmaceutical composition in the preparation of a drug for treating lung cancer, colorectal cancer, or ovarian cancer, said pharmaceutical composition comprising the polypeptide MSE of claim 1.

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