Proximity nano inducer for post-translational modification of protein as well as preparation method and application of proximity nano inducer

Abstract

The invention relates to an adjacent nano inducer for post-translational modification of protein and a preparation method and application thereof, the nano inducer comprises a gold nanocluster and a polypeptide modified on the surface of the gold nanocluster, and the polypeptide comprises a polypeptide targeting a target protein and a polypeptide targeting a modification enzyme. The nano inducer obtained by the invention can effectively enter cells, performs post-translational modification on specific proteins of the cells, and keeps certain activity within at least 72 hours. The nano inducer can induce O-GlcNAc glycosylation, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, acetylation or deacetylation of a target protein, in addition, the nano inducer can also 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] The invention belongs to the field of tumor treatment and relates to a proximity nano-inducer for post-translational modification of proteins and a preparation method and application thereof. Background Art

[0002] Post-translational modifications refer to further modifications of existing proteins to regulate various biological processes. Protein post-translational modifications are usually reversible and mediated by a combination of enzymes that add or remove post-translational modifications at equilibrium. Post-translational modifications and their interactions have direct functional effects on proteins; imbalances in post-translational modifications are associated with a variety of diseases, including cancer. Therefore, a deep understanding of post-translational modifications is very important for understanding life processes, screening disease markers, and identifying drug targets. Selective manipulation of post-translational modifications of target proteins of interest is an effective means to study their functions and disease treatment, but the lack of tools to directly regulate post-translational modifications is a major obstacle to such research.

[0003] Several experimental strategies have been developed to manipulate the post-translational modifications of proteins in cells, such as overexpression, gene knockout or knockdown, and chemical inhibitors or activators of post-translational modification enzymes. However, genetic engineering approaches are currently difficult to implement as therapeutic options, while chemical inhibitors or activators produce extensive changes in the level of protein post-translational modifications, which may complicate the elucidation of biological functions associated with a specific target protein. Recently, heterofunctional small molecules have been developed that can bind the target protein to a relevant ligand and recruit an enzyme through another ligand. Therefore, the post-translational modification of the target protein can be induced by simultaneously linking the target protein and the enzyme through heterofunctional small molecules. This approach largely relies on the development of small molecules that simultaneously target the post-translational modification enzyme and the target protein. In this case, protein fusion tags (such as FKBP12F36V) can only be used for proof-of-concept protein targeting and cannot be used for in vivo treatment. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a proximity nano-inducer for post-translational modification of proteins and a preparation method and application thereof.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a proximity nanoinducer for post-translational modification of proteins, wherein the nanoinducer comprises gold nanoclusters and polypeptides modified on the surface of the gold nanoclusters, wherein the polypeptides modified on the surface of the gold nanoclusters comprise polypeptides targeting target proteins and polypeptides targeting modification enzymes.

[0007] Here, we propose a general strategy to induce specific post-translational modifications of target proteins through nanoinducers. To achieve this goal, peptides with high affinity to post-translational modifying enzymes or targets are first screened through mature phage display technology. Gold nanoclusters are then used as nanoplatforms to attach different targeting peptides for intracellular delivery. Here, we use this nanoinducer technology to demonstrate specific O-linked β-N-acetylglucosamine modification, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, and acetylation and deacetylation of target proteins. Our nanoinducers use nanoscale drugs to induce two proteins to approach or bind to each other to act. "Proximity" is an academic term. For example, the commonly said "proximity chemical inducer" uses small molecule drugs to induce protein-protein binding.

[0008] These heterofunctional nanoinducers are dose-dependent, rapid, and selective for post-translational modifications of target proteins, thereby precisely regulating cellular processes. In addition, we generated heterofunctional nanoinducers to achieve two different types of post-translational modifications of one specific target protein or specific post-translational modifications of two different target proteins.

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

[0010] Among them, 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., and 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 numerical range can be selected, so they will not be listed here one by one.

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

[0012] Among them, the specific point values ​​in (1-3) can be selected as 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 numerical range can be selected and will not be listed here one by one.

[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 to 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] Among them, SEQ ID No.1 is a polypeptide targeting X-linked inhibitor of apoptosis protein XIAP, SEQ ID No.2 is a polypeptide targeting E3 ubiquitin ligase MDM2 protein, SEQ ID No.3 is a polypeptide targeting kinase AKT protein, SEQ ID No.4 is a polypeptide targeting HER2 protein, SEQ ID No.5 is a polypeptide targeting BAX protein, and SEQ ID No.6 is a polypeptide targeting P53 protein.

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

[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, the role played by the polypeptide may be different. In one system, the polypeptide acts as a polypeptide targeting the target protein, while in another system, it may act as a polypeptide targeting the modifying enzyme.

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

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

[0031] (2) Mixing the gold nanocluster suspension with the polypeptide to obtain.

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

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

[0034] Preferably, the method for preparing the gold nanoclusters comprises: mixing a glutathione solution with a tetrachloroauric acid solution, and then mixing with isopropanol, and centrifuging to obtain the gold nanoclusters.

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

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

[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 as 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 as 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 numerical range can be selected, and they will not be listed here one by one.

[0039] Preferably, the volume ratio of the first mixed liquid to isopropanol is (0.8-1):(1-2), wherein the specific point 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., wherein the specific point 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., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here one by one.

[0040] Preferably, the first mixed liquid and isopropanol are mixed at a temperature of 15-25° C. and a time of 1-5 min.

[0041] The temperature can be selected as 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, etc., and the time can be selected as 1min, 1.5min, 2min, 2.5min, 3min, 3.5min, 4min, 4.5min, 5min, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described here one by one.

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

[0043] The rotation speed can be selected as 12000rpm, 12100rpm, 12200rpm, 12300rpm, 12400rpm, 12500rpm, 12600rpm, 12700rpm, 12800rpm, 12900rpm, 13000rpm, and the time can be selected as 10min, 11min, 12min, 13min, 14min, 15min, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described here one by one.

[0044] In a third aspect, the present invention provides a use of the proximity nano-inducer for post-translational modification of a protein according to the first aspect in the preparation of a tumor therapeutic agent.

[0045] Preferably, the tumor treatment comprises targeted tumor treatment.

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

[0047] (1) The present invention adopts a simple method to obtain the nano-inducer, which is easy to operate. In addition, the nano-inducer obtained by the method of the present invention can effectively enter cells to exert its effects.

[0048] (2) The nano-inducer can enter the cell and take effect in a short time, and perform post-translational modification on the specific target protein. The nano-inducer obtained by the present invention can continue to take effect in the cell and be reused.

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

[0050] (4) Nanoinducers can simultaneously induce two different types of post-translational modifications of a specific target protein or specific post-translational modifications of 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 and affect the proliferation of tumor cells.

[0052] (6) The nano-inducer obtained by the present invention has high biological safety and provides a drug that is more suitable for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Transmission electron microscopy image of nanoinducer X-OGnGNCs. The scale bar is 5 nm, and the scale bar in the inset is 1 nm.

[0054] Figure 2 Figure 2 is the glycosylation modification of XIAP by X-OGnGNCs, where (a) shows the glycosylation level of XIAP in HCT116 tumor cells treated with different concentrations of X-OGnGNCs (upper panel) and the protein levels of OGT and GAPDH in whole cell lysates (WCL; lower panel), and (b) shows the image of the tumor collected on the 30th day from HCT116 tumor-bearing mice after treatment with X-OGnGNCs.

[0055] Figure 3Ubiquitination modification of HER2 by nanoinducer H-MDnGNCs targeting HER2 and MDM2, where (a) is a transmission electron microscopy image of H-MDnGNCs with a scale bar of 5 nm and a scale bar of 1 nm in the inset; (b) shows the HER2 protein level in SKOV3 tumor cells treated with different concentrations of H-MDnGNCs; and (c) is an image of the tumor collected on the 18th day from SKOV3 tumor-bearing mice after treatment with H-MDnGNCs.

[0056] Figure 4 Figure 3. Deubiquitination modification of BAX by nanoinducer B-USnGNCs targeting BAX and USP11, where a is a transmission electron microscopy image of B-USnGNCs with a scale bar of 5 nm and a scale bar of 1 nm in the inset; b is the BAX protein level in HepG2 tumor cells treated with different concentrations of B-USnGNCs; and c is an image of the tumor collected on the 26th day from HepG2 tumor-bearing mice after treatment with B-USnGNCs.

[0057] Figure 5 Phosphorylation modification of MDM2 by nanoinducer M-AKnGNCs targeting MDM2 and AKT, where (a) is a transmission electron microscopy image of M-AKnGNCs, with a scale bar of 5 nm and a scale bar of 1 nm in the inset; (b) shows the phosphorylation level of MDM2 protein in A549 tumor cells treated with different concentrations of M-AKnGNCs; and (c) is an image of the tumor collected on the 30th day from A549 tumor-bearing mice after treatment with M-AKnGNCs.

[0058] Figure 6 Dephosphorylation modification of AKT by nanoinducer A-PPnGNCs targeting AKT and PP1, where a is a transmission electron microscopy image of A-PPnGNCs, with a scale bar of 5 nm and a scale bar of 1 nm in the inset; b is the phosphorylation level of AKT protein in SKOV3 tumor cells treated with different concentrations of A-PPnGNCs; and c is an image of the tumor collected on the 18th day from SKOV3 tumor-bearing mice after treatment with A-PPnGNCs.

[0059] Figure 7 Acetylation modification of AKT by nanoinducer A-P3nGNCs targeting AKT and P300, where a is a transmission electron microscopy image of A-P3nGNCs, with a scale bar of 5 nm and a scale bar of 1 nm in the inset; b is the acetylation level of AKT protein in A549 tumor cells treated with different concentrations of A-P3nGNCs; and c is an image of the tumor collected on the 30th day from A549 tumor-bearing mice after treatment with A-P3nGNCs.

[0060] Figure 8 Deacetylation modification of P53 by nanoinducer P-SInGNCs targeting P53 and SIRT1, where a is a transmission electron microscopy image of P-SInGNCs, with a scale bar of 5 nm and a scale bar of 1 nm in the inset; b is the acetylation level of P53 protein in HCT116 tumor cells treated with different concentrations of P-SInGNCs; and c is an image of the tumor collected on the 30th day from HCT116 tumor-bearing mice after treatment with P-SInGNCs.

[0061] Fig. 9 Deubiquitination and acetylation modification of P53 by nanoinducer P-US-P3nGNCs targeting P53, USP11 and P300, where a is the transmission electron microscopy image of P-US-P3nGNCs, with a scale bar of 5 nm; b is the acetylation level of P53 protein in HCT116 tumor cells treated with different concentrations of P-US-P3nGNCs; and c is an image of the tumor collected on the 30th day from HCT116 tumor-bearing mice after treatment with P-US-P3nGNCs.

[0062] Fig.10 Figure 3. Acetylation modification of P53 and AKT by nanoinducer PA-P3nGNCs targeting P53, AKT and P300, where a is the transmission electron microscopy image of PA-P3nGNCs, with a scale bar of 5 nm; b is the acetylation level of P53 and AKT proteins in A549 tumor cells treated with different concentrations of PA-P3nGNCs; and c is an image of the tumor collected on the 30th day from A549 tumor-bearing mice after treatment with PA-P3nGNCs. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0064] Example 1

[0065] This embodiment provides a nano inducer X-OGnGNCs, and the method for preparing the nano inducer comprises:

[0066] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0068] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

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

[0070] (5) Add the polypeptides FLTAVVDQFC (1 μmol) and AVPIAQKC (1 μmol) to the above solution, and then heat the mixture to 37°C and gently stir for 12 hours. Centrifuge and resuspend in PBS to obtain a nanoinducer.

[0071] Example 2

[0072] This embodiment provides a nano inducer H-MDnGNCs, and the method for preparing the nano inducer comprises:

[0073] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0075] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

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

[0077] (5) Add the polypeptides LTVSPWYC (1 μM) and cdwwplafeallr (1 μM) to the above solution, and then heat the mixture to 37°C and gently stir for 12 hours. Centrifuge and resuspend in PBS to obtain the nanoinducer.

[0078] Example 3

[0079] This embodiment provides a nano inducer B-USnGNCs, and the method for preparing the nano inducer comprises:

[0080] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0082] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

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

[0084] (5) The peptides CMRPEIWIAQELRRIGDEFNA (1 μM) and CAEGEFYKLKIRTPR (2 μM) were added to the above solution, and then the mixture was heated to 37°C and gently stirred for 12 hours. After centrifugation, the mixture was resuspended in PBS to obtain the nanoinducer.

[0085] Example 4

[0086] This embodiment provides a nano inducer M-AKnGNCs, and the method for preparing the nano inducer comprises:

[0087] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0089] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

[0090] (4) centrifuging the mixed solution and redispersing 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, and then heat the mixture to 37°C and gently stir for 12 hours. Centrifuge and resuspend in PBS to obtain the nanoinducer.

[0092] Example 5

[0093] This embodiment provides a nano inducer A-PPnGNCs, and the method for preparing the nano inducer comprises:

[0094] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0096] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

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

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

[0099] Example 6

[0100] This embodiment provides a nano inducer A-PPnGNCs, and the method for preparing the nano inducer comprises:

[0101] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0103] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

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

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

[0106] Example 7

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

[0108] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0110] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

[0111] (4) centrifuging the mixed solution and redispersing 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 gently stir for 12 hours. Centrifuge and resuspend in PBS to obtain the nanoinducer.

[0113] Example 8

[0114] This embodiment provides a nano inducer P-US-P3nGNCs, and the method for preparing the nano inducer comprises:

[0115] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0117] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

[0118] (4) centrifuging the mixed solution and redispersing 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, and then heat the mixture to 37°C and gently stir for 12 hours. Centrifuge and resuspend in PBS to obtain the nanoinducer.

[0120] Example 9

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

[0122] (1) Freshly prepared L-GSH (50 mM, 300 μL) and HAuCl4 (50 mM, 200 μL) aqueous solutions were mixed and stored in 3 mL of ultrapure water;

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

[0124] (3) The reaction mixture was cooled to room temperature and then mixed with 5 mL of isopropanol;

[0125] (4) centrifuging the mixed solution and redispersing 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, and then heat the mixture to 37°C and gently stir for 12 hours. Centrifuge and resuspend in PBS to obtain the nanoinducer.

[0127] Test Example 1

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

[0129] Test method: Tumor cells were planted in a 6-well plate and cultured for 24 hours, and then different nano-inducers were added and incubated with tumor cells for another 24 hours. After the treatment, the cells were washed three times with PBS and then lysed and collected in RIPA lysis buffer containing 1% (v / v) protease inhibitor PMSF. The total protein concentration was determined using a BCA kit, and an immunoblotting experiment was performed to test the modification level of the target protein. The results showed that the nano-inducers can effectively change the modification level of the target protein. The specific results are shown in Figure 2 Figure a and Figure 3-10 Figure b in .

[0130] The mice were injected subcutaneously with tumor cells (5×10 6 cells / mouse, for specific tumor cell types, see the attached figure for instructions), to make a tumor-bearing mouse model. 3 At 15 mg / kg of nano-inducer was injected into the tail vein and then injected once every three days for a total of three injections. The tumor volume of the mice was observed after the drug administration, and the tumor images of the mice were photographed and recorded at the end of the treatment. The specific end time of the treatment period is shown in the attached figure.

[0131] For O-linked β-N-acetylglucosamine modification, we screened peptides with high affinity for O-linked β-N-acetylglucosamine transferase OGT. Gold nanoclusters were modified with peptides targeting OGT and X-linked inhibitor of apoptosis protein XIAP to form a nanoinducer (X-OGnGNC). Both 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 nanoinducers targeting E3 ubiquitin ligase MDM2, deubiquitinating enzyme USP11, kinase AKT, phosphatase PP1, acetyltransferase P300, deacetylase SIRT1 for specific ubiquitination of 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 the present invention uses the above-mentioned embodiments to illustrate a proximity nano-inducer for post-translational modification of proteins and its preparation method and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0133] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

Claims

1. A proximity nanoinducer for post-translational modification of proteins, characterized in that: The nano-inducer comprises gold nano-clusters and polypeptides modified on the surface of gold nano-clusters. The polypeptides modified on the surface of gold nano-clusters comprise polypeptides targeting target proteins and polypeptides targeting modified enzymes.

2. The proximity nanoinducer for post-translational modification of proteins according to claim 1, characterized in that: The molar ratio of the gold nanoclusters to the polypeptide modified on the surface of the gold nanoclusters is (0.5-2):(0.5-3); Preferably, the molar ratio of the polypeptide targeting the target protein to the polypeptide targeting the modifying enzyme is (1-3):(1-3).

3. The proximity nanoinducer for post-translational modification of proteins according to claim 1 or 2, characterized in that: The amino acid sequence of the polypeptide targeting the target protein includes any one of SEQ ID No.1 to SEQ ID No.6 or a combination of at least two of them.

4. The proximity nanoinducer for post-translational modification of a protein according to any one of claims 1 to 3, characterized in that: The amino acid sequence of the polypeptide targeting the modified enzyme includes any one of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 7 to SEQ ID No. 11, or a combination of at least two of them.

5. A method for preparing a proximity nano-inducer for post-translational modification of a protein according to any one of claims 1 to 4, characterized in that: The preparation method comprises: (1) dispersing the gold nanoclusters in water to obtain a gold nanocluster suspension; (2) Mixing the gold nanocluster suspension with the polypeptide to obtain.

6. The method for preparing a proximity nano-inducer for post-translational modification of a protein according to claim 5, characterized in that: The mixing temperature in step (2) is 25-37° C. and the mixing time is 12-24 hours.

7. The method for preparing a proximity nano-inducer for post-translational modification of a protein according to claim 5 or 6, characterized in that: The preparation method of the gold nanoclusters comprises: mixing a glutathione solution with a tetrachloroauric acid solution to obtain a first mixed solution; mixing the first mixed solution with isopropanol, and centrifuging to obtain the gold nanoclusters.

8. The method for preparing a proximity nano-inducer for post-translational modification of a protein according to claim 7, characterized in that: The temperature of mixing the glutathione solution and the tetrachloroauric acid solution is 65-75°C and the time is 22-26 hours; Preferably, the molar ratio of glutathione to tetrachloroauric acid is (2.8-3.2):(1.8-2.2); Preferably, the volume ratio of the first mixed liquid to isopropanol is (0.8-1):(1-2); Preferably, the first mixed solution and isopropanol are mixed at a temperature of 15-25° C. and a time of 1-5 min. Preferably, the centrifugal speed is 12000-13000 rpm and the time is 10-15 min.

9. Use of a proximity nano-inducer for post-translational modification of a protein according to any one of claims 1 to 4 in the preparation of a tumor therapeutic agent.

10. The use according to claim 9, characterized in that: The tumor treatment includes targeted tumor treatment.

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