A PROTAC polypeptide targeting the ZMYND8 protein and its application

Abstract

The present invention provides a PROTAC polypeptide targeting ZMYND8 protein and its application, belonging to the technical field of polypeptides. The amino acid sequence of the PROTAC polypeptide is shown as SEQ ID NO.1. This PROTAC polypeptide has a strong binding ability with ZMYND8 protein. By using a nano-selenium delivery system to deliver the PROTAC polypeptide into leukemia cells, it can effectively inhibit cell proliferation. At the same time, the PROTAC polypeptide has the ability to target and degrade intracellular ZMYND8 protein. The PROTAC polypeptide can be applied to the preparation of drugs for preventing and / or treating malignant blood diseases or the preparation of reagents for degrading ZMYND8 protein. The PROTAC polypeptide drug targeting ZMYND8 can provide a new treatment strategy for the targeted treatment of malignant blood diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein polypeptides, and particularly relates to a proteolysis-targeting chimera (PROTAC) polypeptide targeting ZMYND8 protein and its application. Background Art

[0002] Acute myeloid leukemia (AML) is one of the common hematological malignancies in humans, and its incidence shows an increasing trend year by year. In the treatment of AML, traditional chemotherapy and allogeneic hematopoietic stem cell transplantation are the main treatment methods. Especially the progress of hematopoietic stem cell transplantation technology has improved the survival rate of young and low-to-medium-risk AML patients to a certain extent. However, problems such as the significant toxic side effects brought by chemotherapy, the high recurrence rate of the disease, and the poor tolerance of elderly patients to treatment are still the main obstacles restricting the long-term survival of AML patients. In view of this, developing a new AML treatment strategy with high targeting, better efficacy, and lower side effects is of great significance for improving the quality of life and survival rate of AML patients.

[0003] ZMYND8 (zinc finger MYND-type containing 8) is a transcriptional regulatory protein. Its N-terminal contains a plant homeodomain (PHD), a bromodomain (BRD), and a Pro-Trp-Trp-Pro (PWWP) chromatin-binding domain, and a MYND-type zinc finger domain for protein-protein interaction is contained at the C-terminal. The N-terminal PHD-BRD-PWWP domain of ZMYND8 recognizes several acetyl and methyl lysines on histones in vitro

[0004] Residues. As a histone "reader", ZMYND8 can recognize various post-translational histone modifications and certain specifically mutated histones, and is involved in cell transcriptional regulation. Studies have shown that ZMYND8 may play a key role in the tumorigenesis of breast cancer, prostate cancer, colorectal cancer and cervical cancer. Cao et al. found that in AML cells, ZMYND8 binds to the leukemia-specific enhancer of MYC and / or the enhancer of IRF8, and can regulate the expression of MYC and / or IRF8, thereby maintaining the growth of AML cells. It was also found that ZMYND8 only regulates MYC and IRF8 in AML (if IRF8 is expressed), and this pathway does not exist in other solid tumors. It is proved that ZMYND8 is a transcription regulator dependent on AML. Therefore, ZMYND8 can be used as a target for the treatment of AML. Currently, there is no use of small molecule inhibitors or targeted degradation drugs against the ZMYND8 protein at home and abroad. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a PROTAC polypeptide targeting the ZMYND8 protein. The polypeptide has a high binding ability to the ZMYND8 protein, effectively degrades the ZMYND8 protein, and plays an anti-cancer role, filling the blank of PROTAC drugs for the transcriptional regulatory protein ZMYND8.

[0006] In the first aspect of the present invention, a PROTAC polypeptide targeting ZMYND8 is provided, and the amino acid sequence of the PROTAC polypeptide is as shown in SEQ ID NO: 1.

[0007] Preferably, the binding constant of the PROTAC polypeptide to the ZMYND8 protein is 324 nM.

[0008] In the second aspect of the present invention, the application of the PROTAC polypeptide in the preparation of drugs for preventing and / or treating leukemia is provided.

[0009] Preferably, the PROTAC polypeptide is used in the preparation of drugs for inhibiting the proliferation of acute myeloid leukemia cells.

[0010] Preferably, the acute myeloid leukemia cells are MOLM-13, THP-1, MV4-11, M07e, NB4 or primary cells from the bone marrow samples of acute myeloid leukemia patients.

[0011] In the third aspect of the present invention, the application of the PROTAC polypeptide in the preparation of reagents for degrading the ZMYND8 protein is provided.

[0012] In the fourth aspect of the present invention, a drug is provided, which comprises the PROTAC polypeptide and a pharmaceutically acceptable excipient or carrier.

[0013] After adding suitable excipients, the drug can be prepared into pharmaceutically acceptable drug preparations, such as oral preparations or injection preparations. Exemplarily, oral preparations include capsules, tablets, granules or other liquid preparations, such as mixtures, suspensions, syrups, aromatic waters, etc. Exemplarily, the excipients that can be added include stabilizers, suspending agents, pH regulators, flavoring agents, coloring agents, disintegrants, fillers, solubilizers, excipients, humectants, thickeners, wetting agents, penetrants, diluents, flocculants, etc.

[0014] Preferably, the carrier is a nano-delivery system.

[0015] More preferably, the nano-delivery system is nano-selenium, nano-gold or liposome.

[0016] Even more preferably, when the nano-delivery system is nano-selenium, the drug is prepared according to the following steps:

[0017] Mix the polypeptide, an aqueous solution of selenium salt and a solution of chitosan, then carry out a water bath, and then add vitamin C and react to obtain the product.

[0018] Preferably, the molar mass ratio of the PROTAC polypeptide, selenium salt and vitamin C is 1:8-12:70-90.

[0019] Preferably, the mass concentration of the chitosan solution is 0.3-0.8%, and the dosage ratio of the chitosan solution to the polypeptide is 150 μL:1 mg.

[0020] Preferably, after mixing the PROTAC polypeptide, the chitosan solution and the selenium salt, mix them at 45-55 °C, add vitamin C, continue to react for 15-25 minutes, stabilize at room temperature for more than 2 hours, and then store at 4 °C.

[0021] The PROTAC polypeptide targeting ZMYND8 provided by the present invention has an amino acid sequence as shown in SEQ ID NO: 1. The present invention detects the binding ability of the PROTAC polypeptide to the ZMYND8 protein through isothermal titration calorimetry experiments. The binding constant of the PROTAC polypeptide to the ZMYND8 protein is 324 nM, indicating that the PROTAC polypeptide has a strong binding ability to the ZMYND8 protein. Delivering the PROTAC polypeptide into AML cells through the nano-selenium delivery system can effectively inhibit the proliferation of cells and target the degradation of the ZMYND8 protein in AML cells.

[0022] The PROTAC polypeptide provided by the present invention can effectively reduce the tumor burden in an in vivo AML mouse model and prolong the survival time of AML mice.

[0023] Wherein the AML animal model is an NSG mouse model injected with MOLM-13-Luc via the tail vein.

[0024] The PROTAC polypeptide provided by the present invention can effectively promote apoptosis of AML cells in primary cells of ex vivo bone marrow samples of AML patients.

[0025] It can be seen that the PROTAC polypeptide can be applied to the preparation of drugs for preventing and / or treating leukemia or reagents for degrading ZMYND8 protein.

[0026] The present invention provides a drug for preventing and / or treating leukemia, comprising a PROTAC polypeptide and a delivery system. The ZMYND8 PROTAC@NanoSe drug prepared with nano-selenium as the delivery system can provide a new treatment strategy for malignant blood diseases including multiple myeloma, lymphoma, acute myeloid leukemia, etc., filling the gap of drugs for polypeptide-targeted degradation targeting ZMYND8 globally. Description of the Drawings

[0027] Figure 1 It is the design result of the polypeptide targeting ZMYND8 provided by the present invention, where A is a schematic diagram of the crystal structure of ZMYND8 homodimer; B and C are the results of protein-assisted design of the ZMYND8 polypeptide. D is the result of high-performance liquid chromatography characterization of the drug polypeptide. E and F are the results of affinity detection of the ZMYND8 PROTAC drug polypeptide with ZMYND8 protein and MDM2 protein respectively.

[0028] Figure 2 It is the detection result of the in vitro proliferation inhibition activity of the ZMYND8 PROTAC drug, where A is the detection of the ability of the drug to inhibit the proliferation of cancer cells; B is the result of detecting the degradation of ZMYND8 protein in cancer cells by the drug using Western blot, and C represents the result of detecting the degradation of ZMYND8 protein by the ZMYND8 PROTAC drug using Western blot; D and E are the statistical analysis of the immunoblot in Figure C.

[0029] Figure 3 It is the induction of apoptosis of primary cells in bone marrow samples of AML patients by the ZMYND8 PROTAC drug in vitro;

[0030] Figure 4 It is the detection of the drug effect in animal experiments, where A is a schematic diagram of the establishment of an AML mouse model and drug administration treatment. B is the effect of the control group, reverse peptide and ZMYND8 PROTAC on tumor growth. C is the statistical analysis result of the AML tumor burden in mice in the control group, reverse peptide and ZMYND8 PROTAC groups. D is the survival of mice in the control group, reverse peptide and ZMYND8 PROTAC groups. Detailed Embodiments

[0031] The following specific embodiments illustrate the implementation manners of the present invention, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0032] The present invention provides a PROTAC polypeptide targeting ZMYND8, and the amino acid sequence of the PROTAC polypeptide is shown as SEQ ID NO: 1: QYDANCKKEDTTYITYGGG SGGGTSFEQFWAWLWP.

[0033] The present invention has no special limitation on the source of the polypeptide, and the well-known polypeptide sources in the art can be adopted. In the embodiments of the present invention, the polypeptide is synthesized by the solid-phase synthesis method of polypeptides. The present invention has no special limitation on the solid-phase synthesis method of polypeptides, and the well-known polypeptide synthesis methods in the art can be adopted, such as Fmoc polypeptide synthesis. Fmoc-protected amino acids are purchased from Gil Biochemical, and HBTU and HIBT condensing agents are from Suzhou Haofan Biotechnology.

[0034] In the present invention, the binding ability of the PROTAC polypeptide to the ZMYND8 protein is detected by isothermal titration calorimetry experiment, and the binding constant of the PROTAC polypeptide to the ZMYND8 protein is preferably 324 nM. The polypeptide binds to the ZMYND8 protein and the MDM2 protein, induces the ubiquitination of the ZMYND8 protein by the MDM2 protein, and realizes the result of mediating the degradation of ZMYND8 in acute myeloid leukemia. Figure 1 E, F. The crystal structure of the ZMYND8 homodimer is as Figure 1 shown in A.

[0035] In the present invention, the PROTAC polypeptide is delivered into acute myeloid leukemia cells by nano-selenium, and it is found that cell proliferation is inhibited. The IC of the polypeptide drug for MOLM-13 acute myeloid leukemia cells for 24 hours 50 is 0.44 μM, and the IC for 48 hours 50 is 0.18 μM. The IC of the polypeptide drug for THP-1 acute myeloid leukemia cells for 24 hours 50 is 0.81 μM, and the IC for 48 hours 50 is 0.33 μM. The IC of the polypeptide drug for MV4-11 acute myeloid leukemia for 24 hours 50 is 0.51 μM, and the IC for 48 hours 50 is 0.21 μM. The IC of the polypeptide drug for M07e acute myeloid leukemia for 24 hours 50 is 0.20 μM, and the IC for 48 hours 50 is 0.09 μM. The IC of the polypeptide drug for NB4 acute myeloid leukemia for 24 hours 50 is 0.28 μM, and the IC for 48 hours 50 is 0.14 μM.

[0036] In view of the function of PROTAC polypeptide in targeting and binding to inhibit the proliferation of acute myeloid leukemia cells, the present invention provides the application of PROTAC polypeptide in the preparation of drugs for preventing and / or treating acute myeloid leukemia.

[0037] In the present invention, acute myeloid leukemia preferably includes one or more of the following acute myeloid leukemia cells: MOLM-13, THP-1, MV4-11, M07e or NB4.

[0038] In view of the function of PROTAC polypeptide in degrading intracellular ZMYND8 protein, the present invention provides the application of PROTAC polypeptide targeting ZMYND8 protein in the preparation of reagents for degrading ZMYND8 protein.

[0039] The present invention provides a drug for preventing and / or treating acute myeloid leukemia, comprising PROTAC polypeptide and a delivery system.

[0040] The present invention does not impose special restrictions on the type of the delivery system, and the well-known delivery systems in the art can be used, such as gold nanoparticles, liposomes, selenium nanoparticles or other well-known nano-delivery systems in the art. In the embodiments of the present invention, taking selenium nanoparticles as the delivery system as an example, the preparation method and drug efficacy of the drug are illustrated. The present invention does not impose special restrictions on the preparation method of the selenium nanoparticle-polypeptide drug, and the well-known method of coupling selenium nanoparticles with proteins in the art can be used. The drug has been experimentally verified to be non-toxic at the cellular level and animal level and has high safety.

[0041] The following combines examples to detail the PROTAC polypeptide targeting ZMYND8 protein provided by the present invention, its application and the effect of preventing and treating acute myeloid leukemia, but they cannot be understood as limiting the protection scope of the present invention.

[0042] Description of experimental raw materials

[0043] The test materials are divided into the synthesis part: Fmoc amino acids were purchased from Shanghai Gil Biochemical Co., Ltd., and DIEA, HOBT, and HBTU were purchased from Sigma-Aldrich (a company under Merck Life Sciences). The selenium nanoparticle preparation part: sodium selenite, chitosan and vitamin C were purchased from Aladdin Reagent Co., Ltd.

[0044] Nomenclature

[0045] Se represents a single selenium nanoparticle delivery system;

[0046] ZMYND8 PROTAC@NanoSe represents the finished product conjugated with the polypeptide drug and the selenium nanoparticle delivery system;

[0047] Reverse peptide represents the finished product conjugated with the polypeptide negative control and the nano-selenium delivery system;

[0048] Control represents the blank control;

[0049] Vinculin represents the focal adhesion protein and serves as an internal reference in Western blotting;

[0050] Chx represents Cycloheximide, a protein synthesis inhibitor.

[0051] Example 1

[0052] The PROTAC polypeptide targeting the ZMYND8 protein was obtained by means of protein-aided design, as shown in Figure 1 B and C.

[0053] The polypeptide drug was synthesized according to the amino acid sequence using the solid-phase peptide synthesis method. The synthesis method was general Fmoc peptide synthesis. Fmoc-protected amino acids were purchased from GL Biochem, and the HBTU and HIBT condensing agents were from Suzhou Haofan Biotechnology. The synthesis method was as follows:

[0054] 1) Deprotection: The Fmoc-protected column and monomer must be treated with a basic solvent (piperidine) to remove the protecting group of the amino group.

[0055] 2) Activation and crosslinking: The carboxyl group of the next amino acid is activated by an activator. The activated monomer reacts with the free amino group to crosslink and form a peptide bond. A large amount of super-concentrated reagent is used in this step to drive the reaction to completion. Cycle: These two steps are repeated until the synthesis is completed.

[0056] 3) Elution and deprotection: The polypeptide is eluted from the column, and its protecting group is removed by a deprotecting agent (TFA) to obtain the crude product.

[0057] The amino acid sequence of the PROTAC polypeptide is QYDANCKKEDTTYITYGGG SGGGTSFEQFWAWLWP (SEQ ID NO: 1).

[0058] The high-performance liquid chromatography characterization results of the PROTAC polypeptide are shown in Figure 1 D.

[0059] Example 2

[0060] Preparation of ZMYND8 PROTAC@NanoSe drug

[0061] The coupling method is as follows: 1 mg of polypeptide is dissolved in 1 ml of pure water, 200 μl of 50 mM sodium selenite solution and 600 μl of 0.5% chitosan solution are added, and the mixture is shaken and mixed until the solution becomes clear. Then it is placed in a water bath at 50 °C preheated for 1 min. Next, 1.6 ml of 50 mM vitamin C solution is added to the above preheated mixed solution. After mixing, it is continuously placed in a water bath at 50 °C for reaction for 20 min and then taken out to obtain nano-selenium drug-loaded particles. After cooling to room temperature, the polypeptide drug ZMYND8 PROTAC@NanoSe conjugated with nano-selenium can be obtained.

[0062] Example 3

[0063] Cell-level experiments of ZMYND8 PROTAC@NanoSe drug

[0064] 1. Cell culture

[0065] The culture conditions for MOLM-13, THP-1, MV4-11, M07E, and NB4 cells are complete medium of 90% RPMI 1640 + 10% FBS. The cells are cultured in a constant temperature incubator at 37 °C with 5% CO 2 at a saturated humidity. The cell culture density is maintained at 4×10 5 ~2×10 6 / ml.

[0066] 2. Experiment on the ability of the drug to inhibit cancer cell proliferation

[0067] The ability of the drug to inhibit cancer cell proliferation was analyzed by the Cell Counting Kit-8 detection method.

[0068] The basic principle of the CCK8 cell viability detection method is as follows: This reagent contains a compound WST-8 (whose chemical name is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt). Inside the cell, with the assistance of a specific coupling reagent, WST-8 will be reduced by dehydrogenases in the mitochondria to generate a yellow water-soluble formazan product. The concentration of this formazan product is directly proportional to the viability of the cells. Specifically, the more active cells there are, the more formazan product is generated, and the darker yellow the test solution will be. Conversely, if there are fewer live cells, the generated formazan product will decrease, and the color of the solution will be correspondingly lighter. By measuring the color depth of the solution, the activity level of the cells can be indirectly reflected. This method is not only simple to operate but also highly sensitive and is suitable for evaluating the activity of various types of cells.

[0069] When using CCK8 to detect cell viability, the specific experimental steps are as follows: First, inoculate the cells. The AML cells are counted and diluted to 5×10 5The density of cells was adjusted to 90 μl per well and seeded into a 96-well plate. A drug group, a control group (without drug addition), and a background blank control group (complete medium without cells) were set up, with 3 replicates for each concentration. Next was the drug treatment. Different concentration gradients of drugs were added to the drug group, and the cells were continuously cultured for 24 h or 48 h. After the drug treatment, the cell viability was detected using CCK8. 10 μl of CCK8 reagent was added to each well, and the 96-well plate was gently tapped to mix evenly. Then it was placed in a constant temperature incubator and cultured at 37 °C for 2 h. After the incubation, a full-wavelength enzyme-linked immunosorbent assay detector was used to measure the absorbance of each well at wavelengths of 450 nm and 690 nm. After obtaining the absorbance readings, the corrected absorbance value of each well was obtained by calculating the difference between OD 450 and OD 690 . Finally, the cell activity index was evaluated according to a specific calculation formula.

[0070] After drug treatment, the inhibitory proliferation effect of the finished product representing the conjugated polypeptide drug and nanoselenium delivery system on acute myeloid leukemia cells was detected and calculated.

[0071] The results are shown in Figure 2 A. The ZMYND8 PROTAC@NanoSe drug showed dose-dependent growth inhibitory effects in MOLM-13, THP-1, MV4-11, M07e, and NB4 cells, while the naked nanoselenium particles had no effect on cancer cells. The half-maximal inhibitory concentration (IC 50 ) values of the ZMYND8 PROTAC@NanoSe drug on MOLM-13, THP-1, MV4-11, M07e, and NB4 cells at 24 hours were IC 50 being 0.44 μM, 0.81 μM, 0.51 μM, and 0.20 μM, respectively.

[0072] 3. To study the ability of the ZMYND8 PROTAC@NanoSe drug to degrade the ZMYND8 protein, western blotting was used to analyze ZMYND8. The specific experimental procedure was as follows:

[0073] First, AML cells were seeded into a 12-well plate at a density of 5×10 5 / ml. Then different concentrations of drugs were added to the cells, and a control group without drug addition was set up. The cells were cultured in an incubator at 37 °C with 5% CO 2Cultivate for 24 h. Centrifuge the cells in each well at 300 g for 5 min to collect them, wash them 3 times with PBS, centrifuge and discard the supernatant. Add 45 μl of RIPA lysis buffer containing the protease inhibitor PMSF to each cell sample, mix well by shaking and place on ice for 30 min for lysis. After completely lysing the cells, centrifuge at 12,000 rpm at 4 °C for 15 min. Then, pipette 5 μl of the lysis supernatant from each sample and quantify the protein sample using a BCA quantification kit. Add loading buffer to the remaining lysis supernatant, mix well and place in a metal bath at 100 °C for 10 min to completely denature the protein sample. After preparing the protein sample, add the sample and the prestained standard protein marker to the loading wells of the SDS-PAGE gel, and make up the sample volume of each well according to the quantification result, and then perform electrophoresis to separate the proteins. Transfer the proteins on the SDS-PAGE gel to a PVDF membrane using the rapid semi-dry transfer method. Then, block the membrane with 1% BSA in TBST at room temperature for 1 h. After blocking, incubate the membrane with the specific primary antibody solution on a shaker at 4 °C overnight. The next day, wash the membrane incubated overnight 3 times with TBST solution, and then prepare the corresponding secondary antibody solution with 1% BSA in TBST solution. Pour the prepared secondary antibody solution into the membrane incubated with the corresponding species of primary antibody, and incubate at room temperature on a horizontal shaker for 1 h. After the secondary antibody incubation, continue to wash the membrane 3 times with TBST. Finally, use the ECL luminescent solution for chemiluminescent detection of the target protein. The obtained protein bands are analyzed by gray scale using Image J software and statistically analyzed in GraphPad 8.0.

[0074] The ZMYND8 PROTAC@NanoSe drug induces the degradation of ZMYND8 protein in a dose-dependent manner ( Figure 2 B–E) and a time-dependent manner.

[0075] 4. The ZMYND8 PROTAC@NanoSe drug can induce apoptosis of primary cells in the bone marrow samples of AML patients

[0076] To verify the efficacy of ZMYND8 PROTAC@NanoSe in AML patient samples, bone marrow samples from AML patients were collected. After obtaining the bone marrow samples from AML patients, the bone marrow samples were first transferred to 15 ml centrifuge tubes in a laminar flow hood, PBS and erythrocyte sedimentation fluid were added in a ratio of 1:1:1, and the mixture was gently pipetted and mixed well, then left standing at room temperature for 25 min. After sedimentation, the upper layer of liquid was aspirated into a new centrifuge tube, and the precipitate was discarded. The upper layer of liquid was centrifuged at a centrifugal force of 1000 revolutions per minute for 5 min. After centrifugation, the supernatant in the tube was removed and discarded, while the cell sediment at the bottom of the tube was resuspended and uniformly mixed with phosphate buffer (PBS). Next, three volumes of erythrocyte lysate were added to the centrifuge tube, shaken gently, and then the tube was left standing at room temperature for 15 min to further lyse the remaining erythrocytes. After the erythrocyte lysis was completed, centrifugation was performed at 1000 rpm, and the supernatant was discarded to obtain a cell pellet. At this time, if there were still erythrocytes remaining, the lysis could be repeated once. Finally, the cell pellet after erythrocyte lysis was washed 3 times with PBS, and the cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum (FBS), and then transferred to cell culture flasks. Then, these culture flasks were placed in an incubator at 37 °C and 5% CO 2 for culturing.

[0077] After obtaining the bone marrow samples from AML patients, erythrocytes were removed through erythrocyte sedimentation and erythrocyte lysis treatments, the cells were resuspended in RPMI 1640 medium containing 10% FBS, and inoculated into 24-well plates at a density of 5×10 5 / ml, and an empty nanoselenium (Se) negative control equivalent to a concentration of 1.0 μM of ZMYND8 PROTAC@NanoSe was set up. In the drug groups, concentration gradients of 0.25 μM, 0.5 μM, and 1.0 μM were set, and they were added to the bone marrow cells of AML patients respectively. After 24 h of drug treatment, apoptosis was detected using the PI-AnnexinV apoptosis detection kit.

[0078] As Figure 3 shown: Empty nanoselenium (Se) did not induce apoptosis in patient bone marrow cells, while ZMYND8 PROTAC drugs at different concentrations were able to induce apoptosis in primary bone marrow cells.

[0079] Example 4

[0080] Verify the effect of ZMYND8 PROTAC@NanoSe drug on inhibiting tumor growth at the animal level

[0081] To evaluate the therapeutic effect of ZMYND8 PROTAC@NanoSe drug in vivo, a mouse model of acute myeloid leukemia was established. Female NSG mice at 5 weeks of age were used. These mice were housed in an SPF-level animal laboratory and were in a 12-hour day-night cycle environment. After being housed for 1 week, MOLM-13-Luc cells were injected via the tail vein, and each mouse received approximately 1×10 4 cells. Seven days after successful injection of AML cells, potassium D-luciferin was used as the substrate for luciferase to detect the AML tumor burden in the model mice. A sterile DPBS buffer was used to dissolve and prepare a substrate stock solution at a concentration of 15 mg / ml, and it was mixed well. The stock solution was filtered through a 0.2-μm filter to sterilize it. When imaging the mice, the 15 mg / ml potassium D-luciferin was injected into the mice via intraperitoneal injection at a dose of 200 μl / mouse, and after reacting for about 10 minutes, the mice were anesthetized by inhaled isoflurane. The anesthetized mice were placed into the LuminaXRMS Series III small animal imager to detect the bioluminescence intensity, and the AML tumor burden was evaluated based on the bioluminescence intensity of each mouse.

[0082] The correct structure of a polypeptide determines its function, and only a polypeptide with the correct sequence can play its role. To better verify the effect of the ZMYND8 PROTAC polypeptide on AML cells, the present invention synthesized a reverse peptide whose amino acid composition is exactly the same as that of the ZMYND8 PROTAC polypeptide but with the sequence exactly opposite to that of the ZMYND8 PROTAC polypeptide. Therefore, the reverse peptide can be used as an optimal negative control for the ZMYND8 PROTAC polypeptide. The present invention prepared nanoselenium particles loaded with the reverse peptide using the same preparation method and named it Reverse Peptide@NanoSe.

[0083] The mice successfully modeled were randomly divided into three groups: a control group (Vehicle), a reverse peptide group (Reverse Peptide@NanoSe, labeled as Reverse Peptide), and a drug group (ZMYND8 PROTAC@NanoSe, labeled as ZMYND8 PROTAC), with 5 mice in each group. Administration of the drugs began. The drug doses of the reverse peptide and the ZMYND8 PROTAC polypeptide were both 5 mg / kg (mouse body weight), and the control group was given an equal amount of normal saline. The administration method was intraperitoneal injection, once every other day for 14 consecutive days. Imaging was performed once a week during the drug administration period to evaluate the tumor burden and the survival of the mice.

[0084] As Figure 4As shown in A - D, neither the control group nor the reverse peptide had a therapeutic effect on tumor growth. The ZMYND8 PROTAC@NanoSe drug showed high efficacy in the MOLM - 13 xenograft model, prolonging the survival of AML mice.

[0085] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A PROTAC polypeptide targeting ZMYND8 protein, characterized in that The amino acid sequence of the PROTAC polypeptide is shown in SEQ ID NO:

1.

2. Use of the PROTAC polypeptide according to claim 1 in the preparation of a medicament for preventing and / or treating leukemia.

3. The application according to claim 2, characterized in that: The PROTAC polypeptide is used to prepare a drug for inhibiting the proliferation of leukemia cells.

4. The use according to claim 3, characterized in that: The leukemia cells are MOLM-13, THP-1, MV4-11, M07e, NB4 or primary AML cells from bone marrow samples of patients with acute myeloid leukemia.

5. A drug for preventing and / or treating leukemia, characterized in that: It comprises the PROTAC polypeptide according to claim 1 and a pharmaceutically acceptable excipient or carrier.

6. The drug according to claim 5, characterized in that: The carrier is nano-selenium, nano-gold or liposome.

7. The drug according to claim 6, characterized in that The carrier is nano-selenium.

8. The drug according to claim 7, characterized in that: The drug is prepared according to the following steps: The PROTAC polypeptide, the aqueous solution of selenium salt and the chitosan solution are mixed and then placed in a water bath, and vitamin C is added and reacted to obtain the product.

9. The drug according to claim 8, characterized in that The molar mass ratio of the PROTAC polypeptide, selenium salt, and vitamin C is 1:8-12:70-90; The mass concentration of chitosan solution is 0.3-0.8%, and the dosage ratio of chitosan solution to PROTAC peptide is 150 μL:1 mg; After mixing the PROTAC polypeptide, chitosan solution and aqueous solution of selenium salt, vitamin C is added at 45-55° C. and the reaction is continued for 15-25 minutes. After stabilization at room temperature, the drug is obtained.

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