A PROTAC polypeptide targeting nuclear export protein 1 and its application

Abstract

The present invention provides a PROTAC polypeptide targeting nuclear export protein 1 and its application, belonging to the technical field of proteolysis-targeting chimeras technology. The amino acid sequence of the PROTAC polypeptide is shown in SEQ ID NO.1. Through the nano-selenium delivery system, the PROTAC polypeptide can be delivered into multiple myeloma cells. The synthesized se-XPO1 PROTAC drug can effectively inhibit cell proliferation and simultaneously significantly degrade the XPO1 protein level in MM cells. Verified by multiple tests, it is suggested that this polypeptide drug can be applied to the treatment of multiple myeloma. Compared with the already marketed small molecule drug selinexor, the PROTAC polypeptide drug has a stronger XPO1 target protein degradation effect and has achieved better drug treatment effects in samples of patients with relapsed and refractory multiple myeloma, filling the gap of PROTAC drugs targeting XPO1 globally.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteolysis-targeting chimeras (PROTACs), and particularly relates to a PROTAC polypeptide targeting exportin 1 (XPO1) and its application. Background Art

[0002] Multiple myeloma (MM) is the third most common hematological malignancy, characterized by the malignant clonal proliferation of plasma cells in the bone marrow, the secretion of a large amount of monoclonal immunoglobulins, and ultimately the damage of multiple organ functions. Among hematological malignancies, the incidence rate is second only to leukemia and lymphoma. The treatment regimen for MM is mainly chemotherapy, and the first-line chemotherapy regimen usually needs to cover proteasome inhibitors. The current first-line treatment regimen is mainly the VRD treatment regimen of proteasome inhibitors, immunomodulators, and dexamethasone. After achieving complete remission, autologous hematopoietic stem cell transplantation is sought for long-term survival. Since bortezomib was approved by the FDA for the treatment of MM in 2003, the 5-year relative survival rate has increased to 51.3%. However, recurrence and refractoriness are still the main problems that MM cannot be cured and needs to face.

[0003] With the continuous in-depth study of MM, new targeted drugs such as monoclonal antibodies (daratumumab targeting CD38, elotuzumab targeting CS-1), CAR-T cell immunotherapy (targets such as BCMA, GPRC5D, CD138), and exportin 1 (XPO1) inhibitors (Selinxor) have all provided possibilities for relapsed and refractory patients. Especially as the world's first oral XPO1 inhibitor, the combination chemotherapy of selinexor and other second-line drugs has shown good therapeutic effects. However, the clinical application of this drug mostly appears as an adjuvant combination drug, and has significant adverse reaction events, such as gastrointestinal reactions, myelosuppression, non-specific side reactions, etc.

[0004] XPO1 is one of the important receptors for nucleocytoplasmic transport. During nuclear export, XPO1 binds to RanGTP. Through conformational changes, the NES recognition site of the XPO1 protein is opened to bind to the cargo protein, forming a stable ternary complex of RanGTP-XPO1-Cargo protein. This complex binds to the nucleoporin complex located on the nuclear pore and is then transported out of the nucleus as a whole. Once in the cytoplasm, RanGTP is dephosphorylated and hydrolyzed to RanGDP. The XPO1-Cargo Protein complex without bound RanGTP will release the cargo protein, achieving the purpose of nuclear export. The XPO1 protein is highly expressed in tumor cells. The nucleocytoplasmic transport of specific proteins mediated by XPO1 is related to the pathogenesis of various tumors. Tumor-related proteins with nucleocytoplasmic shuttle functions include p53, p21, p27, RB, TOP2A, IκB, BRCA1 / 2, FOXO3, APC, etc. When these proteins are localized in the nucleus, they can effectively inhibit cell proliferation. However, when XPO1 is overexpressed, they are degraded in the cytoplasm due to excessive nuclear export and thus lose their tumor-suppressing function. Summary of the Invention

[0005] In view of the above technical problems, the present invention designs and synthesizes a PROTAC targeting XPO1. By targeting the degradation of the XPO1 protein, it prevents nucleocytoplasmic transport shuttle, resulting in the accumulation of tumor suppressor proteins in the nucleus, thereby exerting an anti-tumor effect. Compared with the already marketed small molecule drug Selinexor, the PROTAC of the present invention has a stronger targeted protein degradation effect, filling the gap in the global market for PROTAC drugs targeting XPO1.

[0006] The specific technical solutions provided by the present invention are as follows:

[0007] In the first aspect of the present invention, a PROTAC polypeptide targeting XPO1 is provided, and its amino acid sequence is EMSEFALEMDKIGGGSGGGTSFEQFWAWLWP. The PROTAC polypeptide uses XPO1 (fragment 504-630) as the target protein, mouse double minute 2 homolog (MDM2) as the E3 ubiquitin ligase, and GGGSGGG as the linker. This PROTAC polypeptide has the ability to specifically recognize the XPO1 protein and bind to the XPO1 and MDM2 proteins.

[0008] In the second aspect of the present invention, an application of the PROTAC polypeptide in the preparation of a product for degrading the XPO1 protein is provided.

[0009] In the third aspect of the present invention, an application of the PROTAC polypeptide in the preparation of a drug for preventing and / or treating multiple myeloma is provided.

[0010] Preferably, the PROTAC polypeptide or the nanodelivery system is used to prepare a drug for inhibiting the proliferation of multiple myeloma cells.

[0011] Preferably, the multiple myeloma cells are NCI-H929, MM.1S, RPMI-8226, U226, luc-U266 or primary MM cells from the bone marrow samples of patients with CD38+.

[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. After adding suitable excipients, the drug can be prepared into a pharmaceutically acceptable pharmaceutical preparation, such as an oral preparation or an injection preparation. Exemplarily, the 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.

[0013] Preferably, the carrier is a nanodelivery system.

[0014] More preferably, the nanodelivery system is nano-selenium.

[0015] Preferably, when the nanodelivery system is nano-selenium, the drug is prepared according to the following steps:

[0016] After dissolving the PROTAC polypeptide, add a selenium salt solution, chitosan and vitamin C, and react to obtain the product.

[0017] Selenium, as an important trace element, has significant antioxidant and anticancer activities. And nano-selenium shows great potential in the drug delivery system due to its unique physicochemical and biological properties. Sodium selenite (Na 2 SeO 3 ) used in the present invention is an inorganic selenium compound and can be an important source of nano-selenium, and other selenium-containing substances such as potassium selenite and sodium selenite can be substituted.

[0018] Chitosan belongs to natural polysaccharides and is easily protonated in an acidic environment to form cationic amino groups (-NH3+). This cationic property can interact with negatively charged molecules or particles to form stable complexes. In the drug delivery system, it can increase the stability and bioavailability of the drug and contribute to the intracellular delivery and absorption of the drug. In the present invention, it can be substituted by other substances with cationic properties, such as polyethyleneimine (PEI), polydimethyldiallylammonium chloride, PDADMAC, etc.

[0019] Vitamin C can be used as a reducing agent in pharmaceutical preparations to simulate the reducing environment in vivo, protect drugs from oxidative damage, extend the drug's shelf life, and can be replaced by other reducing substances such as glutathione (GSH), acetic acid, etc.

[0020] Preferably, the molar mass ratio of the PROTAC polypeptide, selenium salt, and vitamin C is 1:8 - 12:70 - 90.

[0021] 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.

[0022] Preferably, for the reaction, the mixture is first mixed and reacted at 45 - 55 °C for 15 - 25 min, then left to stand at room temperature for 3 - 4 h, and finally reacted overnight at 4 °C.

[0023] The present invention provides a PROTAC polypeptide targeting XPO1. The nano - drug delivery system se - XPO1 PROTAC prepared using this polypeptide can be effectively delivered to the MM cell line, effectively degrade XPO1 protein in vitro cell lines, inhibit the proliferation of MM cells, and promote the apoptosis of MM cells.

[0024] The PROTAC polypeptide targeting XPO1 provided by the present invention can effectively reduce the tumor burden in an in - vivo MM mouse model and extend the survival time of MM mice.

[0025] The MM animal model is the tail vein model of NSG mice injected with luc - U266.

[0026] The PROTAC polypeptide targeting XPO1 provided by the present invention can effectively promote the apoptosis of MM cells in CD38 + primary cells of ex - vivo MM patient bone marrow samples.

[0027] The se - XPO1 PROTAC polypeptide provided by the present invention can be used as a targeted polypeptide drug for the treatment of MM. Description of the Drawings

[0028] Figure 1 Schematic diagram for the design of the PROTAC polypeptide sequence; (a) Amino acid sequence of the XPO1 - binding peptide predicted by the virtual saturation mutagenesis method; (b) Mutant sequence of the predicted XPO1 - binding peptide.

[0029] Figure 2 Characterization of XPO1 PROTAC materials; (a) HPLC identification of the purity of XPO1 PROTAC - Cys; (b) MS identification of the molecular weight of XPO1 PROTAC - Cys.

[0030] Figure 3Characterization of XPO1 PROTAC materials; (a) Transmission electron microscopy observation of the morphology of se-XPO1 PROTAC (scale bar: 50 μm); (b) Particle size detection of se-XPO1 PROTAC; (c) Zeta potential detection of se-XPO1 PROTAC.

[0031] Figure 4 Characterization of XPO1 PROTAC materials; (a) Absorption peaks of Cys-XPO1 PROTAC polypeptides at 280 nm on an HPLC column at different concentrations; (b) Standard curve of Cys-XPO1 PROTAC polypeptides.

[0032] Figure 5 Stability of XPO1 PROTAC and se-XPO1 PROTAC; (a) Release of free polypeptide after adding DTT to se-XPO1 PROTAC; (b) Stability of Free peptide (XPO1 PROTAC-Cys) and se-XPO1 PROTAC in chymotrypsin.

[0033] Figure 6 Uptake of se-XPO1 PROTAC by MM cells; (a) Flow cytometry analysis of the uptake of se-XPO1 PROTAC by MM cell lines; (b) Laser confocal imaging directly showing the uptake of se-XPO1 PROTAC by MM cell lines (scale bar: 10 μm).

[0034] Figure 7 Inhibition of the activity of se-XPO1 PROTAC and nano-se on MM cells; (a) Activity and IC 50 results of MM cell lines; (b) Activity and IC 50 results of U937 cell lines; (c) Activity and IC 50 results of HUVEC control cell lines.

[0035] Figure 8 Statistical analysis of the inhibitory effect of Nano-se on the activity of all cell lines.

[0036] Figure 9 Statistical analysis of the inhibitory effect of se-reverse PROTAC on the activity of all cell lines.

[0037] Figure 10 Degradation of target proteins by se-XPO1 PROTAC in MM and control cell lines; (a) Degradation and statistical analysis of XPO1 target proteins in MM cell lines; (b) Degradation and statistical analysis of XPO1 target proteins in U937 cell lines.

[0038] Figure 11DC of se-XPO1 PROTAC in MM cell lines 50 。

[0039] Figure 12 Degradation of target proteins by se-reverse PROTAC and nano-se; (a) Degradation of XPO1 target protein by se-reverse PROTAC and statistical analysis; (b) Degradation of XPO1 target protein by nano-se and statistical analysis.

[0040] Figure 13 Degradation of XPO1 target protein by se-XPO1 PROTAC after Carfizomib blockade; (a) Selection of working concentration of proteasome inhibitor carfizomib; (b) Degradation of XPO1 target protein by se-XPO1 PROTAC after Carfizomib blockade and statistical analysis.

[0041] Figure 14 Degradation of XPO1 target protein by se-XPO1 PROTAC after CHX blockade; (a) Degradation of XPO1 target protein and statistics in RPMI 8226 cell line; (b) Degradation of XPO1 target protein and statistics in U266 cell line.

[0042] Figure 15 Flow cytometry results of the pro-apoptotic effects of se-XPO1 PROTAC and nano-se on MM cells

[0043] Figure 16 Statistical analysis of the pro-apoptotic effects of se-XPO1 PROTAC and nano-se on MM cells

[0044] Figures 17 - 18 Fluorescence imaging results of se-XPO1 PROTAC and different treatment groups of NSG mice (n = 6)

[0045] Figure 19 Evaluation of the treatment effects of NSG mice in each treatment group; (a) Quantitative analysis and statistics of the total fluorescence intensity of NSG mice in each treatment group (Total Flux = Dorsal Flux + Ventral Flux; MEAN ± SD, n = 6); (b) Body weight statistics of NSG mice in each treatment group (MEAN ± SD, n = 6); (c) Survival curves and analysis of MM mice in different groups (n = 5); * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents no statistical significance).

[0046] Figure 20In vivo toxicity assessment of se-XPO1 PROTAC and selinexor; (a) Assessment of acute liver and kidney toxicity indicators (ALT, AST, BUN, Cr) in normal C57 mice after drug treatment in each treatment group; (b) Hematological toxicity analysis (hemoglobin / Hb, platelet / PLT, white blood cell / WBC, etc.) in normal C57 mice after long-term treatment with drugs in each treatment group; (c) Changes in mouse body weight during acute toxicity assessment; (d) Changes in mouse body weight during chronic toxicity assessment; (e) HE staining to evaluate the toxicity of each treatment group to each organ (scale bar: 50 μm); (MEAN ± SD, n = 5; * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents no statistical significance).

[0047] Figure 21 The killing effect of se-XPO1 PROTAC and selinexor on MM patient samples in vitro.

[0048] Figure 22 Statistical analysis of apoptotic cells in MM patient samples by flow cytometry.

[0049] Figure 23 The killing effect of se-XPO1 PROTAC and selinexor on PBMCs from healthy donors.

[0050] Figure 24 Statistical analysis of apoptotic cells in PBMCs by flow cytometry. Detailed implementation mode

[0051] The present invention can be further elaborated by the following detailed description in conjunction with the accompanying drawings, but the present invention is not limited to the scope of the described embodiments. For the experimental methods without specific conditions indicated in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0052] The meanings of the substances used in the embodiments of the present invention are as follows:

[0053] XPO1 represents the target protein exportin 1; PROTAC represents proteolysis targeting chimera technology; Nano-se represents a nano-selenium delivery system; Se-XPO1 PROTAC represents the finished polypeptide drug conjugated with nano-selenium; Se-XPO1 REVERSEPROTAC represents the finished reverse polypeptide drug conjugated with nano-selenium; Selinexor represents selinexor, a small molecule inhibitor targeting XPO1; GAPDH represents glyceraldehyde-3-phosphate dehydrogenase, used as an internal reference in WB and qPCR experiments; Carfil represents Carfilzomib, a proteasome inhibitor; CHX represents Cycloheximide, a protein production inhibitor.

[0054] Sodium selenite, chitosan, and vitamin C used in the present invention were all purchased from Aladdin Reagents.

[0055] The present invention provides a PROTAC polypeptide targeting XPO1, and the amino acid sequence of the polypeptide is EMSEFALEMDKIGGGSGGGTSFEQFWAWLWP.

[0056] The present invention has no special limitation on the synthesis method of the polypeptide, and the well-known solid-phase synthesis method of polypeptides in the art can be used. There is also no special limitation on the solid-phase synthesis method of polypeptides, and the well-known polypeptide synthesis methods in the art can be used, such as Fmoc polypeptide synthesis. The synthesis of the PROTAC polypeptide of the present invention was completed by Nanjing Genscript Biotechnology Co., Ltd.

[0057] The present invention provides a nanodelivery system including the polypeptide, labeled as se-XPO1 PROTAC. The present invention has no special limitation on the type of the delivery system, and the well-known delivery systems in the art can be used, such as inorganic metals, organic fibers, liposomes, or other nanodelivery systems recognized in the art. In the examples of the present invention, taking the nanoselenium delivery system as an example, the raw materials used are sodium selenite, chitosan, and vitamin C. The present invention has no special limitation on the preparation method of the nanoselenium-polypeptide drug, and the well-known method of coupling nanoselenium with proteins in the art can be used. The nanoselenium-polypeptide drug has been experimentally proven to have no toxic manifestations at the cellular level, animal level, and ex vivo patient samples, and has high safety.

[0058] The following combines specific examples to detail the PROTAC polypeptide drug targeting XPO1 provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0059] Example 1

[0060] Design and synthesis of PROTAC polypeptide and nanoselenium-coupled polypeptide drug

[0061] 1. PROTAC polypeptide

[0062] The amino acid sequence of the XPO1 PROTAC polypeptide targeting the NES domain of XPO1 designed using computer-aided design means (Rosseta) is EMSEFALEMDKIGGGSGGGTSFEQFWAWLWP, as shown in SEQ ID NO.1 ( Figure 1 ).

[0063] To conjugate the Nano - Se carrier, the present invention designs to modify the Cys residue at the N - terminus of the polypeptide, obtaining the Cys - XPO1 PROTAC polypeptide, whose amino acid sequence is CEMSEFALEMDKIGGGSGGGTSFEQFWAWLWP, as shown in SEQ ID NO.2.

[0064] To make the interpretation and inference of experimental results more accurate and reliable, the present invention also designs the reverse peptide Reverse - XPO1 PROTAC as a control group, whose amino acid sequence is PWLWAWFQEFSTGGGSGGGIKDMELAFESME, as shown in SEQ ID NO.3.

[0065] 2. Nano - Se conjugated polypeptide drug

[0066] The present invention selects Nano - Se as the polypeptide drug carrier to assist XPO1 PROTAC to enter tumor cells and play its role. The preparation method of the Se - XPO1 PROTAC drug is as follows:

[0067] 1) Weigh 2 mg of Cys - XPO1 PROTAC polypeptide powder, dissolve it in 800 μL of sterile ddH 2 O, quickly add 100 μL of sodium selenite solution (50 mM), 300 μL of chitosan solution (mass concentration of 0.5%), and 800 μL of VC solution (50 mM), mix well, and the final volume is 2 mL.

[0068] 2) React the mixture in a water bath at 50 °C for 20 min, let it stand at room temperature for 4 h, and then react overnight at 4 °C and store.

[0069] 3) Filter the prepared drug through a 0.22 - μm filter to sterilize it, and then the Nano - Se conjugated polypeptide drug se - XPO1PROTAC can be obtained. Calculate the obtained mass concentration as 1 mg / mL and the molar concentration as 250 μM.

[0070] It should be noted that the above - fixed dosages and reaction parameters are only exemplary selections of the present invention, and are not special limitations on the raw material dosages and reaction conditions in the preparation process of the present invention. When preparing the Nano - Se conjugated polypeptide drug provided by the present invention, the molar mass ratio of PROTAC polypeptide, selenium salt, and vitamin C can vary within the range of 1:8 - 12:70 - 90. The mass concentration of the chitosan solution can be 0.3 - 0.8%. The reaction conditions can be selected to react the mixture at 45 - 55 °C for 15 - 25 min, then let it stand at room temperature for 3 - 4 h, and then react overnight at 4 °C. The Nano - Se conjugated polypeptide drug with the expected effect of the present invention can be prepared under these dosage ranges or reaction conditions, and those skilled in the art can specifically select according to the actual situation during preparation.

[0071] Example 2

[0072] 1. Characterization of Cys-XPO1 PROTAC polypeptide:

[0073] Weighed 1 mg of Cys-XPO1 PROTAC, dissolved it in 200 μL of sterile ddH 2 O, and immediately loaded it for analysis. The specific parameters were UV = 214 nm.

[0074] Through the Expasy website (https: / / web.expasy.org / compute_pi / ), the theoretical molecular weight (MW) of the designed Cys-XPO1 PROTAC polypeptide was accurately calculated to be 3554.95 kD. The crude product of Cys-XPO1 PROTAC polypeptide was obtained by the Fmoc solid-phase peptide synthesis method, and then the crude product was prepared and purified using preparative HPLC.

[0075] To verify whether the purity and molecular weight of the synthesized polypeptide were correct, for the purified Cys-XPO1 PROTAC polypeptide, analytical HPLC was used to verify that the purity of the target polypeptide end product was > 95%( Figure 2 a).

[0076] At the same time, MS was used to identify whether the synthesized polypeptide was the target polypeptide. The result showed that the relative molecular weight of the obtained free polypeptide was 3554.8 kD, which was basically consistent with the calculated molecular weight( Figure 2 b).

[0077] 2. Characterization of se-XPO1 PROTAC polypeptide

[0078] 1) Electron microscopy: Took 100 μL of the prepared se-XPO1 PROTAC drug, diluted it with ddH 2 O, dropped the sample onto a copper grid, fixed it for 15 min, and observed it with a transmission electron microscope.

[0079] The results were as Figure 3 shown in a. The particle size of the se-XPO1 PROTAC polypeptide was about within 100 nm, belonging to nanoscale materials and having the conditions to penetrate cell membranes. And the nano-polypeptide was in the form of scattered and evenly distributed particles, and the particle diameter was about 50 nm.

[0080] 2) Particle size and potential: Took 100 μL of the prepared se-XPO1 PROTAC drug, diluted it to 2 mL with ddH 2 O, added the sample to a Malvern cuvette, and used Zetasizer software for detection and data analysis.

[0081] Generally speaking, the obtained hydrated particle size will be larger than the diameter of the nanoparticles themselves, and the result is shown as 87.32 nm( Figure 3 b), and the average result of 3 detections is (89.74 ± 2.10) nm.

[0082] In addition, for the field of nanomaterials, if the Zeta potential of nanoparticles in the aqueous phase is higher than +30 mV or lower than -30 mV, then the dispersion system is relatively stable. Since chitosan used in the preparation process of this nanomaterial has cationic properties, as shown in Figure 3 c, the potential of this polypeptide is +44.5 mV, and the average result of 3 measurements is (+44.9350 ± 0.40) mV, indicating its certain stability in the aqueous phase distribution.

[0083] 3. Quantification of Cys-XPO1 PROTAC and construction of standard curve

[0084] For the quantification of Cys-XPO1 PROTAC, high performance liquid chromatography (HPLC) was used for detection. The compatible chromatographic column was a C18 reverse phase chromatographic column (Waters XBridge TM 3.5 μm, 4.6 × 150 mm), the temperature was 40 °C, and ultraviolet analysis method was used. The specific parameters were UV = 214 / 280 nm, and the mobile phase was acetonitrile.

[0085] 1) The specific program of HPLC settings was as follows: A was pure methanol, B was ultrapure water, C was 10% methanol-water, D was acetonitrile (mobile phase); 0 - 2 min: 5% acetonitrile, 95% water; 2 - 33 min: 5% acetonitrile to 95% acetonitrile, 95% water to 5% water; 33 - 38 min: 95% acetonitrile to 5% acetonitrile, 5% water to 95% water; 38 - 43 min: 5% acetonitrile, 95% water.

[0086] 2) The standard curve of Cys-XPO1 PROTAC polypeptide was constructed using HPLC. Polypeptide solutions with different concentrations of XPO1 PROTAC were prepared, and the absorption peaks of the polypeptide solutions at 280 nm under different concentration conditions were detected ( Figure 4 a), and the absorption peak areas were calculated using Origin software to construct the standard curve of the standard product ( Figure 4 b).

[0087] 4. Drug loading characterization of se-XPO1 PROTAC polypeptide and stability of se-XPO1 PROTAC polypeptide

[0088] After the se-XPO1 PROTAC drug was prepared, HPLC was used to characterize the drug loading of the polypeptide.

[0089] 1) Excessive DTT was added to se-XPO1 PROTAC, and the free XPO1 PROTAC was reduced from the nano-se carrier. The peak area was detected and calculated by HPLC to obtain its drug loading and efficiency. After preparation, se-XPO1 PROTAC is a nano-system drug. However, after adding excessive reducing agent DTT, the free XPO1 PROTAC was reduced from the nano-se carrier and was very easily detected by HPLC( Figure 5 a).

[0090] 2) Detection of the stability of Cys-XPO1 PROTAC in chymotrypsin:

[0091] (1) Prepare a chymotrypsin (from human liver) solution with a concentration of 10 units / mL;

[0092] (2) Weigh 2 mg of Cys-XPO1 PROTAC, dilute it with ddH 2 O and add it to the chymotrypsin solution at a ratio of 1:1;

[0093] (3) Incubate at room temperature for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h;

[0094] (4) Add acetonitrile at a ratio of 1:1 to mix well when the time is up, and store at -20 °C;

[0095] (5) Perform HPLC quantitative detection.

[0096] 3) Detection of the stability of se-XPO1 PROTAC in chymotrypsin:

[0097] (1) Prepare a chymotrypsin (from human liver) solution with a concentration of 10 units / mL;

[0098] (2) Mix se-XPO1 PROTAC and chymotrypsin solution at a ratio of 1:1;

[0099] (3) Incubate at room temperature for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h;

[0100] (4) Add acetonitrile at a ratio of 1:1 to mix well when the time is up, and store at -20 °C;

[0101] (5) Perform HPLC quantitative detection.

[0102] As is well known, free polypeptides have poor stability and are easily degraded by proteases in the body. Therefore, the present invention evaluated the stability of free XPO1 PROTAC and se-XPO1 PROTAC under different conditions. The results are as Figure 5As shown in Figure b, the free XPO1 PROTAC polypeptide has extremely poor in vitro stability under the condition of chymotrypsin and may be hydrolyzed within about 20 minutes, which is also the biggest defect of PROTAC drugs designed based on polypeptides. However, the stability of the se-XPO1 PROTAC conjugated with the nano-se carrier far exceeds that of the free polypeptide, and even when incubated with chymotrypsin for more than 12 hours, the stability remains above 75%.

[0103] Example 3

[0104] Effect of se-XPO1 PPROTAC on cells

[0105] 1. Uptake of se-XPO1 PPROTAC in cells:

[0106] Prepare cells: MM.1S, RPMI 8226; prepare drugs: Cy5-labeled se-XPO1 PROTAC;

[0107] Prepare drugs: Cy5-NHS, 50 mM sodium selenite, 0.5% chitosan, 50 mM VC solution;

[0108] Set time points: 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h;

[0109] Experimental steps: (1) Incubate MM.1S and RPMI 8226 cells with the drug (2 μM) at time gradients;

[0110] (2) Prepare cell fixative: 4% paraformaldehyde + 0.3% Triton X;

[0111] (3) Centrifuge the cells, resuspend the cell pellet with the fixative, and incubate at room temperature for 30 min;

[0112] (4) After washing with PBS, stain with DAPI for 10 min;

[0113] (5) Wash with PBS and take confocal microscopy images.

[0114] MM.1S and RPMI 8226 cells were co-cultured with the fluorescently labeled polypeptide drug Cy5-se-XPO1 PROTAC (1.0 μM), and the incubation times were set to 0 h, 2 h, 4 h, 8 h, and 12 h. After the incubation, some of the cells were used for flow cytometry detection, and the other part was used for laser confocal microscopy detection. The results are as follows Figure 6As shown in a, for both MM.1S and RPMI 8226 cells, the fluorescence signal of Cy5-se-XPO1 PROTAC uptake gradually increased with time. After 12 h of co-incubation, approximately 70% of the cells were Cy5-positive.

[0115] To visually demonstrate the process of se-XPO1 PROTAC penetrating the membrane and entering MM cells, the present invention conducted laser confocal detection. As Figure 6 shown in b, within 2 h of incubating with MM cells, se-XPO1 PROTAC could rapidly enter MM cells and showed a time-dependent delivery effect. By 12 h, almost all of the fluorescent polypeptide drugs had entered the cells, which was consistent with the results of our flow cytometry detection.

[0116] All in all, the results of the cell uptake experiment indicate that the nano-se vector provided by the present invention can effectively deliver XPO1 PROTAC into MM cells and mediate the further function of the PROTAC drug in targeted protein degradation.

[0117] 2. Effects of se-XPO1 PPROTAC on the proliferation activity of MM cells:

[0118] 1) When conducting experiments to detect cell viability, the MM cell lines NCI-H929, MM.1S, RPMI 8226, U266, the AML control cell line U937, and the normal control cell line HUVEC were selected for in vitro cell experiments;

[0119] 2) Cells were seeded into 96-well plates at a density of 2×10 5 cells / mL. 90 μL of cell suspension was added to each well, and 10 μL of drugs at different concentrations (0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM) were added to each well of cells for treatment. A blank control group (PBS), a single nano-selenium group (se), a reverse peptide group (reverse PROTAC), and a medium background group (media) were set up;

[0120] 3) After 24 h and 48 h of treatment, 10 μL of CCK-8 reagent was added to each well and incubated in the 37 °C incubator in the dark for 2 h;

[0121] 4) After color development, the absorbance values of each well were measured at wavelengths of 450 nm and 690 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0122] 5) Calibrate the absorbance value of each well according to formula (1-1), and calculate the cell viability according to formula (1-2):

[0123] A = OD 450 -OD690 (1-1)

[0124] Cell viability (%) = (A (experimental group) - A (background group) / A (control group) - A (background group)) × 100% (1-2)

[0125] 6) Use GraphPad software to process the data to obtain the IC corresponding to each cell line 50 .

[0126] The results are as Figure 7 shown in a. se-XPO1 PROTAC showed dose-dependent growth inhibitory effects in all MM cell lines, and the 24h IC 50 was all around 1-4 μM. The specific results are as follows: The IC of the polypeptide drug against the cell line NCI-H929 cell line 50 was 1.47 μM and 0.93 μM respectively. The IC against the MM.1S cell line 50 was 2.31 μM and 1.18 μM respectively. The IC against the RPMI-8226 cell line 50 was 3.87 μM and 2.87 μM respectively. The IC against the U266 cell line 50 was 1.13 μM and 0.39 μM respectively. Detection was carried out in the acute lymphoblastic leukemia cell line (U937), and the obtained IC 50 was 5.41 μM and 4.90 μM respectively, indicating that the PROTAC polypeptide drug has a significant effect on inhibiting tumor growth activity.

[0127] In addition, non-MM human histiocytic lymphoma cells (U937) were selected for verification, and it was found that se-XPO1 PROTAC could also inhibit tumor cell growth, and the IC 50 was maintained at around 5 μM ( Figure 7 b). As a normal control cell line, se-XPO1PROTAC showed no obvious growth inhibitory effect in human umbilical vein endothelial cells (HUVEC) whether it was 24h or 48h, indicating that the polypeptide drug has no obvious killing effect on normal cells and has a certain safety ( Figure 7 c).

[0128] In addition, the present invention also detected the proliferation activity of single nano-selenium particles (nano-se) on all cell lines. The statistical results whether it was 24h or 48h showed that nano-se had an inhibitory effect on the activity of MM and other cell lines, further indicating that the carrier selected by the present invention has reliable safety ( Figure 8 ).

[0129] To complete the verification, the present invention uses the same preparation method to couple the designed and synthesized reverse PROTAC with nano-se. In the verification of the MM cell line, the statistical results at both 24 h and 48 h show that se-reversePROTAC has no obvious inhibitory effect on cell activity in the low concentration range, and only shows some tumor cell killing effects when used at high concentrations. Figure 9 )

[0130] 3. Degradation of XPO1 protein by se-XPO1 PROTAC

[0131] To study the degradation ability of the se-XPO1 PROTAC drug on XPO1 protein, WB experiments were used for verification, and the following three experiments were designed in sequence:

[0132] 1) The degradation of XPO1 protein by the PROTAC polypeptide drug is concentration-dependent ( Figures 10 - 12 ):

[0133] (1) Cell preparation: NCI-H929, MM.1S, RPMI-8226, U266, U937.

[0134] (2) Sample treatment: Cells were seeded into 6-well plates at a density of 1×10 6 cells / mL, 900 μL of cell suspension was added to each well, and then 100 μl of the corresponding drug concentration (PBS, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM) was added for treatment.

[0135] (3) Protein extraction: After 24 h, the cells were collected, lysed, and the protein was extracted.

[0136] (4) The total protein content in each sample was detected by the BCA quantification method, and the remaining samples were added with 5×Loading Buffer and boiled at 100 °C for 10 min to completely denature the protein.

[0137] (5) SDS-PAGE electrophoresis: The voltage was set to 80 V, and it took about 20 min until the bromophenol blue reached the separating gel. Then the voltage was adjusted to 120 V, and it took about 60 min until the bromophenol blue reached about the end of the separating gel, and the electrophoresis was stopped.

[0138] (6) Membrane transfer: In all Western Blot experiments of the present invention, PVDF membranes were used, and the membranes needed to be activated with methanol for 1 - 2 min before membrane transfer. On the membrane transfer clamp, arrange in order: filter paper, PVDF membrane, gel, filter paper. Set the membrane transfer current to 400 mA and the membrane transfer time to 2 h.

[0139] (7) Blocking: The PVDF membrane was immersed in a blocking solution containing 1% BSA and incubated on a horizontal shaker at room temperature for 1 h to remove the influence of non-specific adsorption.

[0140] (8) Primary antibody incubation: Dilution solutions of XPO1 and GAPDH antibodies were prepared and then incubated on a horizontal shaker at 4 °C overnight to achieve the purpose of antibody recognition of specific antigens.

[0141] (9) Secondary antibody incubation: A species-specific HRP-labeled secondary antibody (anti-mouse or anti-rabbit) was prepared according to the source species of different primary antibodies and incubated on a horizontal shaker at room temperature for 1 h.

[0142] (10) Membrane washing: After incubation with the primary and secondary antibodies, the membrane was washed on a horizontal shaker with 1×TBST for 10 min, three times in total.

[0143] (11) Development: A developing solution was prepared, the PVDF membrane was completely wetted, and exposure was performed using a chemiluminescence imager. The obtained data and images were saved.

[0144] (12) Analysis and plotting using Image J and GraphPad software.

[0145] The function of the PROTAC polypeptide designed in the present invention is to bind to the XPO1 protein at the XPO1 POI end, and bind to the E3 ubiquitin ligase MDM2 at the other end MDM2POI. Through MDM2, the XPO1 protein is ubiquitinated and labeled, and finally degraded by the proteasome. Therefore, the present invention needs to verify the biological function of se-XPO1 PROTAC at the protein level. According to the CCK-8 detection results, a drug concentration range of 0.25 μM, 0.5 μM, 1.0 μM, 2.0 μM, and 4.0 μM was finally selected for treatment. MM.1S, NCI-H929, RPMI 8226, U266, and U937 cells were treated with drugs at different concentrations, and proteins were collected after 24 h for WB experiment verification.

[0146] The results are as Figure 10 shown in a. se-XPO1 PROTAC has a significant XPO1 protein-targeted degradation effect on multiple MM cell lines, and shows a drug concentration dependence. Especially in MM.1S and NCI-H929 cells, drug concentrations of only 0.5 μM and 0.25 μM showed obvious degradation effects, and there were statistical differences (p < 0.01; p < 0.001). Compared with U937 cells, the PROTAC polypeptide drug needs to be about 2 μM to exert a degradation effect (p < 0.001), as Figure 10 shown in b. In addition, the present invention quantified and calculated the half-maximal degradation concentration (DC50 ) Each Western blot (WB) experiment was repeated three times. As Figure 11 shown, the DC50 values of each MM cell line for se-XPO1 PROTAC were MM.1S (667.7 nM), NCI-H929 (219.0 nM), RPMI 8226 (744.2 nM), and U266 (875.2 nM), respectively. The above results indicate that se-XPO1 PROTAC has broad applicability to the XPO1 protein degradation function of MM cell lines with different genetic backgrounds and can be achieved at relatively low concentrations.

[0147] Similarly, to verify that the degradation of XPO1 protein in MM cell lines was specifically caused by se-XPO1 PROTAC, the XPO1 protein levels after treatment with se-reverse PROTAC were also verified. Proteins were extracted after treating with 4 μM reverse peptide drug for 24 h, and WB experiments were performed. The results are as Figure 12 shown in Fig. a. se-reverse PROTAC did not have the function of targeting and degrading XPO1 protein. In addition, to prove the safety of the nano-se vector, this study also separately verified whether nano-se had the same protein degradation effect on different MM cell lines and used the same experimental method for verification. The results are as Figure 12 shown in Fig. b. Even at a working concentration of 4 μM, nano-se still had no XPO1-targeted degradation effect on multiple MM cell lines. The above results fully demonstrate that the designed se-XPO1 PROTAC of the present invention has the ability to specifically degrade XPO1 protein, and the selected nano-se vector has a certain degree of safety.

[0148] 2) The degradation effect of the drug on XPO1 protein after adding the proteasome inhibitor (Carfilzomib) is as follows:

[0149] (1) Treating samples: RPMI-8226 and U266 cells were seeded into 6-well plates at a density of 1×10 6 cells / mL. 900 μL of cell suspension was added to each well, and then 100 μl of the corresponding drug concentration (PBS, 0.5 μM, 1 μM, 0.5 μM + 5 nM Carfi, 1 μM + 5 nM Carfi) was added for treatment.

[0150] (2) The WB procedure was the same as above.

[0151] The results are as Figure 13As shown in a , after treating with carfizomib at different concentrations for 24 h, the XPO1 protein was not degraded, and 5 nM was finally selected as the experimental drug concentration. According to the previous WB experimental results, in the control group, RPMI 8226 and U266 cell lines were treated with se-XPO1 PROTAC drugs at low concentration (0.5 μM) and high concentration (1.0 μM). In the experimental group, 5 nM carfifizomib was added to inhibit the function of the proteasome. After 24 h, proteins were collected for WB experiment verification. The results are as Figure 13 shown in b . Se-XPO1 PROTAC could significantly degrade the XPO1 protein in RPMI 8226 and U266. However, after adding carfilzomib, the degradation of the XPO1 protein was significantly reduced, and the expression of the XPO1 protein was effectively reversed, with statistical differences.

[0152] These results further indicate that the degradation of the XPO1 protein by PROTAC drugs depends on the proteasome pathway.

[0153] 3) After adding the protein synthesis inhibitor (Cycloheximide, CHX), the time-dependent degradation effect of the drug on the XPO1 protein is as follows:

[0154] (1) Treat the samples: Seed RPMI-8226 and U266 cells into 12-well plates at a density of 1×10 6 cells / ml. Add 900 μL of cell suspension to each well, and then add 100 μL of the corresponding drug concentration (50 μg / mL CHX, 1 μM PROTAC + 50 μg / mL CHX). Set time gradients of 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h.

[0155] (2) Collect samples according to the time, and the WB procedure is the same as above.

[0156] The experimental purpose of this part of the study is to use CHX to block the protein synthesis in cells, so as to judge the degradation rate of the XPO1 protein in MM cell lines only under the action of se-XPO1 PROTAC. According to the literature review, the effective working concentration of CHX finally used for se-XPO1 PPROTAC was determined to be 50 μg / mL. In the study, a separate CHX control group and a CHX + se-XPO1 PROTAC (1 μM) drug group were set up. RPMI 8226 and U266 cells were treated with time gradients, and proteins were collected and verified by WB experiment at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively.

[0157] The results are as Figure 14As shown, in the case of inhibiting protein production in MM cells, the targeted degradation of XPO1 protein was enhanced in a time-dependent manner, showing obvious protein degradation at 8 h, and being basically completely degraded at 24 h.

[0158] 4. Effects of se-XPO1 PPROTAC on apoptosis of MM cells:

[0159] To study the pro-apoptotic effect of se-XPO1 PROTAC drug on myeloma cells, flow cytometry (FCS) was used for verification:

[0160] 1) Treating samples: Seed NCI-H929, MM.1S, RPMI 8226, U266, and U937 cells into 6-well plates at a density of 5×10 5 cells / mL. Add 450 μL of cell suspension to each well, and then add 50 μL of the corresponding drug (PBS, nano-se, 1 μM, 2 μM, 4 μM) for treatment;

[0161] 2) After 24 h, collect the cells, centrifuge at 300 g for 5 min, and discard the supernatant;

[0162] 3) Resuspend the cells with 100 μL of FCS buffer. Add 5 μL of AnnexinV and 10 μL of PI to each sample, and incubate in the dark for 15 min;

[0163] 4) Detect with a flow cytometer and collect 1×10 4 cells;

[0164] 5) Analyze the data with FlowJo software and draw a statistical chart with GraphPad software.

[0165] The results are as Figure 15 shown. The empty vector nano-se had no obvious pro-apoptotic effect on MM cells (no statistical difference). se-XPO1 PROTAC could effectively enter MM cells and cause a concentration-dependent pro-apoptotic effect, and the statistical difference was significant. However, high-concentration empty vector nano-se did not induce apoptosis of MM cells ( Figure 15 and Figure 16 ). For the U937 control cell line, only when the drug concentration was 4 μM, the apoptotic effect of tumor cells was statistically different. However, during the detection of the normal control cell line HUVEC, it was found that se-XPO1 PROTAC had no obvious apoptotic induction effect (p > 0.05).

[0166] Example 5

[0167] Verify the effect of se-XPO1 PROTAC drug at the animal level.

[0168] 1. Establishment of a mouse model of multiple myeloma:

[0169] 6-week-old female NSG mice were used, and 200 μL of luc-U266 cells (a total of 2×10 6 cells) were injected into the tail vein. Small animal in vivo imaging was performed on day 18 after injecting the tumor cells. Before imaging, 200 μL of the substrate (potassium fluorescein) (concentration 15 mg / kg) needed to be injected intraperitoneally. Imaging should be carried out as soon as possible within 15 - 30 minutes after injecting the substrate to prevent the substrate from being metabolized. The mice were anesthetized with isoflurane gas, and both the dorsal and ventral sides of the mice were imaged to observe whether tumors formed.

[0170] The present invention successfully constructed a luc-U266 xenograft mouse model in vivo, and tumors formed on day 18. After random grouping, 30 mice were divided into 5 groups, and drug treatment was given every other day starting from day 19. It should be noted that selinexor was administered by gavage, and the dose was the same as that of the other drug groups.

[0171] 2. Grouped drug treatment:

[0172] Thirty mice were randomly divided into 5 groups, namely PBS (control group), nano-se (empty vector group), reversePROTAC (reverse peptide group), selinexor (selinexor, a small molecule drug), and se-XPO1 PROTAC (polypeptide treatment group), with 6 mice in each group. All drugs were used at the same dose (5 mg / kg), injected once every other day, and the administration method was intraperitoneal injection, with a drug volume of 200 μL; Selinexor was administered by gavage at the same dose. The treatment lasted for 16 days, with a total of 8 doses. Small animal in vivo imaging was performed every 4 - 5 days during the treatment period. The mice were sacrificed on day 35 after the treatment ended.

[0173] According to the results of multiple imaging, the se-XPO1 PROTAC group had good therapeutic effects, and there were significant statistical differences compared with other groups ( Figures 17 - 18 and Figure 19 a).

[0174] 3. Recording of mouse body weight and survival curve ( Figure 19 b and 19c):

[0175] The method for establishing the animal model and grouped treatment was the same as before. During the treatment period, mouse imaging was not required. After 16 days of treatment for each group according to the above method, with a total of 8 doses, the normal death time of the mice was recorded as the survival endpoint, and at the same time, the activity and body weight changes of the mice were observed and monitored.

[0176] 4. Evaluation of drug toxicity experiments:

[0177] 1) Acute toxicity: 6-week-old female C57 mice were used. The drugs were grouped into PBS (control group), nano-se (empty vector group), reverse PROTAC (reverse peptide group), selinexor (small molecule drug), and se-XPO1 PROTAC (polypeptide treatment group). The drugs were administered once a day for 3 days, with a total of 3 doses (15 mg / kg), and the administration method was the same as described above. The mice were sacrificed on day 4 after the treatment, and the whole blood of the mice was sent for routine blood test, and the organs were subjected to HE staining.

[0178] 2) Chronic toxicity: The mouse model was established as described above. The drugs were administered once every other day for 20 days, with a total of 10 doses (5 mg / kg), and the administration method was the same as described above. The mice were sacrificed on day 21 after the treatment, and the serum of the mice was sent for liver function (ALT, AST) and renal function (BUN, Cr) index detection.

[0179] 5. HE staining method: This part of the experiment was completed by Wuhan Sevier Biotechnology Co., Ltd.

[0180] As Figure 20 shown, single nano-selenium particles have no therapeutic effect on tumor growth, selinexor has a certain tumor inhibitory effect, and se-XPO1 PROTAC shows good efficacy in the luc-U266 xenograft model. To evaluate the safety of the se-XPO1 PROTAC drug, the same treatment was performed on immunocompetent C57BL / 6 mice in the xenograft model. In this model, the se-XPO1 PROTAC drug did not show obvious toxicity in organs such as the hematopoietic system, liver, spleen, and kidney. Existing targeted small molecule drugs such as selinexor cannot produce a degradation effect on the XPO1 protein, so the inhibitory tumor effect shown in animal experiments is not obvious. The polypeptide drug provided by the present invention can solve this problem.

[0181] Example 6

[0182] Verify the effect of the Se-XPO1 PROTAC drug at the patient sample level.

[0183] 1. Extraction of patient primary cells

[0184] 1) Confirm patient information and aspirate the bone marrow sample into a 15 ml centrifuge tube;

[0185] 2) Add specimen, normal saline / PBS, and erythrocyte sedimentation fluid in a ratio of 1:1:1;

[0186] 3) Let the specimen sediment for 25 min, take the supernatant and discard the precipitate;

[0187] 4) Centrifuge 300 g of the supernatant for 5 min, discard the supernatant, and resuspend the precipitate in PBS to 1 mL.

[0188] 5) Add erythrocyte lysate at a ratio of 1:3, let it stand for 10 min and then centrifuge, discard the supernatant. If there are still many red blood cells, repeat this step.

[0189] 6) Prepare the medium: 90% RPMI-1640 + 10% FBS, resuspend the cell precipitate after centrifugation, transfer it to a T25 culture flask, and incubate at 37 °C and 5% CO 2 incubation.

[0190] 2. Sorting of CD38-positive cells in multiple myeloma

[0191] This invention uses a CD38 magnetic bead sorting kit (purchased from Miltenyi) for sorting. The formula of MACS buffer used in the process is: PBS + 2 mM EDTA + 1% FBS, stored at 4 °C.

[0192] 1) Antibody labeling:

[0193] (1) Count the primary cells, collect the cells, centrifuge at 300 g for 10 min, and discard the supernatant;

[0194] (2) Add 80 μL of buffer and 20 μL of CD38-Biotin to 1×10 7 cells;

[0195] (3) Gently blow to mix the cell suspension evenly and incubate in a 4 °C refrigerator for 10 min;

[0196] (4) Add 2 mL of buffer to wash 1×10 7 cells, centrifuge at 300 g for 10 min, and discard the supernatant;

[0197] (5) Add 80 μL of buffer and 20 μL of Anti-Biotin magnetic beads to 1×10 7 cells;

[0198] (6) Gently blow to mix the cell suspension evenly and incubate in a 4 °C refrigerator for 15 min;

[0199] (7) Add 2 mL of buffer to wash 1×10 7 cells, centrifuge at 300 g for 10 min, and discard the supernatant;

[0200] (8) Resuspend the precipitate in 500 μL of buffer.

[0201] 2) Magnetic bead sorting

[0202] (1) The sorting column is adsorbed on the magnetic stand, and the column is infiltrated with 3 mL of buffer, and wait naturally until it ends;

[0203] (2) Add the cell suspension into the sorting column, wait until the cells are completely infiltrated, and wait naturally until it ends;

[0204] (3) Wash the column with 3 mL of buffer each time, and repeat 3 times;

[0205] (4) Remove the sorting column and add 5 mL of buffer to wash the column;

[0206] (5) Collect the sorted cells in a culture flask and observe the morphology and count under the microscope.

[0207] After sorting out CD38+ primary MM cells from the patient's bone marrow samples and treating them with nano-se, se-XPO1 PROTAC for 24 h, and selinexor for 48 h respectively, apoptosis was measured by flow cytometry. The results showed that there was no difference in the statistical analysis of the apoptosis rates between the nano-se empty vector group and the control group. The se-XPO1 PROTAC treatment group could significantly induce apoptosis of primary MM cells, while selinexor could not induce obvious apoptosis of MM cells. This result further confirmed that the apoptosis-inducing effect activated by se-XPO1 PROTAC was MM-targeted. Most patients could induce apoptosis of primary MM cells at a drug concentration of 4 μM, and it had obvious statistical significance, as Figure 21 、 Figure 22 shown.

[0208] 3. Extraction of PBMC from healthy donors:

[0209] 1) Confirm the information of healthy donors, and draw 10 mL of whole blood into a 15 mL centrifuge tube;

[0210] 2) Transfer 10 mL of whole blood into a 50 mL centrifuge tube, add 10 mL of PBS solution for 1:1 dilution, and mix gently;

[0211] 3) Take two 15 mL centrifuge tubes, first add 5 mL of Ficoll solution, and then gently add the diluted 10 mL of blood along the tube wall to the upper layer of Ficoll. Be sure to be gentle to avoid mixing the two solutions together;

[0212] 4) Centrifuge at 2000 rpm for 20 min, and set the acceleration and deceleration to rise 1 and fall 0;

[0213] 5) After centrifugation, stratified cells can be obtained. The middle white film layer is PBMC, and suck out the cells in this layer with a pipette;

[0214] 6) Add 10 mL of PBS, centrifuge at 1500 rpm for 10 min, discard the supernatant, and then add complete medium for washing;

[0215] 7) Finally, add 5 - 10 mL of complete medium to resuspend the cells and incubate at 37 °C and 5% CO2.

[0216] 4. Cell treatment and detection

[0217] 1) Centrifuge the sorted cells at 300 g for 5 min and discard the Buffer;

[0218] 2) Add cell culture medium to resuspend and divide into 6 groups: PBS, se, selinexor (100 nM), selinexor (200 nM), PROTAC (2 μM), PROTAC (4 μM);

[0219] 3) Add 900 μL of cells + 100 μL of drug to each group and seed in 6 - well plates;

[0220] 4) Collect the cells after 24 h and detect apoptosis by flow cytometry (the experimental method is the same as before).

[0221] After treating PBMC under the same experimental conditions, apoptosis was measured by flow cytometry. The results are as Figures 23 - 24 , and there was no significant difference in the apoptosis rate between the se - XPO1 PROTAC treatment group and the nano - se empty vector group. When the concentration of se - XPO1 PROTAC was increased to 4 μM, a slight pro - apoptotic effect was shown, further confirming that the synthesized se - XPO1 PROTAC polypeptide drug has certain target selectivity and no obvious killing effect on normal cells.

[0222] Based on the above results, the nano - se provided by the present invention can significantly improve the stability of the XPO1 PROTAC polypeptide and achieve effective tumor cell - targeted delivery; in addition, the se - XPO1 PROTAC polypeptide can significantly reduce the XPO1 protein level in MM cells and activate the downstream signaling pathway through the p53 pathway, playing a role in killing tumor cells; the se - XPO1 PROTAC polypeptide shows good MM killing effects in both in vitro and in vivo experiments and is expected to become a potential targeted drug for the treatment of MM.

[0223] 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 nuclear export protein 1, characterized in that The amino acid sequence of the PROTAC polypeptide is EMSEFALEMDKIGGGSGGGTSFEQFWAWLWP.

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

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

4. The drug according to claim 3, characterized in that The carrier is a nano delivery system.

5. The drug according to claim 4, characterized in that The nano delivery system is nano selenium.

6. The drug according to claim 5, characterized in that The drug is prepared according to the following steps: After the PROTAC polypeptide is dissolved, a solution of selenium salt, chitosan and vitamin C are added and reacted to obtain the product.

7. The drug according to claim 6, 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 the chitosan solution is 0.3-0.8%, and the dosage ratio of the chitosan solution to the polypeptide is 150 μL: 1 mg; The reaction is to firstly mix the mixture at 45-55°C for 15-25 minutes, then stand at room temperature for 3-4 hours, and finally react at 4°C.

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