Cell-penetrating fusion peptide S517-pe and application thereof
By designing a cell-penetrating fusion peptide S517-pe to target the O-GlcNAc modification site of GLUD1, the problems of selective targeting and drug permeability in liver cancer treatment were solved, achieving a highly efficient inhibitory effect on liver cancer cells.
Patent Information
- Application Number
- CN202511540651.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-27
- Publication Date
- 2026-02-27
AI Technical Summary
Existing treatments for liver cancer have significant side effects and lead to drug resistance in cancer cells. Furthermore, the low permeability of cell membranes to drugs results in low treatment efficiency, and there is a lack of selective targeted intervention strategies for liver cancer cells.
A cell-penetrating fusion peptide, S517-pe, was designed to specifically target the O-GlcNAc modification site S517 of the GLUD1 protein, interfering with its glycosylation modification process, inhibiting the stability and function of key oncogenic proteins, arresting the cell cycle, inducing apoptosis, and enhancing immune clearance.
It significantly inhibits the proliferation and migration of liver cancer cells, providing a new and highly effective drug for targeted therapy of liver cancer, and has potential for clinical application.
Smart Images
Figure CN121574953A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to a cell-penetrating fusion peptide and its application in pharmaceuticals. Background Technology
[0002] Primary liver cancer ranks sixth in incidence and third in cancer-related mortality worldwide. Hepatocellular carcinoma (HCC) accounts for approximately 80% of clinical cases, making it the dominant type of primary liver cancer. Therefore, in-depth research into the molecular mechanisms of liver cancer development and progression, and the discovery of specific prognostic biomarkers, are of crucial clinical significance for early diagnosis, disease monitoring, and personalized treatment. Simultaneously, identifying new therapeutic targets holds promise for developing more effective targeted therapies and treatment regimens, potentially overcoming the current limitations in liver cancer treatment and bringing new hope to patients.
[0003] Glycosylation is a highly regulated enzymatic process, referring to the post-translational modification in which oligosaccharide chains are covalently linked to specific amino acid residues in proteins under the action of glycosyltransferases. The main types include N-linked (asparagine site) and O-linked (serine / threonine site) glycosylation. These glycan structures play important biological functions through mechanisms such as mediating molecular recognition, regulating protein stability and activity, and participating in immune responses. Abnormal expression of these glycans is closely related to various pathological states such as tumor progression and neurodegenerative diseases. In liver cancer, abnormally upregulated O-GlcNAc modification levels not only affect cellular signal transduction and metabolic remodeling but also participate in regulating various abnormal biological behaviors of liver cancer, such as progression, metastasis, and drug resistance. Therefore, targeted intervention of O-GlcNAc modification holds promise as an effective anti-liver cancer treatment strategy. However, existing intervention strategies all alter the overall O-GlcNAc modification level, lacking selectivity and specificity for substrate proteins, easily disrupting the overall dynamic homeostasis and affecting normal cellular biological functions. Based on this, targeted blocking of target protein O-GlcNAc modification may become a novel O-GlcNAc modification intervention strategy.
[0004] Currently, cancer treatment mainly relies on the combined use of surgery, radiotherapy, and chemotherapy. However, these therapies are often accompanied by significant side effects, and cancer cells are prone to developing drug resistance. A major challenge in cancer treatment is the low permeability of cell membranes to therapeutic drugs, highlighting the urgent need to develop more efficient drug delivery systems.
[0005] Cell-penetrating peptides (CPPs) are a class of short-chain polypeptides containing 5-30 amino acid residues that can independently cross the cell membrane and enter the cell through interaction with the exposed plasma membrane. Therefore, CPPs can act as transport proteins, transporting large molecules such as peptides, nanoparticles, or oligonucleotides across the cell membrane into the cell. Because CPPs are endogenous or nearly endogenous proteins, they possess good biocompatibility and immunogenicity, making them relatively easy to apply in vivo. In recent years, CPPs have been well-established as promoters of intracellular delivery and have been developed to incorporate targeted specificity, demonstrating great potential for targeted delivery of anticancer drugs. Summary of the Invention
[0006] The purpose of this invention is to study a cell-penetrating fusion peptide modified with the target protein O-GlcNAc and to investigate its application value in the targeted therapy of hepatocellular carcinoma, with the aim of developing new and highly effective targeted drugs for the treatment of hepatocellular carcinoma.
[0007] Based on research, the present invention provides the following technical solution:
[0008] 1. Cell-penetrating fusion peptide S517-pe, amino acid sequence YGRKKRRQRRRAYTMERSARQIMR (SEQ ID No. 3).
[0009] 2. The application of the cell-penetrating fusion peptide S517-pe in the preparation of drugs for treating hepatocellular carcinoma.
[0010] Furthermore, the drug for treating hepatocellular carcinoma is a drug that inhibits the proliferation and migration of liver cancer cells.
[0011] This invention has found that the O-GlcNAc modification level of glutamate dehydrogenase 1 (GLUD1) is significantly upregulated in liver cancer tissues; mass spectrometry identification shows that serine residue 517 (S517) of GLUD1 protein is a potential O-GlcNAc modification site.
[0012] Cell-penetrating fusion peptides designed based on the peptide segments containing glycosylation sites can specifically compete for glycosylation sites of tumor-associated proteins, interfere with their normal glycosylation modification process, thereby inhibiting the stability and function of key oncogenic proteins, reversing tumor metabolic reprogramming, and destroying the protective glycocalyx barrier. Ultimately, they exert anti-cancer effects through multiple mechanisms such as cell cycle arrest, apoptosis induction, and enhanced immune clearance.
[0013] Based on the peptide segment containing the O-GlcNAc modification site S517 of the GLUD1 protein, this invention designed and synthesized three cell-penetrating fusion peptides. Experiments have confirmed that the cell-penetrating fusion peptide S517-pe can specifically target and inhibit the O-GlcNAc modification of GLUD1, significantly inhibiting the proliferation and migration of liver cancer cells, and can be used to prepare drugs for the treatment of hepatocellular carcinoma.
[0014] The beneficial effects of this invention are as follows: This invention provides a cell-penetrating fusion peptide S517-pe and its application in the preparation of drugs for treating hepatocellular carcinoma, providing a new targeted drug for the treatment of hepatocellular carcinoma and having the potential to be promoted to clinical diagnosis and treatment. Attached Figure Description
[0015] Figure 1 The results show the quantitative analysis of O-GlcNAc modification levels of GLUD1 in 42 pairs of human liver cancer and adjacent normal tissues by Western blot grayscale values (A) and the detection of O-GlcNAc modification levels of GLUD1 in 6 pairs of human liver cancer and adjacent normal tissues by succinylated wheat germ agglutinin (sWGA) assay (B), where N represents adjacent normal tissue and T represents liver cancer tissue.
[0016] Figure 2 The image shows that S517 was identified as the O-GlcNAc glycosylation modification site of GLUD1 by immunoprecipitation tandem mass spectrometry (IP-MS), and the sequence DIVHSGLAYTMERSAR in the figure represents amino acid residues 504-519 in the GLUD1 protein.
[0017] Figure 3 The screening of cell-penetrating fusion peptides that inhibit GLUD1 O-GlcNAc modification is shown: A is a schematic diagram of the amino acid sequences of three cell-penetrating fusion peptides; B is a sWGA experiment screening of cell-penetrating fusion peptides that can inhibit GLUD1 O-GlcNAc modification, where con, 1#, 2#, and 3# represent blank control, S517-Peptide-1#, S517-Peptide-2#, and S517-Peptide-3#, respectively; C is a schematic diagram of the amino acid sequences of the cell-penetrating fusion peptide S517-Pe (i.e., S517-Peptide-3#) and its mutant A517-Pe.
[0018] Figure 4 The study demonstrated that the inhibition of GLUD1 O-GlcNAc-modified cell-penetrating fusion peptide S517-Pe inhibited the proliferation of liver cancer cells, where vehicle represents the blank control.
[0019] Figure 5The study showed that the cell-penetrating fusion peptide S517-Pe, modified with GLUD1 O-GlcNAc, inhibited the migration of liver cancer cells. In the figure, A represents the results of the Tranwell assay, B represents the results of the scratch assay, and vehicle represents the blank control. Detailed Implementation
[0020] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the preferred embodiments of this invention are described in detail below. Experimental methods in the preferred embodiments that do not specify specific conditions are generally performed under conventional conditions.
[0021] Example 1. Identification of GLUD1 O-GlcNAc modification sites
[0022] Forty-two pairs of human liver cancer and adjacent normal tissue samples were extracted, and the O-GlcNAc modification level of GLUD1 was detected by Western blot and sWGA experiments. It was found that the O-GlcNAc modification level of GLUD1 was significantly upregulated in liver cancer tissues. Figure 1 ).
[0023] Endogenous GLUD1 protein was enriched using magnetic beads, and IP-MS analysis revealed that serine residue 517 (S517) in the GLUD1 protein is a potential O-GlcNAc modification site. Figure 2 ).
[0024] Example 2. Design and screening of cell-penetrating fusion peptides that inhibit GLUD1 O-GlcNAc modification
[0025] The sequence adjacent to the S517 site of the O-GlcNAc modification site of GLUD1 was analyzed, and three peptides containing the S517 site were extracted and fused with the cell-penetrating peptide TAT (amino acid sequence YGRKKRRQRRR) to obtain three cell-penetrating fusion peptides. Figure 3 A):
[0026] S517-Peptide-1#: The amino acid sequence is YGRKKRRQRRR RSARQIMRTAMK (SEQ ID No. 1).
[0027] S517-Peptide-2#: The amino acid sequence is YGRKKRRQRRR HSGLAYTMERSAR (SEQ ID No. 2).
[0028] S517-Peptide-3#: The amino acid sequence is YGRKKRRQRRR AYTMERSARQIMR (SEQ ID No. 3).
[0029] The three cell-penetrating fusion peptides were subjected to sWGA experiments. The results showed that S517-Peptide-3# specifically targeted GLUD1 O-GlcNAc modification and exhibited the most significant inhibitory effect on GLUD1 O-GlcNAc modification. Figure 3 B). In subsequent experiments, the cell-penetrating fusion peptide S517-Peptide-3# was named S517-pe.
[0030] Based on the above sWGA experimental results, the S residue (S517) in the S517-pe sequence was mutated to an A residue (A517), causing it to lose its ability to inhibit GLUD1 O-GlcNAc modification. The resulting mutant was named A517-pe and served as a negative control. Figure 3 C).
[0031] Example 3. Inhibition of the proliferation of liver cancer cells by GLUD1 O-GlcNAc-modified cell-penetrating fusion peptide.
[0032] Human hepatocellular carcinoma cells PLC / PRF / 524h were treated with 20 μM cell-penetrating fusion peptide S517-pe, with its mutant A517-pe (20 μM) as a negative control and PBS as a blank control. EdU cell proliferation assay was used to detect whether S517-pe could affect the proliferation of hepatocellular carcinoma cells by inhibiting GLUD1 O-GlcNAc modification.
[0033] EdU cell proliferation assay results showed that S517-pe treatment significantly reduced the proliferation ability of liver cancer cells, while the negative control A517-pe treatment did not show a significant inhibitory effect. Figure 4 ).
[0034] Example 4. Inhibition of cell-penetrating fusion peptide modified with GLUD1 O-GlcNAc to inhibit the migration of liver cancer cells.
[0035] PLC / PRF / 5 cells were treated with the cell-penetrating fusion peptide S517-pe (20 μM), with its mutant A517-pe (20 μM) as a negative control and PBS as a blank control. Then, the Transwell assay and scratch assay were used to detect whether S517-pe could affect the migration of liver cancer cells by inhibiting GLUD1 O-GlcNAc modification.
[0036] Both Transwell and scratch assays showed that S517-pe treatment significantly reduced the migration ability of liver cancer cells, while the negative control A517-pe treatment did not show a significant inhibitory effect. Figure 5 ).
[0037] The above results indicate that the cell-penetrating fusion peptide S517-pe can specifically target and significantly inhibit the O-GlcNAc modification of GLUD1, and can significantly inhibit the proliferation and migration of liver cancer cells, and can be used to prepare drugs for the treatment of hepatocellular carcinoma.
[0038] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. Cell-penetrating fusion peptide S517-pe, with the amino acid sequence YGRKKRRQRRRAYTMERSARQIMR.
2. The use of the cell-penetrating fusion peptide S517-pe according to claim 1 in the preparation of a medicament for treating hepatocellular carcinoma.
3. The application as described in claim 2, characterized in that: The drug used to treat hepatocellular carcinoma is one that inhibits the proliferation and migration of liver cancer cells.