Polypeptide MSE for enzyme-responsive targeted degradation of EGFR protein and anti-tumor application thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular is an enzyme-responsive polypeptide MSE for targeted degradation of EGFR protein and its anti-tumor application. Background Art

[0002] The epidermal growth factor receptor (EGFR) is a transmembrane protein and a tyrosine kinase receptor with tyrosine kinase activity. It is a member of the epidermal growth factor receptor (HER) family, which also includes HER2, HER3, and HER4. Upon binding to its ligand, EGFR forms a dimer and is subsequently activated by phosphorylation. Activated EGFR is recognized by multiple downstream signaling molecules, including JAKs, PI3K, and Ras. These signals are then transmitted to the cell nucleus through various signaling pathways, including the PI3K-AKT-mTOR and Ras-Raf-MEK pathways, thereby regulating various cellular functions, including proliferation, apoptosis, and angiogenesis. EGFR is widely expressed in various tumor cell types, including colorectal cancer, lung cancer, and breast cancer, and cancer progression is inextricably linked to EGFR mutations. Therefore, EGFR has become a potential therapeutic target for cancer. To date, three generations of specific tyrosine kinase inhibitors have been developed, 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 to the ligand. However, some cancer patients often have EGFR mutations, and EGFR mutants often exhibit ligand-independent activity. Therefore, antagonizing ligand-receptor interactions cannot completely block intracellular signal transduction. Moreover, the above-mentioned drugs are only commonly used in the treatment of lung cancer or breast cancer in clinical practice, and are less commonly used in the treatment of other types of cancer. Therefore, with the emergence of the above-mentioned targeted drugs, although the survival of cancer patients has improved, the treatment prospects still face great challenges.

[0003] Peptide sequences have been rationally modified and designed to give them the ability to bind to specific targets. Compared to small molecules, they have a larger binding surface area and are less susceptible to drug resistance. To achieve precise drug release and improve their biostability, researchers have designed in situ peptide self-assembly. This technology relies on hydrogen bonds caused by aromatic amino acid residues to induce the spontaneous conversion of peptides into more complex polymers. Physiochemical biosignals such as enzymes, pH, reactive oxygen species (ROS), and reduced glutathione (GSH) can serve as stimulus-response signals to induce the spontaneous conversion of self-assembly precursors to achieve high-level drug enrichment, offering a promising approach to addressing these challenges. Summary of the Invention

[0004] The present invention synthesizes an enzyme-responsive self-assembling polypeptide, which has a module that precisely targets and binds to EGFR. It utilizes MMPs and self-assembles nanofibers through specific interactions, and then targets and binds to the membrane protein EGFR overexpressed in tumor cells. Matrix metalloproteinases (MMPs) are overexpressed in a variety of tumor tissues, but not expressed or expressed at very low levels in corresponding normal tissues. After binding to EGFR, the nanofibers generated by in situ self-assembly on the cell surface are internalized into lysosomes under the action of lysosomal sorting sequences, thereby degrading EGFR. We have verified that it can effectively induce EGFR degradation, thereby leading to tumor cell death, and exert anti-tumor effects in mice. This method of targeted degradation of membrane proteins through enzyme-responsive self-assembly is expected to provide new ideas for the treatment of cancer.

[0005] The present invention adopts the following technical solutions:

[0006] The present invention provides an enzyme-responsive polypeptide MSE for targeted degradation of EGFR protein. 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 used in the preparation of anti-tumor drugs.

[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 co-localizes and binds with the EGFR protein on the cell membrane surface to degrade EGFR.

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

[0011] The present invention provides an application of a pharmaceutical composition in the preparation of an anti-tumor drug, wherein 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 present invention has the following beneficial effects:

[0014] The polypeptide MSE of the present invention utilizes the membrane protein EGFR and MMPs overexpressed in tumor cells and self-assembles nanofibers through specific interactions, accurately targeting and degrading the EGFR of tumor cells. After being enzymatically cleaved by matrix metalloproteinases (MMPs) overexpressed in tumors, it was demonstrated by electron microscopy that the self-assembled nanofibers can promote their endocytosis into lysosomes under the action of lysosomal sorting sequences, thereby degrading EGFR. The in situ self-assembly strategy effectively improves the tissue selectivity of MSE and the accumulation capacity of the diseased site, thereby improving the safety and effectiveness of MSE in degrading EGFR. We have verified that it can effectively induce EGFR degradation both in vivo and in vitro, thereby leading to tumor cell death. This method of targeted degradation of membrane proteins through enzyme-responsive self-assembly is expected to provide new ideas for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the structural composition of MSE.

[0016] Figure 2 Reverse-phase high performance liquid chromatography analysis of MSE (A) and mass spectrometry analysis of MSE (B).

[0017] Figure 3 MSE is aggregate-free in aqueous solution and is transformed into a unique nanofilament structure after incubation with MMP-2.

[0018] Figure 4 This is a fluorescence image showing that MSE can successfully bind to and co-localize with the EGFR receptor on the cell surface 6 hours after being taken up by cells.

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

[0020] Figure 6 Results of cell live-dead staining experiments; A. H226 cells, B. HCT-116 cells.

[0021] Figure 7 Fluorescence detection results of intracellular reactive oxygen species levels; A. H226 cells, B. HCT-116 cells.

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

[0023] Figure 9In vivo antitumor effect of MSE in a nude mouse model; A. Nude mouse images and tumor growth curve; B. Nude mouse tumor images and weight.

[0024] Figure 10 Results of the biosafety evaluation of MSE in the nude mouse model; A. AST, ALT, and UREA level detection; B. Body weight changes of nude mice in each group. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific examples, but the present invention is not limited thereto in any way.

[0026] First, the enzyme-responsive self-assembling peptide MSE was successfully synthesized, and the self-assembly process was verified by electron microscopy. Next, the fluorescence colocalization of MSE was verified by microscopy. The results demonstrated that H226 had good uptake of MSE and that it colocalized with the EGFR protein on the cell membrane surface. Its in vitro anti-tumor activity was then verified using various assays. The effects of different concentrations of MSE on the growth and proliferation of various cancer cells and normal cells were evaluated using the CCK-8 assay. The results showed that MSE at certain concentrations inhibited cancer cell proliferation, and this effect was concentration-dependent, with higher concentrations resulting in lower cell viability. However, this effect was not observed in normal 293T cells, likely due to the relatively low EGFR expression in 293T cells. Furthermore, the reported EGFR-binding peptide GE11 showed no significant anti-proliferation effect on either cancer or normal cells at the same concentration. Cell live-dead staining and plate cloning experiments also verified the in vitro anti-tumor ability of MSE. In the reactive oxygen species experiment, cancer cells underwent a significant change in green fluorescence enhancement. We have reason to infer that MSE is very likely to promote cell apoptosis through the reactive oxygen species signaling pathway, thereby exerting its drug effect.

[0027] To investigate the effects of MSE on EGFR, we next examined its ability to selectively degrade EGFR protein. Western blot results demonstrated time- and concentration-dependent degradation of EGFR by MSE, confirming its EGFR degradation. Subsequent nude mouse tumorigenesis experiments demonstrated significant inhibition of tumor growth, confirming the drug's in vivo antitumor activity. Furthermore, changes in nude mouse body weight and biochemical markers demonstrated its good biosafety profile.

[0028] The present invention proposes a 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. It is hoped that the new strategy of the present invention can provide a new perspective for the treatment of cancer.

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

[0030] The enzyme-responsive precursor MSE (YHWYGYTPQNVI-NPGY-KLVFF-PLGYLG-EEE) consists of five modules: EGFR binding peptide module, lysosomal sorting module, self-assembly module, enzyme response module, and hydrophilic module ( Figure 1 The peptide was synthesized using the solid-phase FMOC method, which involves adding amino acids one by one from the carboxyl end to the amino end. After the synthesis is complete, the side chain protecting groups are removed and the peptide is cleaved from the resin. It was purified by high-performance liquid chromatography and identified by mass spectrometry. The results showed ( Figure 2 ), MSE was successfully synthesized, purified by HPLC, and identified by mass spectrometry with a molecular weight of 3595.03Da.

[0031] Transmission electron microscopy (TEM) was used to examine the morphology of the enzyme reaction MSE before and after the addition of MMP-2. The results showed that ( Figure 3 ), MSE was aggregate-free in aqueous solution and transformed into a unique nanofilament structure after incubation with MMP-2 at a final concentration of 1 μg / mL at 37°C for 2 h.

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

[0033] To evaluate the uptake of MSE in vitro, cellular uptake experiments were performed.

[0034] We used 60 μM MSE labeled with Cy3 dye, which can excite red fluorescence, to verify whether the drug taken up by H226 cells binds to the EGFR receptor. The results showed that ( Figure 4 ), there was an obvious co-localization, which indicated that MSE could successfully bind to the EGFR receptor on the cell surface after being taken up by cells for 6 hours.

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

[0036] To more intuitively demonstrate the efficacy advantage of MSE, GE11 was used for comparison. GE11 (YHWYGYTPQNVI) is a widely reported and validated EGFR ligand that can specifically bind to EGFR.

[0037] The inhibitory effects of these two drugs on tumor cells were verified by treating cells with drug concentration gradient for 24 hours and then performing CCK8 cell activity detection. The results showed that ( Figure 5), MSE had a strong killing effect on H226, HCT-116, and SKOV3 cells in a dose-dependent manner, but had no significant killing effect on 293T cells. Further analysis of the cytotoxic effects of GE11 on tumor cells revealed no significant changes in the viability of the aforementioned tumor cells.

[0038] In order to further verify the killing effect of drugs on tumor cells more intuitively, a cell live-death staining experiment was performed ( Figure 6 ). The cells were seeded in a 48-well plate. After the cells were fully attached, 60 μM of the drug was added for treatment. PBS was used as the control group and the cells were cultured in the incubator for 24 hours before staining. Calcein is a calcium ion fluorescent indicator. Calcein-AM has good membrane penetration ability. After entering the cell, a strong green fluorescence is generated. It is only used to label living cells. PI cannot pass through the cell membrane of living cells and is excluded from living cells, but it can pass through damaged cell membranes and embed into the nuclear DNA structure to emit red fluorescence. Based on this principle, the above two dyes can be used in combination to distinguish and identify living cells from dead cells. The results showed that in the two cell lines H226 and HCT-116, the number of living cells in the MSE-treated group was significantly reduced, while the number of dead cells increased compared with the control group.

[0039] Next, in order to explore whether MSE causes damage to tumor cells through the reactive oxygen signaling pathway, the level of reactive oxygen species (ROS) was detected. Reactive oxygen species are substances with oxidative activity in cells. When produced excessively, they will cause oxidative stress and damage to the cells. DCFH-DA is an indicative probe that does not emit fluorescence itself and can freely pass through the cell membrane. After entering the cell, it is hydrolyzed to form DCFH under the catalysis of intracellular esterases and retained in the cell. ROS can oxidize non-fluorescent DCFH to generate DCF that can emit green fluorescence. Based on this principle, the level of reactive oxygen species in the cell can be determined by observing the fluorescence intensity, and then it can be judged whether the drug has damaged the tumor cells through the reactive oxygen signaling pathway. The cells were seeded in a 48-well plate. After the cells were fully attached, 60μM of the drug was added for treatment, and PBS was used as the control group. After 24 hours of incubation, staining was performed. The results showed that ( Figure 7 ), in both H226 and HCT-116 cell lines, the reactive oxygen species content in the MSE-treated group was significantly increased compared with the control group.

[0040] Example 4 Effect of Selective Degradation on EGFR Expression:

[0041] We explored the differences in EGFR degradation induced by MSE in HCT-116 cells. The CCK-8 results showed that the IC50 of MSE in H226 and HCT-116 cells was approximately 60 μM, respectively. Therefore, we added MSE to a final concentration of 60 μM to H226 cells, collected cells at different time points, extracted total protein, and performed western blot. The results showed that ( Figure 8 A) Drug exposure 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] In addition, the effect of different concentrations of MSE on EGFR protein degradation was studied. Different concentrations of MSE diluted with DMEM medium were added to H226 cells, and then total protein was collected after 24 hours for western blot. The results showed 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 anti-tumor effect of MSE:

[0044] By subcutaneously injecting HCT-116 cells, a xenograft tumor model was established in 9 nude mice, which were then randomly divided into 3 groups. Each nude mouse in the experimental group was injected with 50 μl of MSE or GE11 solution at a concentration of 1.5 μM / kg, while the control group was injected with an equal amount of normal saline for 8 consecutive days. During this period, the weight and volume of the nude mice were measured every two days. The results showed that both the MSE and GE11 experimental groups had varying degrees of inhibitory effects on tumor growth, but the inhibitory effect of the MSE experimental group was significantly stronger ( Figure 9 A). After taking pictures and weighing the tumors of the three groups, it was found that the tumor volume and size of the MSE experimental group were significantly smaller than those of the control group ( Figure 9 B).

[0045] The three biochemical indicators of ALT, AST and UREA were tested by extracting serum. The results showed that ( Figure 10 ), there was no significant difference between the experimental group and the control group ( Figure 10 A). We also analyzed the weight changes of nude mice and found that the weight of mice remained stable in both the experimental and control groups ( Figure 10 B).

Claims

1. A polypeptide MSE for enzyme-responsive targeted degradation of EGFR protein, characterized in that: The amino acid sequence of the polypeptide MSE is YHWYGYTPQNVINPGY-KLVFFPLGYLGEEE.

2. Use of the polypeptide MSE according to claim 1 in the preparation of anti-tumor drugs.

3. The use according to claim 2, characterized in that The tumor is lung cancer, colorectal cancer or ovarian cancer.

4. The use according to claim 2, characterized in that The application is that the polypeptide MSE co-localizes and binds to the EGFR protein on the cell membrane surface to degrade EGFR.

5. The use according to claim 2, characterized in that The application is that the polypeptide MSE promotes tumor cell apoptosis through the reactive oxygen signaling pathway.

6. Use of a pharmaceutical composition in the preparation of an anti-tumor drug, the pharmaceutical composition comprising the polypeptide MSE according to claim 1.

7. The use according to claim 6, characterized in that The tumor is lung cancer, colorectal cancer or ovarian cancer.

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