Arginine methylation nano proximity inducer, preparation method and application thereof
By linking the target protein arginine methyltransferase with the affinity peptide of the target protein through gold nanoclusters, a nano-proximity inducer for arginine methylation was designed, solving the problem of precise regulation of methylation modification of specific proteins and achieving rapid and efficient intracellular targeted delivery and modification.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHENGZHOU UNIV
- Filing Date
- 2026-03-12
- Publication Date
- 2026-06-09
AI Technical Summary
Existing technologies struggle to achieve precise regulation of arginine methylation modification of specific proteins, especially in targeted delivery and modification within cells.
By using gold nanoclusters as linkers to connect the affinity peptides of the target protein arginine methyltransferase with the affinity peptides of the target protein, arginine methylation nano-proximity inducers were designed to achieve specific methylation modification of the target protein.
It achieves rapid and efficient regulation of arginine methylation modification of specific target proteins, overcomes off-target effects, and shows significant in vivo effects, enabling methylation modification of the same target protein in different cell types.
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Figure CN122167589A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology and relates to a protein post-translational modification inducer, specifically to an arginine methylation nano-proximity inducer, its preparation method, and its application. Background Technology
[0002] Post-translational modifications (PTMs) refer to specific chemical modifications that introduce structural changes into existing proteins to regulate various biological processes. They are a crucial dynamic mechanism for regulating cellular proteins. PTMs are diverse, including glycosylation, ubiquitination, phosphorylation, acetylation, and methylation. These PTMs play important roles in various physiological activities of living organisms, regulating multiple biological processes, and their imbalance is associated with various diseases. Among these, protein methylation and demethylation are important post-translational modifications.
[0003] Protein methylation is a form of post-translational modification, generally referring to the methylation of arginine or lysine residues in a protein sequence. Arginine can be methylated once or twice, transferring both methyl groups simultaneously to the same nitrogen atom at the end of the arginine polypeptide to form asymmetric methylarginine, or adding a methyl group to each nitrogen terminus. Lysine can be methylated once, twice, or three times catalyzed by lysine transferases. The biochemical process of removing methyl groups from modified amino acid residues under the action of protein demethylases is a reversible process of protein methylation. Methylation modification is jointly regulated by protein methyltransferases and demethylases, and is a reversible process. Disruption of protein methylation homeostasis can lead to various human diseases.
[0004] Existing technologies disclose the use of chemical inhibitors or activators of overexpression, gene knockout, or gene knockout of post-translational modifying enzymes to manipulate the post-translational modification of proteins in cells. However, since genetic engineering methods are difficult to use as a treatment, it is necessary to develop a tool to precisely regulate protein arginine methylation modification and achieve specific targeting of protein arginine methylation post-translational modification in vivo.
[0005] "Proximity" is an academic term; for example, the commonly used term "proximity chemical inducer" uses small molecule drugs to induce protein-protein binding. Specific bifunctional materials, using a linker as a linker, can simultaneously bind to endogenous effectors and target proteins, allowing the two proteins to approach each other and exert their effects. By selectively regulating the post-translational modification (PTM) of the target protein (POI), this becomes an effective means of studying its function and provides new research ideas for tools that specifically regulate the post-translational modifications of target proteins. Summary of the Invention
[0006] The purpose of this invention is to provide a protein post-translational modification inducer, specifically an arginine methylation nano-proximity inducer, its preparation method, and its application. Based on the concept of heterobifunctional small molecule drugs, this invention uses gold nanoclusters as linkers to connect the affinity peptide of the protein-targeting arginine methyltransferase to the targeting affinity peptide of the target protein, designing an arginine methylation proximity nano-inducer to achieve specific regulation of arginine methylation modification of the target protein.
[0007] To achieve this objective, the present invention adopts the following technical solution:
[0008] In a first aspect, the present invention provides an arginine methylation nano-proximity inducer, the arginine methylation nano-proximity inducer comprising a gold nanocluster and a targeting affinity peptide modified on the surface of the gold nanocluster; the targeting affinity peptide comprises an affinity peptide targeting a target protein and an affinity peptide targeting a protein arginine methyltransferase.
[0009] This invention achieves efficient intracellular delivery by linking gold nanoclusters to the target affinity peptides of the target protein and the arginine methyltransferase, overcoming off-target effects and realizing arginine methylation modification of specific target proteins. The results demonstrate significant effects in vivo, achieving rapid and efficient regulation of post-translational arginine methylation modification of target proteins. The arginine methylation nanoproximity inducer provided by this invention can simultaneously achieve arginine methylation modification of different target proteins in the same cell type or the same target protein in different cell types.
[0010] Preferably, the target protein includes p53 or AKT.
[0011] Preferably, the molar ratio of the gold nanoclusters to the targeting affinity peptide is (1-2):(1-3).
[0012] The specific point values in (1-2) can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.; the specific point values in (1-3) can be 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, etc.; other specific point values within the above range can be selected, and will not be elaborated here.
[0013] Preferably, the molar ratio of the affinity peptide targeting the target protein to the affinity peptide targeting the arginine methyltransferase is (1-2):(1-4).
[0014] The specific point values in (1-2) can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.; the specific point values in (1-4) can be 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, etc.; other specific point values within the above range can be selected, and will not be elaborated here.
[0015] Preferably, the amino acid sequence of the affinity peptide targeting the target protein p53 includes the sequence shown in SEQ ID No. 1.
[0016] SEQ ID No. 1: CREDEDEIEW.
[0017] Preferably, the amino acid sequence of the affinity peptide targeting the target protein AKT includes the sequence shown in SEQ ID No. 2.
[0018] SEQ ID No.2: CYLEAF.
[0019] In this invention, the sequence shown in SEQ ID No. 1 can specifically target the target protein p53, and the sequence shown in SEQ ID No. 2 can specifically target the target protein AKT.
[0020] Preferably, the amino acid sequence of the affinity peptide targeting the protein arginine methyltransferase includes the sequence shown in SEQ ID No. 3.
[0021] SEQ ID No. 3: DATWSPLNHSC.
[0022] This invention uses peptide screening technology to screen out a peptide sequence SEQ ID No. 3 that can bind to PRMT5 (protein arginine methyltransferase 5) with high affinity. It can stably bind to PRMT5 and activate its hydrolytic activity, thereby achieving arginine methylation post-translational modification of the target protein.
[0023] In a second aspect, the present invention provides a method for preparing the arginine methylation nano-proximity inducer according to the first aspect, the preparation method comprising the following steps:
[0024] The arginine methylation nanoproximity inducer is formed by mixing and incubating a suspension of gold nanoclusters with a targeted affinity peptide and self-assembling the nano-proximity inducer.
[0025] The synthetic route of the inducer provided by this invention is simple and has high synthetic efficiency.
[0026] Preferably, the mixed incubation is carried out at a temperature of 35-40℃ (e.g., 35℃, 35.5℃, 36℃, 36.5℃, 37℃, 37.5℃, 38℃, 38.5℃, 39℃, 39.5℃, 40℃, etc.) for 10-18 h (e.g., 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, etc.); other specific point values within the above range can be selected, and will not be elaborated here.
[0027] Preferably, the gold nanoclusters are prepared by reacting a mixture of glutathione solution and tetrachloroauric acid solution.
[0028] Preferably, the gold nanoclusters are prepared by the following method:
[0029] The glutathione solution was mixed with the tetrachloroauric acid solution and reacted. After the reaction was completed, the reaction solution was mixed with isopropanol and centrifuged to obtain the final product.
[0030] Preferably, the molar ratio of tetrachloroauric acid to glutathione is (1-2):(1-3).
[0031] The specific point values in (1-2) can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc.; the specific point values in (1-3) can be 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, etc.; other specific point values within the above range can be selected, and will not be elaborated here.
[0032] Preferably, the temperature of the mixing reaction is 70-75℃ (e.g., 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, etc.), and the time is 20-28 h (e.g., 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, etc.); other specific values within the above range can be selected, and will not be elaborated here.
[0033] Preferably, the centrifugation speed is 12000-13000 rpm, such as 12000 rpm, 12500 rpm, 13000 rpm, etc. Other specific values within this range can be selected, and will not be elaborated here.
[0034] Preferably, the volume ratio of the reaction solution to isopropanol is (1-1.5):(1-2).
[0035] The specific point values in (1-1.5) can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.; the specific point values in (1-2) can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, etc.; other specific point values within the above range can be selected, and will not be elaborated here.
[0036] Thirdly, the present invention provides the application of the arginine methylation nano-proximity inducer according to the first aspect in the methylation modification of arginine in a target protein.
[0037] Compared with the prior art, the present invention has the following beneficial effects:
[0038] This invention achieves efficient intracellular delivery by linking gold nanoclusters to the target affinity peptides of the target protein and the arginine methyltransferase, overcoming off-target effects and realizing arginine methylation modification of specific target proteins. The results demonstrate significant effects in vivo, achieving rapid and efficient regulation of post-translational arginine methylation modification of target proteins. The arginine methylation nanoproximity inducer provided by this invention enables arginine methylation modification of specific target proteins in different cell types. Attached Figure Description
[0039] Figure 1 This is the result of the affinity test between the peptide DATWSPLNHSC and the PRMT5 protein;
[0040] Figure 2 This is a TEM image of arginine methylation inducers P-PRnGNPs (scale bar is 5 nm).
[0041] Figure 3 This is a particle size distribution diagram of arginine methylation inducers P-PRnGNPs;
[0042] Figure 4 This is a Zeta potential diagram of arginine methylation inducers P-PRnGNPs;
[0043] Figure 5 This is an immunofluorescence image of the uptake of arginine methylation inducers P-PRnGNPs in tumor cells;
[0044] Figure 6 This is an immunoblot image verifying the methylation of the cellular target protein p53 by the arginine methylation inducer P-PRnGNPs at different concentrations.
[0045] Figure 7 This is an immunoblot image verifying the methylation of the cellular target protein p53 by the arginine methylation inducer P-PRnGNPs at different time points;
[0046] Figure 8 This is a cell viability curve of tumor cells after treatment with the arginine methylation inducer P-PRnGNPs.
[0047] Figure 9 This is a graph showing the cell viability of tumor cells after treatment with the arginine methylation inducer A-PRnGNPs.
[0048] Figure 10 This is a graph showing the change in tumor volume in tumor-bearing mice after injection of the arginine methylation inducer P-PRnGNPs.
[0049] Figure 11 This is a tumor tissue image of a tumor-bearing mouse on day 28 after injection of the arginine methylation inducer P-PRnGNPs (the scale bar in the image is 1 cm).
[0050] Figure 12 This is an HE staining image of major organs and tumor tissues in tumor-bearing mice on day 28 after injection of the arginine methylation inducer P-PRnGNPs (scale bar in the image is 100 µm). Detailed Implementation
[0051] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.
[0052] Example 1
[0053] This embodiment provides an arginine methylation inducer (abbreviated as P-PRnGNPs), the preparation method of which is as follows:
[0054] (1) Preparation of gold nanoclusters: 300 μL of tetrachloroauric acid aqueous solution (50 mM) and 200 μL of glutathione aqueous solution (50 mM) were added to 3 mL of deionized water and stirred at 70 °C for 24 h; 5 mL of isopropanol was added, mixed evenly, and centrifuged at 13000 rpm for 12 min to obtain the gold nanoclusters;
[0055] (2) The obtained gold nanoclusters were dispersed in 3 mL of deionized water, and the peptide CREDEDEIEW (2 μmol) targeting p53 and the peptide DATWSPLNHSC (2 μmol) targeting PRMT5 were added. The mixture was stirred thoroughly at 37 °C for 15 h to obtain the arginine methylation inducer P-PRnGNPs.
[0056] Example 2
[0057] This embodiment provides an arginine methylation inducer (abbreviated as A-PRnGNPs), the preparation method of which is as follows:
[0058] (1) Preparation of gold nanoclusters: 300 μL of tetrachloroauric acid aqueous solution (50 mM) and 200 μL of glutathione aqueous solution (50 mM) were added to 3 mL of deionized water and stirred at 75 °C for 20 h; 5 mL of isopropanol was added, mixed evenly, and centrifuged at 12000 rpm for 15 min to obtain the gold nanoclusters;
[0059] (2) The obtained gold nanoclusters were dispersed in 3 mL of deionized water, and 2 μmol of CYLEAF (targeting AKT) and 2 μmol of DATWSPLNHSC (targeting PRMT5) were added. The mixture was stirred thoroughly at 39 °C for 12 h to obtain the arginine methylation inducer A-PRnGNPs.
[0060] Test Example 1
[0061] The affinity of the peptide DATWSPLNHSC for PRMT5 protein was investigated.
[0062] Surface plasmon resonance (SPR) experiments were performed using the Biacore 8K+ system to analyze the interaction between the peptide sequence DATWSPLNHSC and the PRMT5 protein, and fitting curves were plotted and kinetic parameters were calculated. The test results are as follows: Figure 1 As shown, there is a specific interaction between the peptide DATWSPLNHSC and the human PRMT5 protein, and its equilibrium dissociation constant (KD) is 0.299 μM.
[0063] Test Example 2
[0064] The physicochemical properties of the arginine methylation inducers P-PRnGNPs prepared in Example 1 were characterized and determined:
[0065] (1) Morphological observation: The morphology was observed and the size was counted using a transmission electron microscope (TEM). The test results are as follows: Figure 2 As shown, the particle size of the arginine methylation inducer P-PRnGNPs provided by this invention is 1-3 nm, according to the results.
[0066] (2) The particle size was detected by dynamic light scattering (DLS), and the results are as follows: Figure 3 As shown.
[0067] (3) Its Zeta potential was detected by dynamic light scattering (DLS), and the results are as follows: Figure 4 As shown.
[0068] Test Example 3
[0069] Verify the ability of arginine methylation inducer P-PRnGNPs to enter cells:
[0070] The arginine methylation inducer P-PRnGNPs or the PRMT5-targeting peptide DATWSPLNHSC prepared in Example 1 were respectively linked to Cy5.5 as detection samples. Free Cy5.5 was used as a control group, and HCT116 tumor cells were used as the research object. Free Cy5.5, free PRMT5-targeting peptides loaded with Cy5.5, and P-PRnGNPs loaded with Cy5.5 were co-incubated with tumor cells for 0 h, 1 h, 3 h, and 6 h. After incubation, immunofluorescence imaging was performed on each group. Figure 5 As shown in the figure, compared with free Cy5.5 dye, the targeted peptide treatment group with fluorescent dye only showed a weak fluorescence signal after co-incubation with cells for 6 h, while HCT116 cells co-incubated with the inducer with fluorescent dye showed a strong fluorescence signal, and the intracellular fluorescence signal increased in a time-dependent manner. This indicates that the arginine methylation inducer provided by the present invention can be effectively delivered into cells, while the simple PRMT5 targeted peptide cannot be effectively delivered into cells.
[0071] Test Example 4
[0072] Verify the methylation modification effect of arginine methylation inducers P-PRnGNPs on target proteins in cells:
[0073] Using HCT116 tumor cells as the research object, HCT116 tumor cells were cultured for 24 h in a medium containing graded concentrations of the methylation inducer P-PRnGNPs prepared in Example 1. The cells were washed three times with PBS buffer, and then lysed with RIPA lysis buffer containing 1% protease inhibitor (PMSF) and 1% phosphatase inhibitor. The tumor cell proteins were collected, centrifuged, and the supernatant was used for enrichment of the target protein using magnetic beads and followed by elution and denaturation. Western blotting and SDS-PAGE electrophoresis were then performed. The protein signal was transferred to a PVDF membrane, blocked, and incubated with antibody. Finally, the arginine methylation effect of the target protein was detected by chemiluminescence staining. The results are as follows: Figure 6 As shown.
[0074] The same method described above was used to investigate the effects of culturing HCT116 tumor cells in a medium containing 12.5 μM of the methylation inducer P-PRnGNPs prepared in Example 1 for different time periods. Cells were washed three times with PBS buffer, lysed with RIPA lysis buffer containing 1% protease inhibitor (PMSF) and 1% phosphatase inhibitor, and the tumor cell proteins were collected. The supernatant was collected by centrifugation, and the target protein was enriched using magnetic beads and eluted for denaturation. Western blotting and SDS-PAGE electrophoresis were performed, and the protein signal was transferred to a PVDF membrane for blocking and antibody incubation. Finally, the arginine methylation effect of the target protein was detected by chemiluminescence staining. The results are as follows: Figure 7 As shown.
[0075] Depend on Figure 6 and Figure 7 The results show that the methylation inducer can upregulate the level of symmetrical dimethylation of arginine in the intracellular p53 protein, and exhibits good concentration- and time-dependent effects. This indicates that the arginine methylation inducer provided by this invention can successfully achieve arginine methylation modification of the target protein in cells.
[0076] Test Example 5
[0077] To investigate the effects of arginine methylation inducers P-PRnGNPs and A-PRnGNPs on tumor cells, and to verify the methylation modification effect of arginine methylation inducers on the target protein:
[0078] HCT116 tumor cells were seeded in 96-well plates. After cell attachment, the cells were treated with different concentrations of arginine methylation inducers P-PRnGNPs or A-PRnGNPs and cultured for 24 hours. The original culture medium was removed, and the cells were washed twice with PBS buffer. 100 μL of CCK-8 working solution (CCK-8 to basal medium volume ratio 1:10) was added to each well of the 96-well plate, and the cells were incubated for 1 hour. After incubation, the absorbance at 450 nm was measured using a small microplate reader, and cell viability was calculated. Results are as follows: Figure 8 (P-PRnGNPs) and Figure 9 As shown in (A-PRnGNPs).
[0079] Depend on Figure 8 and Figure 9 The results show that the arginine methylation inducer provided by the present invention can effectively deliver peptides into tumor cells and methylate the arginine in the p53 and AKT proteins in tumor cells, ultimately leading to the proliferation of tumor cells.
[0080] Test Example 6
[0081] To investigate the effects of arginine methylation inducers P-PRnGNPs and A-PRnGNPs on a mouse model of tumors, in order to verify the methylation modification effect of arginine methylation inducers on the target protein:
[0082] Male BALB / c nude mice were subcutaneously injected with HCT116 tumor cells (5 × 10⁶ cells per mouse). 6 HCT116 tumor-bearing mouse models were established using 100 cells. P-PRnGNPs (30 mg / kg) were injected into the tail vein three times, with each injection five days apart. A control group was also set up (injected with the same volume of physiological saline, with each injection five days apart). During this period, the weight and tumor volume of the mice were recorded every other day. After 28 days, all mice were sacrificed, and the tumors and organs were collected for further analysis.
[0083] Tumor volume changes in HCT116 tumor-bearing mice after treatment with P-PRnGNPs and saline are as follows: Figure 10 As shown, tumor images collected on day 28 are as follows. Figure 11 As shown, the arginine methylation inducer provided by the present invention can methylate arginine in p53 and AKT proteins in tumor cells, thereby promoting the proliferation of tumor cells and the growth of tumor tissue.
[0084] HE staining of major organs and tumors collected on day 28 is as follows: Figure 12 As shown, there was no significant difference between the two groups, indicating that the arginine methylation inducer provided by this invention has excellent biocompatibility.
[0085] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.
[0086] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.
[0087] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.
Claims
1. A nano-proximity inducer for arginine methylation, characterized in that, The arginine methylation nanoproximity inducer includes a gold nanocluster and a targeting affinity peptide modified on the surface of the gold nanocluster; the targeting affinity peptide includes an affinity peptide targeting the target protein and an affinity peptide targeting the protein arginine methyltransferase.
2. The arginine methylation nano-proximity inducer according to claim 1, characterized in that, The target protein includes p53 or AKT.
3. The arginine methylation nano-proximity inducer according to claim 1 or 2, characterized in that, The molar ratio of the gold nanoclusters to the targeted affinity peptide is (1-2):(1-3); Preferably, the molar ratio of the affinity peptide targeting the target protein to the affinity peptide targeting the arginine methyltransferase is (1-2):(1-4).
4. The arginine methylation nano-proximity inducer according to claim 2 or 3, characterized in that, The amino acid sequence of the affinity peptide targeting the target protein p53 includes the sequence shown in SEQ ID No.
1.
5. The method for preparing the arginine methylation nano-proximity inducer according to any one of claims 2-4, characterized in that, The amino acid sequence of the affinity peptide targeting the target protein AKT includes the sequence shown in SEQ ID No.
2.
6. The method for preparing the arginine methylation nano-proximity inducer according to any one of claims 1-5, characterized in that, The amino acid sequence of the affinity peptide for the target protein arginine methyltransferase includes the sequence shown in SEQ ID No.
3.
7. The method for preparing the arginine methylation nano-proximity inducer according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: The arginine methylation nanoproximity inducer is formed by mixing and incubating a suspension of gold nanoclusters with a targeted affinity peptide and self-assembling the nano-proximity inducer.
8. The preparation method according to claim 7, characterized in that, The mixed incubation was carried out at a temperature of 35-40℃ for 10-18 h.
9. The preparation method according to claim 7, characterized in that, The gold nanoclusters were prepared by reacting a mixture of glutathione solution and tetrachloroauric acid solution. Preferably, the gold nanoclusters are prepared by the following method: The glutathione solution was mixed with the tetrachloroauric acid solution and reacted. After the reaction was completed, the reaction solution was mixed with isopropanol and centrifuged to obtain the product. Preferably, the molar ratio of tetrachloroauric acid to glutathione is (1-2):(1-3); Preferably, the mixing reaction is carried out at a temperature of 70-75°C for 20-28 hours. Preferably, the centrifugation speed is 12000-13000 rpm; Preferably, the volume ratio of the reaction solution to isopropanol is (1-1.5):(1-2).
10. The application of the arginine methylation nano-proximity inducer according to any one of claims 1-6 in the methylation modification of arginine in the target protein.