And theaflavin-3, 3apos, theaflavin-3, 3apos; use of digallate in the preparation of a medicament for the treatment of multiple myeloma

Abstract

The invention discloses application of theaflavin-3, 3 '-digallate in preparation of a medicine for treating multiple myeloma, and belongs to the technical field of multiple myeloma (MM). The TRIM28 inhibitor theaflavin-3, 3 '-digallate (TF3) obtained through high-throughput drug screening can effectively inhibit the E3 ubiquitinating enzyme activity of TRIM28, reduce TRIM22 degradation and down-regulate IRF4 protein expression, further significantly inhibits MM cell proliferation, can be used as a drug for treating multiple myeloma, and provides a new treatment strategy and drug target for preventing and treating multiple myeloma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the use of theaflavin-3,3'-digallate in the preparation of a medicament for treating multiple myeloma. BACKGROUND

[0002] Multiple myeloma (MM) is a plasma cell neoplastic disease, accounting for about 10% of hematological malignancies, and the incidence is increasing in the growing elderly population. The occurrence of the disease is due to genetic changes in normal plasma cells, forming monoclonal gammopathy of unknown significance. With the driving of secondary mutations and interaction with the bone marrow microenvironment, it further progresses to smoldering multiple myeloma. Finally, due to genomic instability and immune disorders, it evolves into active multiple myeloma. The most prominent clinical manifestations of multiple myeloma include anemia, hypercalcemia, renal failure, and bone lesions. Although new chemotherapy drugs (such as bortezomib and lenalidomide) are used for single or combined treatment, most patients still experience relapse or progression to refractory disease after high-dose chemotherapy, highlighting the grim reality that the disease is currently incurable.

[0003] Multiple myeloma is currently an incurable tumor, and many patients will relapse after achieving complete remission, so it is of great significance to deeply study the pathological mechanism of multiple myeloma and discover novel therapeutic targets. SUMMARY

[0004] The purpose of the present application is to provide the use of theaflavin-3,3'-digallate in the preparation of a medicament for treating multiple myeloma.

[0005] To achieve the above-mentioned purpose, the present application provides a new use of theaflavin-3,3'-digallate: it can be used for preparing a medicament for treating multiple myeloma.

[0006] Further, theaflavin-3,3'-digallate inhibits the ubiquitinase activity of TRIM28, reduces the degradation of TRIM22, down-regulates the expression of IRF4 protein, and thus significantly inhibits the proliferation and survival of MM cells.

[0007] Further, theaflavin-3,3'-digallate inhibits TRIM28, specifically including at least one of a product inhibiting the expression of TRIM28 gene, a product inhibiting the TRIM28 protein, and a product inhibiting the functional activity of the TRIM28 protein.

[0008] Further, the product for inhibiting the expression of TRIM28 gene or the product for inhibiting the expression of TRIM28 protein includes at least one of a polynucleotide, a lentivirus or retrovirus packaged plasmid, a virus, a lipid, and an inhibitory antibody of TRIM28; and the product for inhibiting the functional activity of TRIM28 protein includes at least one of a protein, a polypeptide, and an enzyme for inhibiting the activity of TRIM28 protein.

[0009] The application also provides a pharmaceutical composition for treating multiple myeloma, which at least comprises teaflavin-3,3'-digallic acid or a pharmaceutical excipient thereof.

[0010] Further, the pharmaceutical composition further comprises at least one of a proteasome inhibitor, an immunomodulator, or an anti-CD38 monoclonal antibody, which are combined for treating multiple myeloma.

[0011] The above technical solution has the following beneficial effects: The TRIM28 inhibitor teaflavin-3,3'-digallic acid (TF3) obtained through high-throughput drug screening can effectively inhibit the E3 ubiquitinase activity of TRIM28, reduce the degradation of TRIM22, down-regulate the expression of IRF4 protein, and thus significantly inhibit the proliferation of MM cells, and can be used as a drug for treating multiple myeloma, thereby providing a new treatment strategy and drug target for preventing and treating multiple myeloma. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 : Effect diagram of E3 ligase TRIM28 maintaining the stability of IRF4 by inhibiting the proteasome pathway; Figure 2 : Effect diagram of TRIM28 regulating the proliferation of myeloma cells by stabilizing IRF4 protein; Figure 3 : Effect diagram of TRIM22 directly combining with IRF4 and regulating the degradation rate thereof; Figure 4 : Effect diagram of TRIM28 degrading TRIM22 by inhibiting the proteasome pathway; Figure 5 : Effect diagram of the compound TF3 in treating tumors. DETAILED DESCRIPTION

[0013] To describe the technical content, structural features, purposes and effects of the technical solutions in detail, the following will be described in detail in combination with specific embodiments and the accompanying drawings. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0014] (1) Total protein extraction, western-blot, and immunoprecipitation and pull down analysis After the myeloma cells were washed with ice-cold PBS for 3 times, the cells were lysed with 50 μL of ice-cold lysis buffer containing protease inhibitor cocktail, and incubated on ice for 30 minutes. After centrifugation of the cells at 4°C, 12000 rpm, the supernatant was taken, and the protein was quantified by BCA. After the supernatant was supplemented with RIPA buffer to a uniform concentration, 5xLoading Buffer was added. After 5 minutes of incubation in a 95°C metal bath, 10-100 μg of total protein was used for 8-10% SDS-PAGE electrophoresis according to the molecular weight and expression of the target protein. After 2 hours of transmembrane at 220 mA, 5% skim milk was incubated at room temperature for 2 hours. The primary antibody and the HRP-labeled secondary antibody were used for incubation, respectively. For the immunoprecipitation experiment, the cells were first lysed with IP lysis buffer, and the supernatant was taken after centrifugation. 50 ul was taken as Input, and then added to the washed protein A / G beads. After 2 hours of rotation at 4°C, the cells were centrifuged at 4000 rpm, and the supernatant was taken. The supernatant was equally divided into two parts, and the primary antibody and IgG control were added for incubation at 4°C overnight. The washed protein A / G beads were added for incubation at 4°C for 2 hours. The IP lysis buffer (containing PMSF) was used to wash the beads precipitated at the bottom, and the washing was repeated for 5-6 times. The supernatant was discarded, 30 ul of 2X Loading buffer was added, and the mixture was boiled at 95°C for 5 minutes. The supernatant was taken for electrophoresis. For the pull down experiment, the cells were first lysed with IP lysis buffer, and the supernatant was taken after centrifugation. 30 μL of cell lysate was taken as WCL, and 5xLoading Buffer was added to 30 μL of cell lysate. The protein was denatured at 95°C metal bath for 5 min, and stored at -80°C. The remaining A lysis buffer was added to the washed HA agarose beads for incubation at 4°C overnight. After centrifugation to remove the supernatant, B lysis buffer was added for incubation at 4°C overnight. The IP lysis buffer (containing PMSF) was used to wash the beads precipitated at the bottom, and the washing was repeated for 5-6 times. The supernatant was discarded, 30 ul of 2X Loading buffer was added, and the mixture was boiled at 95°C for 5 minutes. The supernatant was taken for electrophoresis.

[0015] (2) Cell proliferation detection ① Live cell counting a) The number of cells to be tested was counted with a bovine bivalve counting plate, and 96-well plates were plated. According to the cell proliferation rate, 5000 cells were inoculated per well. 200 μL of 1640 whole culture medium was added to the cells, and each cell was plated in 3 replicate wells.

[0016] b) The number of cells inoculated on the first day was recorded as day 0, and the cell counting was performed at the same time point every day.

[0017] c) Once the cell counts for all 5 days are completed, organize and analyze the data from these 5 days, and use GraphPad to plot the cell proliferation curve.

[0018] ② CCK8 cell proliferation experiment a) Count the cells to be tested using a Newton-Bauer counting chamber, then seed the cells in a 96-well plate with 1000 cells per well and add 100 μL of 1640 full culture. Seed each cell type in 3 replicates.

[0019] b) Add 10 μL of CCK-8 staining solution to each well of cells and place the culture plate in an incubator to continue incubation for 2 hours.

[0020] c) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0021] d) Subsequently, CCK-8 dye solution was added to each well at the same time every day, and after incubation for two hours, the absorbance value was measured at a wavelength of 450 nm.

[0022] e) After completing 5 days of testing, organize the data and use GraphPad software to create a line graph to represent the cell proliferation trend.

[0023] ③ EDU Experiment a) Count 2 million cells to be tested and seed them into 6-well plates. Add 2 mL of 1640 complete culture medium and 2 μL of EDU dye, and incubate in an incubator for 2 h.

[0024] b) Remove the supernatant and fix the cells with 4% paraformaldehyde at room temperature for 15 min.

[0025] c) Centrifuge to remove the supernatant, then add washing buffer to the cells to wash them. Repeat the washing process 3 times, each time for 3 minutes.

[0026] d) Add 0.3% Trizon X-100 to the cells and permeate the membrane at room temperature for 10 min.

[0027] e) Repeat step c).

[0028] f) Prepare the Click reaction solution according to the reagent instructions based on the cell concentration to be tested. Then add 200 μL of the reaction solution to each well and incubate at room temperature in the dark for 30 min.

[0029] i. Click Reaction Buffer 430 μL ii. CuSO4 20 μL iii. Azide 1 μL iv. Click Additive Solution 50 μL g) Repeat step c).

[0030] h) Dilute Hoechst dye 1000 times and add to cells, then stain cell nuclei at room temperature for 10 min in the dark.

[0031] i) Repeat step c).

[0032] j) Place cells under an inverted fluorescence microscope and observe the staining.

[0033] (3) Immunofluorescence a) Collect cells in a 1.5 mL EP tube and wash with PBS 3 times.

[0034] b) Fix with 4% paraformaldehyde at room temperature for 10 min.

[0035] c) Wash with PBS 3 times, centrifuge at 3000 rpm for 3 min, and discard the supernatant.

[0036] d) Break the membrane with 0.5% Triton for 15 min.

[0037] e) Wash with PBS 3 times, centrifuge at 3000 rpm for 3 min, and discard the supernatant.

[0038] f) Block with 5% BSA at room temperature for 30 min.

[0039] g) Dilute the primary antibody to the corresponding volume according to the antibody instructions, then add the primary antibody to the cells and incubate overnight in the refrigerator at 4°C.

[0040] h) Wash the cells with PBS solution 3 times, each time at 3000 rpm for 5 min, and remove the supernatant.

[0041] i) Add the fluorescent secondary antibody diluted with 1% BSA to the washed cells, then incubate the cells at room temperature in the dark for 2 h.

[0042] j) Wash the cells with PBS solution 3 times, each time at 3000 rpm for 5 min, and remove the supernatant.

[0043] k) Add DAPI and stain for 10 min in the dark.

[0044] l) Add an anti-quenching agent to the cell sample, take 10 μL of the cell suspension with a pipette and drop it in the center of the glass slide, then gently cover it with a cover glass. To ensure the accuracy of the experimental results, observe under a laser confocal microscope as soon as possible.

[0045] (4) Subcutaneous tumor formation experiment in mice a) Take an appropriate number of ARP-1-shCtrl, shTRIM28, IM-9-shCtrl, and shTRIM28 cells, wash the cells three times with autoclaved PBS to remove as much serum as possible. Resuspend the cells in FBS-free 1640 medium and store on ice for later use.

[0046] b) Inject 1 million cells subcutaneously into NSG mice and then continue feeding them.

[0047] c) One week later, the mouse tumors were observed. Tumor volume was measured using calipers.

[0048] d) When the tumors reached a certain size, the mice were euthanized by cervical dislocation, and the tumor tissue from both sides of the mouse was carefully removed. The tumor tissue was then photographed and recorded, and the weight of each tumor tissue was accurately measured and recorded.

[0049] e) Finally, using GraphPad plotting software, the obtained tumor volume and weight were analyzed, and tumor growth curves and weight change graphs were plotted.

[0050] Depend on Figure 1 It is known that the E3 ligase TRIM28 maintains IRF4 stability by inhibiting the proteasome pathway. Immunoprecipitation combined with mass spectrometry analysis revealed that the E3 ubiquitin ligase TRIM28 is a specific binding protein of IRF4. Figure 1 A). Furthermore, Co-IP and Pull-down experiments revealed that TRIM28 and IRF4 did not bind directly ( Figure 1 BC). Treatment with the protein synthesis inhibitor cyclohexylimide (CHX) showed that TRIM28 knockdown significantly accelerated IRF4 protein degradation (BC). Figure 1 D). The proteasome inhibitor MG-132 can reverse the degradation of IRF4 ( Figure 1 E), subsequently, CO-IP experiments confirmed that TRIM28 knockdown increased IRF4 ubiquitin levels ( Figure 1 F) indicates that TRIM28 maintains IRF4 stability by inhibiting the proteasome pathway.

[0051] Depend on Figure 2 It is known that TRIM28 regulates myeloma cell proliferation by stabilizing the IRF4 protein. To investigate the role of TRIM28 in MM progression, a stable TRIM28 knockdown cell line was constructed in IM-9 and ARP-1 cells. Figure 2 AB). Functional experiments confirmed that knockdown of TRIM28 inhibited myeloma cell growth ( ).Figure 2 CE), in vivo experiments showed that myeloma cells with knocked-down TRIM28 had significantly reduced tumorigenicity in NSG mice ( Figure 2 (FG), indicating that TRIM28 is a key regulator of myeloma cell proliferation.

[0052] Depend on Figure 3 It can be seen that TRIM22 directly binds to IRF4 and regulates its degradation rate. TRIM28 does not directly act as an E3 ligase to degrade IRF4, but rather regulates its stability through other mechanisms. Based on this, it is inferred that TRIM28 may interact with other E3 ligases, inhibiting their degradation of IRF4. Reanalysis of IRF4 mass spectrometry data was conducted, and the E3 ligase TRIM22 was identified. Figure 3 A), Co-IP and IF experiments confirmed that TRIM22 and IRF4 form an endogenous complex in myeloma cells ( Figure 3 BD), Pull-down experiments revealed that TRIM22 and IRF4 bind directly ( Figure 3 E). Furthermore, CHX treatment showed that TRIM2 overexpression significantly accelerated IRF4 protein degradation (E). Figure 3 F).

[0053] Depend on Figure 4 It is known that TRIM28 degrades TRIM22 by inhibiting the proteasome pathway. Next, the Co-IP experiment confirmed that TRIM28 and TRIM22 form an endogenous complex in myeloma cells ( Figure 4 AB), Pull-down experiments revealed a direct binding between TRIM28 and TRIM22 ( Figure 4 C). Furthermore, CHX treatment showed that TRIM28 knockdown significantly slowed TRIM22 protein degradation (C). Figure 4 D), and knocking down TRIM28 reduces the ubiquitination level of TRIM22, suggesting that TRIM28 stabilizes IRF4 protein levels by degrading TRIM22 (D). Figure 4 E).

[0054] The above analysis revealed that the E3 ubiquitin ligase TRIM28 maintains IRF4 stability by inhibiting the proteasome pathway. Stable transgenic IM-9 and ARP-1 cells with TRIM28 knockdown were constructed. Experiments including CCK-8 assays, viable cell counts, EDU, and NSG subcutaneous tumorigenesis showed that TRIM28 knockdown inhibited myeloma cell growth. Mechanistically, TRIM28 mediates the proteasome degradation of the E3 ubiquitin ligase TRIM22, thereby relieving TRIM22's inhibitory effect on IRF4, thus maintaining IRF4 protein stability and promoting the malignant progression of myeloma. Example 1

[0055] CCK8 experiment showed that compound TF3 significantly inhibited myeloma proliferation (A), and had no significant toxicity to normal peripheral blood mononuclear cells (B). Subsequently, the results of CO-IP experiment showed that TF3 directly inhibited the TRIM28 enzyme activity and reduced the ubiquitination level of TRIM22 (C). Figure 5 Figure 5 Figure 5 Example Two

[0056] The spontaneous multiple myeloma mouse model was formed by tail vein injection of vk12598 cells to C57BL / 6J mice, and TF3 treatment for two weeks significantly delayed the progression of myeloma (D-E), and immunofluorescence also showed that TF3 treatment reduced tumor IRF4 expression (F). Figure 5 Figure 5 Example Three

[0057] IM-9 and ARP-1 cells were transplanted subcutaneously into NSG mice, and after three days of treatment with 3TF for two weeks, the subcutaneous tumors were taken and weighed, and the results showed that 3TF treatment significantly inhibited the subcutaneous tumor formation ability of mice (G-H). Figure 5

[0058] It should be noted that the relational terms such as first and second and the like are used merely to distinguish one entity or action from another entity or action, without necessarily requiring or implying that there is any such actual relationship or order between such entities or actions. Also, the terms "comprises", "comprising", or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by an "includes" or "comprising" statement does not exclude the presence of additional elements that are not explicitly listed or inherent to such process, method, article, or apparatus. In addition, in this document, "greater than", "less than", "more than", and the like are understood to not include the number itself; "above", "below", "within", and the like are understood to include the number itself.

[0059] Although the above embodiments have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic inventive concept, so the above description is only an embodiment of the present application, and does not limit the patent protection scope of the present application, and any equivalent structure or equivalent process transformation using the content of the present application specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.​​​​​​

Claims

1. Use of theaflavin-3,3'-bisgallate in the preparation of drugs for the treatment of multiple myeloma.

2. The use according to claim 1, characterized in that, Theaflavins-3,3'-bisgallate inhibit the ubiquitination activity of TRIM28, reduce TRIM22 degradation, and downregulate IRF4 protein expression.

3. The use according to claim 2, characterized in that, Theaflavins-3,3'-bisgallate inhibit TRIM28, specifically including at least one of products that inhibit TRIM28 gene expression, products that inhibit TRIM28 protein, and products that inhibit the functional activity of TRIM28 protein.

4. The use according to claim 3, characterized in that, Products that inhibit TRIM28 gene expression or TRIM28 protein expression include at least one of polynucleotides, plasmids packaged with lentiviruses or retroviruses, viruses, lipids, and TRIM28 inhibitory antibodies; products that inhibit the functional activity of TRIM28 protein include at least one of proteins, peptides, and enzymes that inhibit the activity of TRIM28 protein.

5. A pharmaceutical composition for treating multiple myeloma, characterized in that, The pharmaceutical composition comprises at least theaflavin-3,3'-bisgallate or a pharmaceutical excipient thereof.

6. The pharmaceutical composition according to claim 5, characterized in that, It may also include at least one of a proteasome inhibitor, an immunomodulatory agent, or an anti-CD38 monoclonal antibody.

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