A proximity nano-inducer for post-translational modification of proteins and preparation method and application thereof

Abstract

This invention relates to a proximity nano-inducer for protein post-translational modification, its preparation method, and its application. The nano-inducer comprises gold nanoclusters and peptides modified on the surface of the gold nanoclusters. The peptides include peptides targeting target proteins and peptides targeting modifying enzymes. The nano-inducer obtained by this invention can effectively enter cells, induce post-translational modifications on specific proteins, and maintain a certain level of activity for at least 72 hours. It can induce O-GlcNAc glycosylation, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, acetylation, or deacetylation of target proteins. Furthermore, the nano-inducer can specifically induce two different types of post-translational modifications on one target protein, or induce a specific post-translational modification on two different target proteins.

Description

Technical Field

[0001] This invention belongs to the field of tumor treatment and relates to a proximity nano-inducer for protein post-translational modification, its preparation method, and its application. Background Technology

[0002] Post-translational modifications (PDMs) refer to further modifications of existing proteins to regulate various biological processes. Protein PDMs are generally reversible and, in equilibrium, are mediated by combinations of enzymes that add or remove PDMs. PDMs and their interactions have direct functional effects on proteins; imbalances in PDMs are associated with a variety of diseases, including cancer. Therefore, a deep understanding of PDMs is crucial for understanding life processes, screening disease biomarkers, and identifying drug targets. Selectively manipulating the PDMs of target proteins of interest is an effective means of studying their function and disease treatment; however, the lack of tools for directly regulating PDMs is a major obstacle to such research.

[0003] Several experimental strategies have been developed to manipulate post-translational modifications of proteins within cells, such as overexpression, gene knockout, or chemical inhibitors or activators of post-translational modifying enzymes. However, genetic engineering methods are currently limited as therapeutic approaches, and chemical inhibitors or activators produce a wide range of variations at the level of protein post-translational modification, which can complicate the elucidation of biological functions associated with specific target proteins. Recently, heterofunctional small molecules have been developed that can bind target proteins to relevant ligands and recruit enzymes via another ligand. Therefore, post-translational modifications of the target protein can be induced by simultaneously linking the target protein and enzyme through heterofunctional small molecules. This approach relies heavily on the development of small molecules that simultaneously target post-translational modifying enzymes and target proteins. In this context, protein fusion tags (such as FKBP12F36V) can only be used for proof-of-concept protein targeting and cannot be used for in vivo therapy. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a proximity nano-inducer for protein post-translational modification, its preparation method, and its application.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a proximity nano-inducer for post-translational modification of proteins, the nano-inducer comprising gold nanoclusters and peptides modified on the surface of the gold nanoclusters, wherein the peptides modified on the surface of the gold nanoclusters include peptides targeting target proteins and peptides targeting modifying enzymes.

[0007] Here, we propose a general strategy for inducing specific post-translational modifications of target proteins using nano-inducers. To achieve this, peptides with high affinity for post-translational modifying enzymes or targets are first screened using mature phage display technology. Then, gold nanoclusters are used as a nanoplatform to connect different targeting peptides for intracellular delivery. Here, we utilize this nano-inducer technology to demonstrate specific O-linked β-N-acetylglucosamine modification, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, acetylation, and deacetylation of target proteins. Our nano-inducers utilize nanoscale drugs to induce two proteins to approach or bind, thereby achieving an effect. "Proximity" is an academic term; for example, the commonly used "proximity chemical inducer" uses small molecule drugs to induce protein-protein binding.

[0008] These heterofunctional nano-inducers exhibit dose-dependent, rapid, and selective post-translational modifications of target proteins, thereby precisely modulating cellular processes. Furthermore, we have generated heterofunctional nano-inducers to achieve two different types of post-translational modifications of a specific target protein or specific post-translational modifications of two different target proteins.

[0009] Preferably, the molar ratio of the gold nanoclusters to the peptides modified on the surface of the gold nanoclusters is (0.5-2):(0.5-3).

[0010] The specific point values ​​in (0.5-2) can be selected from 0.5, 0.6, 0.7, 0.8, 0.9, 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 (0.5-3) can be selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0011] Preferably, the molar ratio of the peptide targeting the target protein to the peptide targeting the modifying enzyme is (1-3):(1-3).

[0012] The specific point values ​​in (1-3) can be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0013] Preferably, the amino acid sequence of the polypeptide targeting the target protein includes any one or a combination of at least two of SEQ ID No. 1-SEQ ID No. 6.

[0014] SEQ ID No.1: AVPIAQKC.

[0015] SEQ ID No. 2: cdwwplafeallr.

[0016] SEQ ID No.3: cyleaf.

[0017] SEQ ID No. 4: LTVSPWYC.

[0018] SEQ ID No. 5:CMRPEIWIAQELRRIGDEFNA.

[0019] SEQ ID No. 6: CREDEDEIEW.

[0020] SEQ ID No. 1 is a polypeptide targeting the X-linked apoptosis inhibitor protein XIAP, SEQ ID No. 2 is a polypeptide targeting the E3 ubiquitin ligase MDM2 protein, SEQ ID No. 3 is a polypeptide targeting the kinase AKT protein, SEQ ID No. 4 is a polypeptide targeting the HER2 protein, SEQ ID No. 5 is a polypeptide targeting the BAX protein, and SEQ ID No. 6 is a polypeptide targeting the p53 protein.

[0021] Preferably, the amino acid sequence of the polypeptide targeting the modifying enzyme includes any one or a combination of at least two of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7-SEQ ID No. 11.

[0022] SEQ ID No. 7: FLTAVVDQFC.

[0023] SEQ ID No. 8: CAEGEFYKLKIRTPR.

[0024] SEQ ID No. 9: CRPKRKRKNARVTFAEAAEII.

[0025] SEQ ID No. 10: CKEKRIKELELLLMSTENELKGQQALW.

[0026] SEQ ID No. 11: CSWR.

[0027] SEQ ID No. 7 is a polypeptide targeting O-linked β-N-acetylglucosamine transferase (OGT), SEQ ID No. 8 is a polypeptide targeting deubiquitinase USP11, SEQ ID No. 9 is a polypeptide targeting phosphatase PP1, SEQ ID No. 10 is a polypeptide targeting acetyltransferase P300, and SEQ ID No. 11 is a polypeptide targeting deacetylase SIRT1.

[0028] In different systems, peptides may play different roles. In one system, a peptide may act as a peptide that targets a target protein, while in another system, it may act as a peptide that targets a modifying enzyme.

[0029] In a second aspect, the present invention provides a method for preparing a proximity nano-inducer for protein post-translational modification according to the first aspect, the method comprising:

[0030] (1) Disperse gold nanoclusters in water to obtain a gold nanocluster suspension;

[0031] (2) The gold nanocluster suspension is mixed with the peptide to obtain the final product.

[0032] Preferably, the mixing temperature in step (2) is 25-37°C and the time is 12-24h.

[0033] The temperature can be selected from 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, etc., and the time can be selected from 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0034] Preferably, the preparation method of the gold nanoclusters includes: mixing glutathione solution with tetrachloroauric acid solution, then mixing with isopropanol, and centrifuging to obtain the gold nanoclusters.

[0035] Preferably, the glutathione solution and tetrachloroauric acid solution are mixed at a temperature of 65-75°C for 22-26 hours.

[0036] The temperature can be selected from 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, etc., and the time can be selected from 22h, 22.5h, 23h, 23.5h, 24h, 24.5h, 25h, 25.5h, 26h, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0037] Preferably, the molar ratio of glutathione to tetrachloroauric acid is (2.8-3.2):(1.8-2.2).

[0038] The specific point values ​​in (2.8-3.2) can be selected from 2.8, 2.85, 2.9, 2.95, 3, 3.05, 3.1, 3.15, 3.2, etc., and the specific point values ​​in (1.8-2.2) can be selected from 1.8, 1.85, 1.9, 1.95, 2, 2.05, 2.1, 2.15, 2.2, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0039] Preferably, the volume ratio of the first mixture to isopropanol is (0.8-1):(1-2), where the specific values ​​in (0.8-1) can be selected from 0.8, 0.82, 0.84, 0.86, 0.88, 0.9, 0.92, 0.94, 0.96, 0.98, 1, etc., and the specific values ​​in (1-2) can be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0040] Preferably, the temperature for mixing the first mixture with isopropanol is 15-25°C, and the time is 1-5 minutes.

[0041] The temperature can be selected from 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, etc., and the time can be selected from 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0042] Preferably, the centrifugation speed is 12000-13000 rpm and the time is 10-15 min.

[0043] The rotational speed can be selected from 12000rpm, 12100rpm, 12200rpm, 12300rpm, 12400rpm, 12500rpm, 12600rpm, 12700rpm, 12800rpm, 12900rpm, and 13000rpm, and the time can be selected from 10min, 11min, 12min, 13min, 14min, and 15min, etc. Other specific values ​​within the above range can also be selected, which will not be described in detail here.

[0044] Thirdly, the present invention provides the application of the protein post-translational modification neighbor nano-inducer as described in the first aspect in the preparation of tumor therapeutic agents.

[0045] Preferably, the tumor treatment includes targeted tumor therapy.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] (1) The present invention uses a simple method to obtain nano-inducers, which is easy to operate. Furthermore, the nano-inducers obtained using the method of the present invention can effectively enter cells and exert their effects.

[0048] (2) Nano-inducers can exert their effects in a short time after entering cells, and can perform post-translational modifications on specific target proteins. The nano-inducers obtained in this invention can exert their effects continuously in cells and can be reused.

[0049] (3) The present invention can obtain nano-inducers to perform different protein post-translational modifications: O-linked β-N-acetylglucosamine modification, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, acetylation and deacetylation.

[0050] (4) Nano-inducers can simultaneously perform two different types of post-translational modifications on a specific target protein or specific post-translational modifications on two different target proteins.

[0051] (5) The nano-inducer obtained by the present invention is small in size and can effectively enter the tumor site to affect the proliferation of tumor cells.

[0052] (6) The nano-inducer obtained by the present invention has high biosafety and provides drugs that are more suitable for clinical use. Attached Figure Description

[0053] Figure 1 Transmission electron microscopy image of the nano-inducing agent X-OGnGNCs. Scale bar is 5 nm, and the scale bar of the inset is 1 nm.

[0054] Figure 2 The images show the glycosylation modification of XIAP by X-OGnGNCs. Figure a shows the glycosylation level of XIAP in HCT116 tumor cells treated with different concentrations of X-OGnGNCs (top) and the protein levels of OGT and GAPDH in whole-cell lysates (WCL; bottom). Figure b shows the tumor images collected from HCT116 tumor-bearing mice on day 30 after X-OGnGNCs treatment.

[0055] Figure 3The ubiquitination modification of HER2 by H-MDnGNCs, a nano-inducer targeting HER2 and MDM2, is shown in Figure a. The transmission electron microscope image of H-MDnGNCs is shown in Figure a, with a scale bar of 5 nm and a scale bar of 1 nm for the inset. Figure b shows the HER2 protein level in SKOV3 tumor cells treated with different concentrations of H-MDnGNCs. Figure c shows the tumor images collected on day 18 of SKOV3 tumor-bearing mice after treatment with H-MDnGNCs.

[0056] Figure 4 To illustrate the deubiquitination modification of BAX by the nano-inducer B-USnGNCs targeting BAX and USP11, Figure a shows a transmission electron microscope image of B-USnGNCs with a scale bar of 5 nm and an inset with a scale bar of 1 nm. Figure b shows the BAX protein levels in HepG2 tumor cells treated with different concentrations of B-USnGNCs. Figure c shows an image of tumors collected on day 26 of HepG2 tumor-bearing mice after treatment with B-USnGNCs.

[0057] Figure 5 The phosphorylation modification of MDM2 by the nano-inducer M-AKnGNCs targeting MDM2 and AKT is shown in Figure a. The transmission electron microscope image of M-AKnGNCs is shown in Figure a. The scale bar is 5 nm. The scale bar of the inset is 1 nm. The phosphorylation level of MDM2 protein in A549 tumor cells treated with different concentrations of M-AKnGNCs is shown in Figure c. The tumor image was collected from A549 tumor-bearing mice on day 30 after treatment with M-AKnGNCs.

[0058] Figure 6 The dephosphorylation modification of AKT by A-PPnGNCs, a nano-inducer targeting AKT and PP1, is shown in Figure a. A transmission electron microscope image of A-PPnGNCs, scale bar 5 nm, inset scale bar 1 nm. Figure b shows the phosphorylation level of AKT protein in SKOV3 tumor cells treated with different concentrations of A-PPnGNCs. Figure c shows the tumor images collected on day 18 of SKOV3 tumor-bearing mice after A-PPnGNCs treatment.

[0059] Figure 7 The acetylation modification of AKT by the nano-inducer A-P3nGNCs targeting AKT and P300 is shown in Figure a. Transmission electron microscopy image of A-P3nGNCs, scale bar 5 nm, inset scale bar 1 nm; Figure b. acetylation level of AKT protein in A549 tumor cells treated with different concentrations of A-P3nGNCs; Figure c. image of tumors collected on day 30 of A549 tumor-bearing mice after A-P3nGNCs treatment.

[0060] Figure 8 The deacetylation modification of P53 by P-SInGNCs, a nano-inducer targeting P53 and SIRT1, is shown in Figure a. Transmission electron microscopy image of P-SInGNCs, scale bar 5 nm, inset scale bar 1 nm; Figure b. acetylation level of P53 protein in HCT116 tumor cells treated with different concentrations of P-SInGNCs; Figure c. image of tumors collected on day 30 of HCT116 tumor-bearing mice after P-SInGNCs treatment.

[0061] Figure 9 To illustrate the deubiquitination and acetylation modification of P53 by the nano-inducer P-US-P3nGNCs targeting P53, USP11, and P300, Figure a shows a transmission electron microscope image of P-US-P3nGNCs with a scale bar of 5 nm. Figure b shows the acetylation level of P53 protein in HCT116 tumor cells treated with different concentrations of P-US-P3nGNCs. Figure c shows an image of the tumor collected from HCT116 tumor-bearing mice on day 30 after treatment with P-US-P3nGNCs.

[0062] Figure 10 The acetylation modification of P53 and AKT by PA-P3nGNCs, a nano-inducer targeting P53, AKT, and P300, is shown in Figure a. Transmission electron microscopy image of PA-P3nGNCs, scale bar 5 nm; Figure b. acetylation levels of P53 and AKT proteins in A549 tumor cells treated with different concentrations of PA-P3nGNCs; and Figure c. image of tumors collected from A549 tumor-bearing mice on day 30 after treatment with PA-P3nGNCs. Detailed Implementation

[0063] 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.

[0064] Example 1

[0065] This embodiment provides a nano-inducer X-OGnGNCs, and the preparation method of the nano-inducer includes:

[0066] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0067] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0068] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0069] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0070] (5) Add peptides FLTAVVDQFC (1 μmol) and AVPIAQKC (1 μmol) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0071] Example 2

[0072] This embodiment provides a nano-inducer H-MDnGNCs, and the preparation method of the nano-inducer includes:

[0073] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0074] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0075] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0076] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0077] (5) Add peptides LTVSPWYC (1 μM) and cdwwplafeallr (1 μM) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0078] Example 3

[0079] This embodiment provides a nano-inducer B-USnGNCs, and the preparation method of the nano-inducer includes:

[0080] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0081] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0082] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0083] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0084] (5) Add peptides CMRPEIWIAQELRRIGDEFNA (1 μM) and CAEGEFYKLKIRTPR (2 μM) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0085] Example 4

[0086] This embodiment provides a nano-inducer M-AKnGNCs, and the preparation method of the nano-inducer includes:

[0087] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0088] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0089] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0090] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0091] (5) Add peptides cdwwplafeallr (1 μM) and cyleaf (2 μM) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0092] Example 5

[0093] This embodiment provides a nano-inducer A-PPnGNCs, and the preparation method of the nano-inducer includes:

[0094] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0095] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0096] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0097] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0098] (5) Add peptide cyleaf (1 μM) and CRPKRKRKNARVTFAEAAEII (1 μM) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0099] Example 6

[0100] This embodiment provides a nano-inducer A-PPnGNCs, and the preparation method of the nano-inducer includes:

[0101] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0102] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0103] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0104] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0105] (5) Add peptide cyleaf (1 μM) and CKEKRIKELELLLMSTENELKGQQALW (1 μM) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0106] Example 7

[0107] This embodiment provides a nano-inducer P-SInGNCs, and the preparation method of the nano-inducer includes:

[0108] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0109] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0110] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0111] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0112] (5) Add peptides CREDEDEIEW (1 μM) and CSWR (1 μM) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0113] Example 8

[0114] This embodiment provides a nano-inducer P-US-P3nGNCs, and the preparation method of the nano-inducer includes:

[0115] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0116] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0117] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0118] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0119] (5) Add peptides CREDEDEIEW (1 μM), CAEGEFYKLKIRTPR (1 μM), and CSWR (1 μM) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0120] Example 9

[0121] This embodiment provides a nano-inducer PA-P3nGNCs, and the preparation method of the nano-inducer includes:

[0122] (1) Mix freshly prepared L-GSH (50mM, 300μL) and HAuCl4 (50mM, 200μL) aqueous solutions and store them in 3mL of ultrapure water;

[0123] (2) Heat the mixture to 70°C with gentle stirring for 24 hours;

[0124] (3) Cool the reaction mixture to room temperature and then mix it with 5 mL of isopropanol;

[0125] (4) Centrifuge the mixed solution and redisperse the obtained gold nanoclusters in 3 mL of ultrapure water to obtain a gold nanocluster suspension.

[0126] (5) Add peptides CREDEDEIEW (1 μM), cyleaf (1 μM), and CSWR (1 μM) to the above solution, then heat the mixture to 37°C and stir gently for 12 hours. After centrifugation, resuspend in PBS to obtain the nano-inducer.

[0127] Test Example 1

[0128] The specific steps are as follows: The morphology and size of the nano-inducers prepared in Examples 1-9 are tested using a transmission electron microscope.

[0129] Assay Method: Tumor cells were seeded in 6-well plates and cultured for 24 hours. Different nano-inducers were then added and co-incubated with the tumor cells for another 24 hours. After treatment, the cells were washed three times with PBS and lysed in RIPA lysis buffer containing 1% (v / v) PMSF protease inhibitor, and the cells were collected. Total protein concentration was determined using a BCA kit, and Western blotting was performed to test the modification level of the target protein. Results showed that the nano-inducers effectively altered the modification level of the target protein; detailed results are shown below. Figure 2 Figure a in the middle and Figure 3-10 Figure b in the diagram.

[0130] Tumor cells (5 × 10⁻⁶) were subcutaneously injected into the right abdomen of mice. 6 To create a tumor-bearing mouse model, use individual cells per mouse (see attached diagram for specific tumor cell types). When the tumor volume is 50-100 mm², a tumor-bearing mouse model is established. 3 At the time of administration, 15 mg / kg of the nano-inducing agent was injected via the tail vein, followed by injections every three days for a total of three injections. Tumor volume was observed after treatment, and tumor images were taken at the end of treatment. Specific treatment end times are shown in the attached figures.

[0131] In the area of ​​O-linked β-N-acetylglucosamine modification, we screened peptides with high affinity for the O-linked β-N-acetylglucosamine transferase OGT. Gold nanoclusters were modified with peptides targeting OGT and the X-linked apoptosis inhibitor protein XIAP to form a nano-inducer (X-OGnGNC). In vitro and in vivo studies confirmed that X-OGnGNCs can directly recruit OGT to target and induce O-linked β-N-acetylglucosamine modification of XIAP. Similarly, we developed nano-inducers targeting the E3 ubiquitin ligase MDM2, deubiquitinase USP11, kinase AKT, phosphatase PP1, acetyltransferase P300, and deacetylase SIRT1, which were used for specific ubiquitination of the target protein HER2, deubiquitination of BAX, phosphorylation of MDM2, dephosphorylation of AKT, acetylation of AKT, or deacetylation of P53, respectively.

[0132] The applicant declares that this invention illustrates a proximity nano-inducer for protein post-translational modification, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0133] 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.

[0134] 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 proximity nano-inducer for post-translational modification of proteins, characterized in that, The nano-inducer includes gold nanoclusters and peptides modified on the surface of gold nanoclusters, wherein the peptides modified on the surface of gold nanoclusters include peptides that target target proteins and peptides that target modifying enzymes. The molar ratio of the gold nanoclusters to the peptides modified on the surface of the gold nanoclusters is (0.5-2):(0.5-3); The molar ratio of the peptide targeting the target protein to the peptide targeting the modifying enzyme is (1-3):(1-3); The amino acid sequence of the polypeptide targeting the target protein is the sequence shown in SEQ ID No. 1; The amino acid sequence of the polypeptide targeting the modified enzyme is the sequence shown in SEQ ID No.

7.

2. A method for preparing a proximity nano-inducer for protein post-translational modification according to claim 1, characterized in that, The preparation method includes: (1) Disperse gold nanoclusters in water to obtain a gold nanocluster suspension; (2) The gold nanocluster suspension is mixed with the peptide to obtain the final product.

3. The method for preparing a proximity nano-inducer for protein post-translational modification according to claim 2, characterized in that, The mixing temperature in step (2) is 25-37℃, and the time is 12-24 h.

4. The method for preparing a proximity nano-inducer for protein post-translational modification according to claim 2, characterized in that, The method for preparing the gold nanoclusters includes: mixing glutathione solution and tetrachloroauric acid solution to obtain a first mixture; mixing the first mixture with isopropanol and centrifuging to obtain the final product.

5. The method for preparing a proximity nano-inducer for protein post-translational modification according to claim 4, characterized in that, The glutathione solution and tetrachloroauric acid solution were mixed at a temperature of 65-75℃ for 22-26 hours.

6. The method for preparing a proximity nano-inducer for protein post-translational modification according to claim 4, characterized in that, The molar ratio of glutathione to tetrachloroauric acid is (2.8-3.2):(1.8-2.2).

7. The method for preparing a proximity nano-inducer for protein post-translational modification according to claim 4, characterized in that, The volume ratio of the first mixture to isopropanol is (0.8-1):(1-2).

8. The method for preparing a proximity nano-inducer for protein post-translational modification according to claim 4, characterized in that, The first mixture is mixed with isopropanol at a temperature of 15-25℃ for 1-5 minutes.

9. The method for preparing a proximity nano-inducer for protein post-translational modification according to claim 4, characterized in that, The centrifugation speed is 12000-13000 rpm, and the time is 10-15 min.

10. The use of a proximity nano-inducer for protein post-translational modification according to claim 1 in the preparation of a colorectal cancer therapeutic agent.

11. The application according to claim 10, characterized in that, The treatment of colon cancer includes targeted colon cancer therapy.

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