A short peptide for inhibiting PRDX1 O-GlcNAc modification and its application

Abstract

This invention provides a short peptide for inhibiting PRDX1 O-GlcNAc modification and its application. A short peptide (CPP-G1) containing a cell-penetrating peptide (CPP), fluorescently labeled FITC, and the first 14 amino acids of PRDX1 was chemically synthesized, and a control peptide with an S2 / 3 site mutation (CPP-SA) was constructed. Experiments showed that CPP-G1 effectively entered non-small cell lung cancer (NSCLC) A549 cells and downregulated PRDX1 expression by competitively binding to the O-GlcNAc modification site, thereby enhancing the cells' sensitivity to radiotherapy. In vivo experiments further confirmed that intratumoral injection of the CPP-G1 short peptide combined with radiotherapy significantly inhibited tumor growth, and mice showed good tolerance to the short peptide without significant side effects. This provides a new potential therapeutic approach for radiosensitizing NSCLC.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a short peptide for inhibiting PRDX1O-GlcNAc modification and its application. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide, and radiotherapy is one of its primary treatment methods. However, the resistance of tumor cells to radiotherapy severely limits its effectiveness. Therefore, inhibiting O-GlcNAc modification of PRDX1 has become a potential strategy to enhance radiosensitivity.

[0003] In existing technologies, small molecule inhibitors are mainly used to inhibit OGT, a key enzyme in O-GlcNAc modification, thereby broadly suppressing intracellular glycosylation. However, this strategy has the following drawbacks:

[0004] Firstly, there are the side effects of non-specific inhibition. OGT inhibitors, due to their broad inhibition of cellular glycosylation, may lead to various adverse side effects, including affecting the function and metabolism of normal cells and even causing systemic toxicity. Secondly, the therapeutic effect is limited. Non-specific inhibition may not be effective against specific pathological processes, such as PRDX1 O-GlcNAc modification, resulting in limited therapeutic effects and failing to fully enhance the sensitivity of tumor cells to radiotherapy. Moreover, small molecule inhibitors face challenges in terms of intracellular delivery efficiency and specificity, and may not be able to effectively reach the target site, thus affecting the therapeutic effect. Current technologies lack precise means to regulate PRDX1 O-GlcNAc modification, making it impossible to achieve specific and efficient targeted therapy. Therefore, we have made improvements to this by proposing a short peptide for inhibiting PRDX1 O-GlcNAc modification and its application. Summary of the Invention

[0005] The purpose of this invention is to address the problems raised in the existing background technology. To achieve the above-mentioned objective, this invention provides the following technical solution: a sequence of...

[0006] The FITC-YGRKKRRQRRR-MSSGNAKIGHPAPN (CPP-G1) is a short peptide, where MSSGNAKIGHPAPN is the original 14 amino acid sequences of the PRDX1 protein, capable of O-glycosylation modification. FITC is a fluorescent label used to indicate cellular uptake of the short peptide; YGRKKRRQRRRR (CPP) is the cell-penetrating peptide used to promote the entry of the short peptide into cells; and YGRKKRRQRRR is the cell membrane-penetrating peptide segment of CPP.

[0007] As a preferred technical solution of the present invention, it also includes another short peptide for inhibiting PRDX1 O-GlcNAc modification. The sequence of the short peptide is FITC-YGRKKRRQRRR-MAAGNAKIGHPAPN (CPP-SA), where FITC is a fluorescent marker used to indicate cellular uptake of the short peptide; YGRKKRRQRRR (CPP) is a cell-penetrating peptide used to promote the entry of the short peptide into the cell; the cell membrane-penetrating peptide YGRKKRRQRRR of CPP, and MAAGNAKIGHPAPN is the sequence after the serine (S) at positions 2 and 3 of the first 14 amino acids of the PRDX1 protein is mutated to alanine (A), used to mimic the PRDX1 protein but without O-GlcNAc glycosylation modification, as a negative control.

[0008] As a preferred technical solution of the present invention, the application of short peptides in the preparation of drugs for inhibiting PRDX1 O-GlcNAc modification.

[0009] As a preferred embodiment of the present invention, it comprises a short peptide and a pharmaceutically acceptable carrier or diluent.

[0010] As a preferred embodiment of the present invention, the pharmaceutical composition for inhibiting PRDX1 O-GlcNAc-modified short peptides is used in the preparation of a medicament for treating diseases caused by abnormal PRDX1 O-GlcNAc modification.

[0011] A method for detecting the modification state of PRDX1 O-GlcNAc includes the following steps:

[0012] Step 1: Introduce the short peptide (CPP-G1) of claim 1 or the short peptide (CPP-SA) of claim 2 into the cells or tissues to be tested;

[0013] Step 2: Detect fluorescence signals to confirm whether the short peptide has successfully entered the cell;

[0014] Step 3: Assess the O-GlcNAc modification status of PRDX1 by comparing its binding to O-glycosyltransferase or changes in O-GlcNAc modification levels.

[0015] As a preferred technical solution of the present invention, the application of competitive binding short peptides targeting the O-GlcNAc modification site of PRDX1 in enhancing the radiosensitivity of non-small cell lung cancer is discussed.

[0016] A method for inhibiting PRDX1 O-GlcNAc modification involves contacting a short peptide with cells or tissues to be treated, thereby inhibiting PRDX1 O-GlcNAc modification.

[0017] As a preferred technical solution of the present invention, the method for inhibiting PRDX1 O-GlcNAc modification is used in the preparation of a treatment regimen for treating diseases caused by abnormal PRDX1 O-GlcNAc modification.

[0018] An agent for targeting diseases caused by abnormal PRDX1 O-GlcNAc modification comprises a short peptide and an agent for implementation.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. Specific inhibition, reduced side effects: By designing short peptides containing the S1 / 2 sites on PRDX1, competitive binding to OGT, which normally binds to PRDX1, is achieved, thereby inhibiting O-GlcNAc modification at this site without interfering with the overall cellular glycosylation process. Compared to small molecule inhibitors of OGT, this strategy significantly reduces the risk of adverse side effects caused by broad inhibition of glycosylation.

[0021] 2. Enhanced radiosensitivity: The short peptide can downregulate PRDX1 expression, thereby increasing the sensitivity of non-small cell lung cancer (NSCLC) cells to radiotherapy. This property provides a new sensitization strategy for NSCLC radiotherapy, which is expected to improve radiotherapy efficacy, reduce radiotherapy dose, and thus reduce the side effects of radiotherapy.

[0022] 3. Efficient Delivery and Intracellular Localization: Cell-penetrating peptides (CPPs) are used to efficiently deliver short peptide sequences into cells. The application of fluorescently labeled FITC visualizes the intracellular localization of short peptides, facilitating real-time monitoring of delivery efficiency and distribution. This delivery system ensures an effective intracellular concentration of short peptides, thereby enhancing their biological activity.

[0023] 4. Dose-dependent effect: Treatment with different concentrations of CPP-G1 peptide showed a dose-dependent reduction in PRDX1 O-GlcNAcization and expression. This controllable regulation provides flexible dose adjustment space for clinical applications, which helps to optimize treatment effects and reduce potential toxicity.

[0024] 5. Specificity and accuracy: The first 14 amino acid sequences of PRDX1 were selected as the basis for short peptide design. This ensures that the short peptide can mimic the binding of PRDX1 to glycosyltransferase, while the shorter length improves cell membrane penetration efficiency and specificity, reduces non-specific binding, and thus improves the accuracy and effectiveness of treatment.

[0025] 6. Negative control verification: By designing a CPP-SA short peptide with an S2 / 3 site mutation of A as a negative control, the specific mechanism of action of the CPP-G1 short peptide was further verified, enhancing the reliability and persuasiveness of the experimental results.

[0026] 7. In vivo experiments verifying safety and efficacy: In vivo experiments in the BALB / c nude mouse model showed that mice tolerated the CPP-short peptide well after intratumoral injection, without significant toxicity or lethal side effects. Simultaneously, the combination of the short peptide and radiotherapy significantly inhibited tumor growth, demonstrating the safety and efficacy of this treatment regimen and providing strong experimental evidence for clinical application.

[0027] 8. Potential Clinical Application Value: This technology provides a new treatment strategy for radiosensitization in NSCLC and has potential clinical application value. By specifically inhibiting the O-GlcNAc modification of PRDX1, combined with radiotherapy, it is expected to improve the treatment effect and quality of life of NSCLC patients. Attached Figure Description

[0028] Figure 1 This diagram illustrates how the short peptide can competitively bind to O-glycosylation modification sites by constructing a GFP fusion plasmid, as provided by the present invention.

[0029] Figure 2 This is a schematic diagram illustrating the competitive delivery of short peptides into cells using the cell-penetrating peptide (CPP) provided by the present invention.

[0030] Figure 3 A schematic diagram of the in vivo animal experiment curve of intratumoral injection of CPP-short peptide (5 mg / kg) combined with radiotherapy for sensitization provided by the present invention;

[0031] Figure 4 This is a schematic diagram of an in vivo animal experiment involving intratumoral injection of CPP-short peptide (5 mg / kg) combined with radiotherapy to enhance sensitization, provided by the present invention.

[0032] Figure 5 This is a schematic diagram of the design of cell-penetrating peptide (CPP) coupled with short peptides provided by the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0034] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0035] Example 1: A short peptide with the sequence FITC-YGRKKRRQRRR-MSSGNAKIGHPAPN(CPP-G1), wherein MSSGNAKIGHPAPN is the original 14 amino acid sequences of the PRDX1 protein, capable of O-glycosylation modification. FITC is a fluorescent label used to indicate cellular uptake of the short peptide; YGRKKRRQRRR(CPP) is a cell-penetrating peptide used to promote cell entry of the short peptide; and the CPP is the cell membrane-penetrating peptide YGRKKRRQRRR.

[0036] The present invention also includes another short peptide for inhibiting PRDX1 O-GlcNAc modification. The sequence of the short peptide is FITC-YGRKKRRQRRR-MAAGNAKIGHPAPN (CPP-SA), wherein FITC is a fluorescent label used to indicate cellular uptake of the short peptide; YGRKKRRQRRR (CPP) is a cell-penetrating peptide used to promote the entry of the short peptide into the cell; the cell membrane-penetrating peptide YGRKKRRQRRR of CPP, and MAAGNAKIGHPAPN is the sequence after the serine (S) at positions 2 and 3 of the first 14 amino acids of the PRDX1 protein is mutated to alanine (A), used to mimic the PRDX1 protein but not undergo O-GlcNAc glycosylation modification.

[0037] As a negative control.

[0038] The present invention relates to the use of short peptides in the preparation of drugs for inhibiting PRDX1 O-GlcNAc modification.

[0039] This invention comprises short peptides and pharmaceutically acceptable carriers or diluents.

[0040] The present invention relates to the use of a pharmaceutical composition for inhibiting PRDX1 O-GlcNAc-modified short peptides in the preparation of a medicament for treating diseases caused by abnormal PRDX1 O-GlcNAc modification.

[0041] A method for detecting the modification state of PRDX1 O-GlcNAc includes the following steps:

[0042] Step 1: Introduce the short peptide (CPP-G1) of claim 1 or the short peptide (CPP-SA) of claim 2 into the cells or tissues to be tested;

[0043] Step 2: Detect fluorescence signals to confirm whether the short peptide has successfully entered the cell;

[0044] Step 3: Assess the O-GlcNAc modification status of PRDX1 by comparing its binding to O-glycosyltransferase or changes in O-GlcNAc modification levels.

[0045] The method of the present invention for detecting the modification state of PRDX1 O-GlcNAc is applied in the preparation of reagents for diagnosing diseases caused by abnormal PRDX1 O-GlcNAc modification.

[0046] A method for inhibiting PRDX1 O-GlcNAc modification involves contacting a short peptide with cells or tissues to be treated, thereby inhibiting PRDX1 O-GlcNAc modification.

[0047] The application of the method for inhibiting PRDX1 O-GlcNAc modification in the preparation of a treatment regimen for treating diseases caused by abnormal PRDX1 O-GlcNAc modification.

[0048] An agent for targeting diseases caused by abnormal PRDX1 O-GlcNAc modification comprises a short peptide and an agent for implementation.

[0049] Example 2: A short peptide for inhibiting PRDX1 O-GlcNAc modification was synthesized by chemical synthesis. The short peptide sequence containing the fluorescent label FITC, CPP and the first 14 amino acids of PRDX1 (CPP-G1) or the sequence with the S2 / 3 O glycosylation modification site mutated to A (CPP-SA) was synthesized.

[0050] CPP-G1:FITC-YGRKKRRQRRR-MSSGNAKIGHPAPN;

[0051] CPP-SA:FITC-YGRKKRRQRRR-MAAGNAKIGHPAPN;

[0052] The specific design principle is as follows: The S2 / 3 site of PRDX1 is an important functional site, and its O-glycosylation modification affects the stability of the PRDX1 protein. CPP is a small peptide that can penetrate the cell membrane and is often used to deliver biomolecules such as drugs, proteins, or nucleic acids into cells. Its amino acid sequence is: YGRKKRRQRRR. The first 14 amino acid sequences of PRDX1 are: MSSGNAKIGHPAPN, where the 2nd and 3rd sites are O-glycosylation modification sites. The reason for choosing the first 14 amino acids to simulate the O-glycosylation modification of PRDX1 is that a shorter length helps it penetrate the cell membrane more effectively, thereby improving delivery efficiency and reducing non-specific binding, thus improving the specificity and accuracy of this short peptide in simulating the binding of PRDX1 to glycosyltransferases. CPP-SA is a short peptide with the S2 / 3 site mutated to A, which cannot undergo O-glycosylation modification, and serves as a negative control for subsequent experiments. FITC is added to the N-terminus of this short peptide as a fluorescent signal indicating whether the short peptide has entered the cell.

[0053] To detect whether short peptides can competitively bind to O-glycosylation sites and undergo O-glycosylation modification, plasmids fused with GFP tags (GFP-SA and GFP-G1) were constructed. These plasmids, either of equal mass or co-transfected with O-glycosyltransferase OGT overexpression plasmids, were transfected into the non-small cell lung cancer cell line A549. Cell proteins were extracted 72 hours after transfection. Co-IP assays using a GFP antibody were performed to detect the expression of O-GlcNAc. Figure 1 As shown, the results revealed that the GFP-G1 group had significantly higher levels than the GFP-SA group, the two OGT-transfected groups had significantly higher levels than the untransfected group, and the GFP-G1+OGT group had significantly higher levels than the GFP-SA+OGT group. This indicates that GFP-G1 can competitively bind to O-glycosylation modification sites in the presence of OGT.

[0054] like Figure 2 Different concentrations of short peptides (5 μM, 10 μM, 20 μM) were co-incubated with the non-small cell lung cancer cell line A549 for 20 hours. FITC fluorescence intensity was detected by confocal microscopy, and the fluorescence intensity showed a concentration-dependent increase, indicating that the short peptides were successfully delivered into the cells.

[0055] BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. A549 cells were digested from the cell culture dish using trypsin for accurate cell counting, followed by washing with PBS to remove culture medium and serum. 4 × 10⁶ cells were then... 6 One cell was dissolved in PBS to a total volume of approximately 200 μL and injected into the right hind limb of a mouse. Tumor size was measured weekly using calipers, and tumor volume (V) was calculated using the formula V = (length × width²) / 2. When the tumor size reached 100 mm...3 Mice were randomly divided into different groups, with 5 mice in each group. The above-mentioned CPP-short peptide (5 mg / kg) was injected intratumorally once, followed by another intratumoral injection every other day for a total of 3 times. One day after the last injection of the short peptide, the tumor received a single 15 Gy radiation dose (the day of radiation therapy was counted as day 0). Tumor volume was then measured weekly until a tumor reached 1500 mm². 3 Afterwards, the tumors were removed and weighed. From the injection of the short peptide until the tumor measurement endpoint, mice showed good tolerance to the short peptide at the experimental dose, without causing significant toxic or fatal side effects, demonstrating high safety.

[0056] Experimental Example: In vivo application of short peptides used to inhibit PRDX1 O-GlcNAc modification in tumor therapy

[0057] Experimental Objective

[0058] To evaluate the inhibitory effect of a short peptide (CPP-SA) containing cell-penetrating peptide (CPP) on PRDX1 O-GlcNAc modification in vivo, and its inhibitory effect on tumor growth when combined with radiotherapy.

[0059] Experimental materials

[0060] 1. Animal models:

[0061] BALB / c nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.)

[0062] 2. Cell lines:

[0063] A549 non-small cell lung cancer cell line

[0064] 3. Short peptides:

[0065] Inhibits the modification of PRDX1 O-GlcNAc by the short peptide (CPP-G1):

[0066] FITC-YGRKKRRQRRR-MSSGNAKIGHPAPN

[0067] 4. Reagents and instruments:

[0068] Pancreatic enzymes, PBS solution, vernier calipers, radiotherapy equipment

[0069] 5. Ethical Approval:

[0070] The experimental procedures were approved by the Animal Nursing and Use Ethics Committee of Shandong Cancer Hospital and Shandong First Medical University.

[0071] 1. Establishment of tumor models:

[0072] The A549 cell line was digested from the culture dish and accurately counted.

[0073] Wash the cells with PBS solution to remove the culture medium and serum.

[0074] 4×10 6 One cell was dissolved in approximately 200 μL of PBS and injected into the right hind limb of each mouse.

[0075] The tumor size was measured weekly using calipers, and the tumor volume (V = length × width) was calculated. 2 / 2).

[0076] 2. Experimental Groups:

[0077] When the tumor volume reaches 100mm 3 Mice were randomly divided into different experimental groups, with 5 mice in each group. The experimental groups included:

[0078] Control group (received no treatment)

[0079] Short peptide group (only receiving CPP-G1 short peptide injections)

[0080] Radiation therapy group (receiving radiation therapy only)

[0081] Short peptide + radiotherapy group (receiving CPP-G1 short peptide injection and radiotherapy)

[0082] Negative control group (received only CPP-SA short peptide injections)

[0083] Negative control group (short peptide + radiotherapy group, receiving CPP-SA short peptide injection and radiotherapy)

[0084] 3. Short peptide injection:

[0085] Intratumoral injection of CPP-G1 or CPP-SA short peptide at a dose of 5 mg / kg.

[0086] The injection is given once every other day, for a total of 3 times.

[0087] 4. Radiation therapy:

[0088] On day 1 following the last short peptide injection, the tumor was given a single 15 Gy radiation dose.

[0089] The day you receive radiotherapy is counted as day 0.

[0090] 5. Tumor volume measurement:

[0091] Continue to measure tumor volume weekly and record tumor growth.

[0092] When a tumor reaches 1500mm 3 At that point, the experiment ended, the tumor was removed, and weighed.

[0093] 6. Security Assessment:

[0094] Observe the health status of mice throughout the experiment and record any toxic or fatal side effects.

[0095] Assess the tolerance of mice to short peptides.

[0096] Experimental Results Analysis

[0097] 1. Tumor growth curves: Tumor growth curves of mice in each group were used to compare changes in tumor volume, such as... Figure 3 and Figure 4 As shown.

[0098] 2. Tumor weight analysis: The tumors removed at the end of the experiment were weighed, and the differences between the groups were compared.

[0099] 3. Safety assessment: Analyze the health status of mice to assess the safety and tolerability of the short peptide.

[0100] 4. Statistical analysis: Use appropriate statistical methods (such as Two-way ANOVA with two-sided unpaired Student's t-test) to analyze the data and determine the significant differences between the groups.

[0101] Expected results

[0102] 1. The tumor growth rate in the short peptide CPP-G1+ radiotherapy group was significantly lower than that in other groups.

[0103] 2. The tumor weight in the short peptide CPP-G1+ radiotherapy group was significantly lower than that in other groups.

[0104] 3. Mice tolerated the short peptide well, and no obvious toxic or lethal side effects were observed.

[0105] Experimental conclusions

[0106] This experiment aims to evaluate the inhibitory effect of short peptides modified with PRDX1 O-GlcNAc on tumor growth in vivo, and their therapeutic efficacy when used in combination with radiotherapy. Expected results show that the combination of short peptides and radiotherapy can significantly enhance the efficacy of tumor treatment, and that the short peptides exhibit good safety and tolerability at the experimental dose, providing a potential therapeutic strategy for clinical application.

[0107] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.

Claims

1. A short peptide for inhibiting PRDX1 O-GlcNAc modification, characterized in that, The sequence of the short peptide is FITC-YGRKKRRQRRR-MSSGNAKIGHPAPN; wherein, MSSGNAKIGHPAPN is the original amino acid sequence of the first 14 positions of the PRDX1 protein, which can undergo O-glycosylation modification; FITC is a fluorescent label used to indicate cellular uptake of the short peptide; YGRKKRRQRRR is a cell-penetrating peptide used to promote the entry of the short peptide into the cell.

2. The short peptide according to claim 1, characterized in that, The cell-penetrating peptide segment YGRKKRRQRRR of the short peptide can effectively promote the entry of the short peptide into non-small cell lung cancer cells.

3. A pharmaceutical composition for inhibiting PRDX1 O-GlcNAc-modified short peptides, characterized in that, It comprises the short peptide of claim 1 and a pharmaceutically acceptable carrier.

4. The use of the short peptide according to claim 1 in the preparation of an in vitro reagent to enhance the radiosensitivity of non-small cell lung cancer cells.

5. An in vitro reagent for targeting abnormal PRDX1 O-GlcNAc modification in non-small cell lung cancer, comprising the short peptide of claim 1, and a reagent for detecting cellular uptake of the short peptide or the level of PRDX1 O-GlcNAc modification.

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