Use of small molecule compounds that activate endoplasmic reticulum stress in the preparation of drugs for treating multiple myeloma and high-throughput screening method

Abstract

The application discloses a small-molecule compound for activating endoplasmic reticulum stress in the preparation of a drug for treating multiple myeloma and a high-throughput screening method, and belongs to the technical field of biological medicines. The application first establishes a high-throughput screening method for small-molecule compounds for activating endoplasmic reticulum stress. The method is based on an IRE1 / XBP1 fluorescent reporting system related to an endoplasmic reticulum stress path, can synchronously detect XBP1 splicing activation related to endoplasmic reticulum stress and cell survival rate, realizes rapid screening of a high-throughput compound library, and first proves that a cucurbitacin E obtained through screening promotes myeloma cell death by specifically deteriorating endoplasmic reticulum stress of myeloma cells, and realizes the effect of treating myeloma. The cucurbitacin E shows a strong killing effect on myeloma cells and low toxicity on normal cells, can realize specific killing of myeloma cells, avoids side effects caused by broad-spectrum toxicity of traditional drugs, improves treatment safety, has good drug-making potential and clinical conversion value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of small molecule compounds that activate endoplasmic reticulum stress in the preparation of drugs for treating multiple myeloma and a high-throughput screening method. Background Technology

[0002] Multiple myeloma is an incurable hematologic malignancy characterized by the clonal proliferation of plasma cells in the bone marrow, leading to severe bone disease, renal dysfunction, and immunodeficiency. Despite significant advances in targeted and immunotherapies over the past decade, drug resistance and disease relapse remain major challenges in the clinical treatment of multiple myeloma. Therefore, new drugs or treatment strategies are still needed to treat multiple myeloma.

[0003] Multiple myeloma cells are essentially plasma cells, and a prominent characteristic of them is the massive synthesis of immunoglobulins, a process that places a huge demand on the endoplasmic reticulum (ER) for unfolded protein processing. Unlike normal cells or other cancer cells, multiple myeloma cells are inherently overly dependent on ER function: the abundant production of immunoglobulin heavy and light chains requires efficient ER chaperone mechanisms to ensure proper protein folding. This robust immunoglobulin synthesis leads to a persistent basal ER stress state in multiple myeloma cells. To cope with this inherent protein toxicity stress, multiple myeloma cells have evolved adaptive mechanisms to overactivate the unfolded protein response (UPR)—a conserved signaling pathway that restores ER homeostasis by enhancing protein folding capacity, promoting ER-related degradation of misfolded proteins, and regulating cellular metabolism. However, this adaptive UPR activation also makes multiple myeloma cells extremely sensitive to further induced ER stress. Unlike normal cells with a strong endoplasmic reticulum (ER) stress tolerance reserve, multiple myeloma cells are on the "edge" of ER stress. Even mild additional ER stress can exceed the adaptive capacity of the unfolded protein response (UPR), leading to ER stress-induced cell death. This unique biological vulnerability makes ER stress a highly attractive and selective therapeutic target for multiple myeloma, as drugs that enhance ER stress can preferentially eliminate myeloma cells without harming normal cells with low baseline ER stress levels. Therefore, obtaining compounds that can specifically worsen ER stress holds promise for providing new potential drugs for the treatment of multiple myeloma.

[0004] For a long time, natural products derived from plants, microorganisms, and marine organisms have been a cornerstone of anticancer drug development, with an impressive track record of translating preclinical potential into clinical success. To date, many bioactive compounds extracted from plants, fungi, and marine organisms have revolutionized cancer treatment by targeting key dysregulated cellular pathways in malignant tumors. Notably, many natural products exert their anticancer effects by disrupting protein homeostasis. This inherent ability to exploit the metabolic and stress-adaptive vulnerabilities unique to cancer highlights the potential of natural products as both mechanistic probes for elucidating disease-driving pathways and as priority compounds for developing targeted therapies. Given that multiple myeloma is a disease characterized by abnormal protein synthesis and extremely high basal endoplasmic reticulum stress, natural products represent an underutilized resource for exploring novel drugs that can selectively further exacerbate endoplasmic reticulum stress and induce endoplasmic reticulum stress-mediated cell death. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide the application of small molecule compounds that activate endoplasmic reticulum stress in the preparation of drugs for treating multiple myeloma.

[0008] To solve the above technical problems, the present invention provides the following technical solution: the small molecule compound that activates endoplasmic reticulum stress is cucurbitacin E (CuE) with the structural formula shown in Formula I; Formula I.

[0009] As a preferred embodiment of the application of the small molecule compound that activates endoplasmic reticulum stress according to the present invention in the preparation of a drug for treating multiple myeloma, wherein: the cucurbitacin E triggers endoplasmic reticulum stress in human multiple myeloma cells in a concentration-dependent manner.

[0010] As a preferred embodiment of the application of the small molecule compound that activates endoplasmic reticulum stress as described in this invention in the preparation of a drug for treating multiple myeloma, wherein: the human multiple myeloma cells include the H929 cell line, the RPMI-8226 cell line, and the multiple myeloma.1S cell line.

[0011] As a preferred embodiment of the application of the small molecule compound that activates endoplasmic reticulum stress as described in this invention in the preparation of a drug for treating multiple myeloma, wherein: the cucurbitacin E enhances the phosphorylation levels of IRE1α and eIF2α in human multiple myeloma cells.

[0012] As a preferred embodiment of the application of the small molecule compound that activates endoplasmic reticulum stress as described in this invention in the preparation of a drug for treating multiple myeloma, wherein: the cucurbitacin E upregulates the protein expression of GRP78 and DDIT3 in human multiple myeloma cells in a concentration-dependent manner.

[0013] As a preferred embodiment of the application of the small molecule compound that activates endoplasmic reticulum stress as described in this invention in the preparation of a drug for treating multiple myeloma, wherein: cucurbitacin E has specific anti-multiple myeloma activity.

[0014] Another object of the present invention is to provide a medicament for treating multiple myeloma, said medicament targeting endoplasmic reticulum stress signaling molecules to trigger endoplasmic reticulum stress in cells, comprising cucurbitacin E and other pharmaceutically acceptable excipients.

[0015] Another objective of this invention is to provide a high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress.

[0016] To solve the above-mentioned technical problems, the present invention provides the following technical solution: The screening method is based on the IRE1 / XBP1 signal path, constructing a high-throughput screening platform for the IRE1 / XBP1 reporting system, including, The XBP1 plasmid containing the mNeonGreen fluorescent fragment was transfected into human multiple myeloma cells via lentivirus transfection. Stable expression strains were screened using puromycin to obtain a fluorescent reporter system; The cells in the fluorescence reporter system were treated overnight, and the small molecule compound to be detected was added. After culturing for 24 hours, the fluorescence intensity and cell viability of human multiple myeloma cells were detected by flow cytometry. Small molecule compounds with a fluorescence intensity ratio >2.0 and a cell viability ratio <0.4 were screened to obtain small molecule compounds that activate endoplasmic reticulum stress.

[0017] As a preferred embodiment of the high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress according to the present invention, wherein the human multiple myeloma cells are selected from H929 cells.

[0018] As a preferred embodiment of the high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress according to the present invention, the XBP1 reporter plasmid containing the mNeonGreen fluorescent fragment contains a partially alternatively spliced ​​fragment of the XBP1 gene. The partially alternatively spliced ​​fragment of the XBP1 gene is specifically excised when endoplasmic reticulum stress induces IRE1RNase activity activation, triggering a translation frameshift, thereby achieving the expression of the fluorescent protein mNeonGreen.

[0019] Beneficial effects of this invention: (1) This invention is the first to demonstrate that cucurbitacin E can activate endoplasmic reticulum stress in multiple myeloma cells to exert anti-multiple myeloma activity, and it is clear that it triggers endoplasmic reticulum stress in human multiple myeloma cells in a concentration-dependent manner, can enhance the phosphorylation level of IRE1α and eIF2α in human multiple myeloma cells and upregulate the protein expression of GRP78 and DDIT3 in human multiple myeloma cells in a concentration-dependent manner.

[0020] (2) The cucurbitacin E provided by the present invention exhibits a strong killing effect on multiple myeloma cells, while having very low toxicity to normal cells. It can achieve selective tumor treatment, avoid the side effects caused by the broad-spectrum toxicity of traditional drugs, improve treatment safety, and has good drug development potential and clinical translation value.

[0021] (3) This invention also establishes a high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress. This method is based on the IRE1 / XBP1 fluorescence reporter system, which can simultaneously detect XBP1 splicing activation and cell viability, and achieve rapid and unbiased screening of multiple compounds with reliable screening results. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein: Figure 1 The diagram shows the process flow and results of the high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress in Example 1 of this invention.

[0023] Figure 2 This is a graph showing the results of verifying the endoplasmic reticulum stress activation effect of cucurbitacin E on multiple myeloma cells in Example 2 of this invention.

[0024] Figure 3 The figure shows the results of verifying the specific killing effect of cucurbitacin E on multiple myeloma cells in Example 3 of this invention.

[0025] Figure 4 This is a graph showing the results of the verification of cucurbitacin E inhibiting the growth of multiple myeloma in vivo in Example 4 of the present invention.

[0026] Figure 5 This is a summary diagram of the overall technical solution of the present invention. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] Unless otherwise specified, all raw materials used in the specific embodiments of this invention are commercially available in the art.

[0031] In specific embodiments of the present invention, the human multiple myeloma (multiple myeloma) cell lines (RPMI-8226, H929 and multiple myeloma 1.S) used were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0032] HEK-293T cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin (P / S) in a humidified incubator at 37°C and 5% carbon dioxide.

[0033] All cell lines in our laboratory are routinely tested for mycoplasma contamination, and the mycoplasma test results of the cells used in this study were all negative.

[0034] Unless otherwise specified, the testing and analysis methods used in this invention are all conventional methods in the field.

[0035] Example 1 Reference Figure 1 This embodiment provides a high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress: 1) Construction of the IRE1α-XBP1 fluorescent reporter cell line ( Figure 1 A, Figure 1 B) HEK-293T cells with a density of 80% and good growth status were transfected with plasmids; Add 16.68 µg of DNA, including psPAX2 (6.10 µg), pDM2.G (2.44 µg), and the core plasmid (8.14 µg), to 500 µL of serum-free culture medium and mix thoroughly by pipetting to obtain the plasmid dilution. Add 50 µL of PEI transfection reagent solution to 500 µL of serum-free culture medium, and gently mix by pipetting to obtain the diluted PEI solution. Slowly add 500µL of PEI dilution to 500µL of plasmid dilution, gently pipette to mix, incubate at room temperature for 15 min, and then gently drop the resulting 1000µL mixture into different parts of the HEK-293T cell culture dish, gently shake the dish to mix, and then place the cells in a cell culture incubator for 10 h.

[0036] After the culture was completed, the old culture medium was quickly aspirated by a vacuum pump. 8 mL of preheated 37°C complete culture medium was carefully added along the wall of the dish. After culturing for another 48 hours, the viral supernatant was collected. The virus was centrifuged at 2000 rpm for 10 min to remove dead cells, cell debris, etc. The collected viral supernatant was filtered through a 0.45 µm filter and aliquoted into 2 mL centrifuge tubes to obtain target cells. 0.6×10 6 One target cell was resuspended in 1000µL of complete culture medium and cultured overnight in a 12-well plate. After the cells recovered, 1mL of virus and 1µL of polybrene (8µg / mL) were added directly. The cells were centrifuged at 1800rpm for 50min at room temperature and gently placed in an incubator. After 16h of infection, the cells were centrifuged and the medium was changed. 8mL of preheated fresh complete culture medium was added. 2) After 72 hours of infection (depending on cell status), puromycin was added and screened for 3 days to obtain the H929-XBP1 stable reporter cell line. The fluorescence was significantly increased when verified with a 1 μM thiocarotene (TG) positive control, proving that the reporter system was successfully constructed. Figure 1 A represents the structural principle of this reporter system. By inserting a 26bp intron into the XBP1 sequence, translation normally terminates prematurely and there is no fluorescence. Endoplasmic reticulum stress activates the IRE1α RNase, which removes the intron, restores the reading frame, and expresses mNeonGreen fluorescence. Figure 1B represents the flow cytometry analysis results of reporter gene activity in H929 cells transduced with IRE1 / XBP1 reporter gene lentivirus. The WT group showed no fluorescence, while the XBP1-reporter group showed low fluorescence at baseline (high baseline endoplasmic reticulum stress in multiple myeloma cells). The fluorescence in the TG-treated group was significantly increased, demonstrating the effectiveness of the system constructed in this invention.

[0037] 3) H929-XBP1 reporter cells were cultured at 3 × 10⁻⁶ cells per cell line. 4 Inoculate one well per well into a 96-well plate and incubate overnight.

[0038] After culturing, 2 μM of the natural product compound to be tested was added (in this example, 2880 natural products in the Natural Product Custom Library for HTS (96-well) were used as the test products). Each group was set up with a DMSO control group and a TG (1 μM carotenoid) positive control group. The mixture was cultured at 37°C and 5% CO2 for 24 h. High-throughput detection was performed using flow cytometry with a flow cytometer equipped with a high-throughput sample introduction system. 100 μL was collected from each well, and mNeonGreen fluorescence intensity (reflecting IRE1α-XBP1 activation and XBP1 splicing) and cell viability (reflecting the cytotoxic activity of the compound) were detected simultaneously. The results are as follows Figure 1 As shown in Figure C, the cell viability and reporter gene activity of representative samples (wells) treated with 1 μM carotenoid (TG) for 24 hours are displayed; the dashed line represents the average fluorescence intensity of the reporter gene. Using the DMSO group as a baseline, 15 compounds with fluorescence intensity ratios >2.0 and cell viability ratios <0.4 were screened, all of which showed the ability to target and activate the IRE1α-XBP1 pathway. The results are as follows: Figure 1 As shown in D, the red boxes represent samples with a cell viability normalized ratio <0.4 and a mean fluorescence intensity ratio >2.

[0039] 4) Twelve compounds obtained from step 3) that target and activate the IRE1α-XBP1 pathway were selected, including cucurbitacin E, colchicine, 3-demethylcolchicine, 9-hydroxycamptothecin, quercetin-3-O-glucuronide, cucurbitacin B, irinotecan, vincristine sulfate, vinblastine sulfate, podophyllotoxin, (-)-epipodophyllotoxin, and plumbagin. To verify the reliability of the system, orthosphenic acid was also randomly selected. Three natural compounds, namely, acid, pantethine, and oxkanin, were screened for their ability to activate the IRE1α-XBP1 pathway. Fifteen natural compounds were used to treat H929 cells at 1 μM for 24 h. Total RNA was extracted and reverse transcribed into cDNA. Endogenous XBP1 splicing (XBP1s / XBP1u) was detected according to the method in Example 1. XBP1u (unspliced) and XBP1s (spliced) were distinguished by agarose gel electrophoresis. The XBP1s / XBP1u ratio was calculated using ImageJ software for grayscale analysis. The results are as follows: Figure 1 As shown in Figure E, a distinct XBP1s band and a significantly increased XBP1s / XBP1u ratio were observed, confirming the actual activation of the IRE1α-XBP1 pathway. Finally, cucurbitacin E, the compound with the most significant upregulation of XBP1 splicing, was selected through screening. Its structural formula is shown in Figure 1F. This compound can significantly increase the fluorescence signal, significantly reduce the survival of multiple myeloma cells, and induce endogenous XBP1 splicing, thus being identified as a specific activator targeting the IRE1α-XBP1 pathway.

[0040] Example 2: Verification of the ERS-activating effect of cucurbitacin E on multiple myeloma cells Endoplasmic reticulum (ER) stress activates three parallel unfolded protein response (UPR) pathways mediated by IRE1α, PERK, and ATF6, each with unique molecular characteristics. XBP1 splicing is a hallmark event of IRE1α activation and is crucial for adaptive ER stress signaling. GRP78 (BiP), a major ER chaperone protein, dissociates from UPR sensors to initiate the stress response and is often used to quantify the initiation of ER stress. DDIT3 (CHOP) is a key pro-apoptotic transcription factor induced by long-term ER stress, while eIF2α phosphorylation is a classic PERK-mediated translational repression mechanism in UPR. Given the crucial roles of these molecules in ER stress-induced UPR execution, this embodiment verifies the ERS activation effect of cucurbitacin E on multiple myeloma cells by detecting the above ER stress markers. Specifically: 20 nM, 200 nM, and 2000 nM cucurbitacin E were applied to H929, RPMI-8226, and multiple myeloma .1S cells for 12 h, respectively. Endoplasmic reticulum stress markers were detected by PCR, RT-qPCR, and Western blot.

[0041] PCR testing 120 nM, 200 nM, and 2000 nM cucurbitacin E were applied to H929, RPMI-8226, and multiple myeloma .1S cells for 12 h, respectively. RNA was extracted and cDNA was reverse transcribed.

[0042] Place the required reagents in an ice box beforehand and wait for them to melt completely before adding them to the PCR tubes according to the system requirements in Table 1.

[0043] Table 1 PCR reaction system

[0044] The primer sequences are as follows: XBP1-F:5'-CCTGGTTGCTGAAGAGGAGG-3' XBP1-R:5'-CCATGGGGAGATGTTCTGGAG-3' RPLP0-F:5'-GCTTCCTGAGGGTGTCCGC-3' RPLP0-R:5'-TCCGTCTCCACAGACAAGGCCA-3' Place the 96-well plate into the real-time quantitative PCR instrument and set the reaction program as shown in Table 2: Table 2 PCR reaction procedure

[0045] Prepare an agarose gel by mixing 3g agarose with 70ml TAE and 7ul EB. 10x loading buffer, 1ul loading buffer + 9ul sample mixed and loaded, 100V, 400mA, 1h electrophoresis.

[0046] RT-qPCR detection: Different concentrations of cucurbitacin E (20, 200, 2000 nM) were used to treat three types of multiple myeloma cells—H929, RPMI-8226, and multiple myeloma .1S—for 12 hours. RNA was then extracted, and cDNA was reverse transcribed. Specifically: Place the required reagents in an ice box beforehand and wait for them to completely melt before adding them to the 96-well plate according to the system requirements. The RT-qPCR reaction system is shown in Table 3.

[0047] Table 3 RT-qPCR reaction system

[0048] The primer sequences are as follows: CHOP-F: 5'-ACCAAGGGAGAACCAGGAAAG-3' CHOP-R: 5'-TCACCATTCGGTCAATCAGAGC-3' BIP-F: 5'-GCCTGTATTTCTAGACCTGCC-3' BIP-R: 5'-TTCATCTTGCCAGCCAGTTG-3' RPLPO-F: 5'-GCTTCCTGGAGGGTGTCCGC-3' RPLPO-R: 5'-TCCGTCTCCACAGACAAGGCCA-3' Place the 96-well plate into the real-time quantitative PCR instrument and set the reaction program as shown in Table 4: Table 4 RT-qPCR reaction procedure

[0049] Using RPLPO as an internal control, the relative expression levels of all target genes were expressed as a function of 2. -△CT express.

[0050] Western Blot analysis: After treating H929, RPMI-8226, and multiple myeloma .1S cells with different concentrations of cucurbitacin E (20, 200, 2000 nM) for 12 h, total protein was extracted and the protein concentration was determined according to the BCA kit.

[0051] 10 μg of cell proteins from each group were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes via Western blotting. After blocking the PVDF membranes with 5% BSA, the membranes were incubated overnight at 4°C with primary antibody. The membranes were then washed and co-incubated with secondary antibody for 2 hours. Protein expression was detected using a chemiluminescence analyzer. Finally, Western blotting was used to quantify the proteins.

[0052] The results are as follows Figure 2 As shown, Figure 2 A. To analyze the XBP1 splicing in three multiple myeloma cell lines treated with different concentrations of cucurbitacin E or DMSO for 12 hours, TG was used as a positive control. The expression levels of XBP1s and XBP1u were assessed by PCR and agarose gel electrophoresis. The results showed that cucurbitacin E triggered XBP1 splicing in human multiple myeloma cell lines H929, RPMI-8226, and multiple myeloma.1S in a concentration-dependent manner. Figure 2 A).

[0053] Figure 2 B and Figure 2 C represents the mRNA expression levels of endoplasmic reticulum stress markers DDIT3 (B) and GRP78 (C) after 12 hours of treatment with different concentrations of cucurbitacin E or DMSO in three multiple myeloma cell lines using qPCR. TG was used as a positive control. Data are expressed as mean ± standard deviation of three independent experiments. Significance was indicated using one-way ANOVA and Dunnett's post-hoc test: ns indicates no significant difference; * indicates p ≤ 0.05; ** indicates p ≤ 0.01; *** indicates p ≤ 0.001. The results show that at the mRNA level, cucurbitacin E significantly upregulated the transcriptional expression of DDIT3 and GRP78 with increasing concentration.

[0054] Figure 2 D represents the expression levels of ERS-related protein markers in three multiple myeloma cell lines after treatment with different concentrations of cucurbitacin E or DMSO for 12 hours using Western blotting. TG was used as a positive control, and β-tubulin as an internal control. The figure shows representative results from three independent experiments. Immunoblot analysis of human multiple myeloma cell lines H929, RPMI-8226, and multiple myeloma.1S confirmed that cucurbitacin E significantly enhanced the phosphorylation levels of IRE1α and eIF2α and upregulated the protein expression of GRP78 and DDIT3 in a concentration-dependent manner, collectively indicating that the endoplasmic reticulum stress pathway was strongly activated.

[0055] The above results demonstrate that cucurbitacin E can effectively activate the endoplasmic reticulum stress signaling pathway in multiple myeloma.

[0056] Example 3: Specific killing effect of cucurbitacin E on multiple myeloma cells Given that multiple myeloma cells produce large amounts of immunoglobulins and have significantly elevated basal levels of endoplasmic reticulum stress, making them particularly sensitive to other stress stimuli, this embodiment verifies the specific killing effect of cucurbitacin E on multiple myeloma cells using the following method: CCK8 (Cytotoxicity) Detection The cells were seeded at a density of 20,000 cells per well into 96-well culture plates with 100 μL of cell suspension per well and cultured overnight. The following three groups of experimental wells were set up: wells containing only culture medium (background blank group), wells containing normal cells without drug treatment (negative control group), and wells containing experimental cells with drug intervention (drug treatment group).

[0057] After 72 hours of drug treatment, cell morphology was observed under a microscope. The culture plate was then removed, and 10 μL of CCK-8 was added to each well. After addition, the culture plate was incubated at 37°C for 2 hours.

[0058] The absorbance of the culture plate was measured at 450 nm, and cell viability was calculated as follows: (Drug-treated sample wells - Background blank control wells) / (Sample control wells - Background blank control wells) × 100% Cell surface protein staining Bone marrow mononuclear cells were collected from patients with multiple myeloma and washed with FACS buffer (PBS solution containing 1% fetal bovine serum and 2% multiple myeloma EDTA). The cells were then stained with PE-labeled CD138 antibody (Miltenyi Biotec, Germany) for 30 minutes at 4°C. After incubation, the cells were washed again with FACS buffer and then stained with 7-AAD (Beyotime, Beijing) for 15 minutes in the dark. The washed cells were resuspended in FACS buffer and analyzed by flow cytometry (BD Fortessa). Data were analyzed using FlowJo software.

[0059] Apoptosis detection Multiple myeloma cells were seeded into 24-well culture plates at a density of 20,000 cells per well, with 1 mL of cell suspension per well, and the cells were cultured overnight.

[0060] The following experimental wells were set up: wells containing only normal cells without drug treatment (negative control group), and wells containing experimental cells with drug intervention (drug treatment group).

[0061] After 24 hours of drug treatment, the cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, the cells were collected, resuspended in PBS, and centrifuged again at 300g for 5 minutes to discard the supernatant and collect the cells.

[0062] Resuspend cells in 195 μL Annexin V-FITC binding solution, add 5 μL Annexin-FITC and mix well; add 10 μL LPI staining solution and mix well.

[0063] After incubation on ice in the dark for 20 minutes, fluorescence was detected by flow cytometry.

[0064] Cell cycle detection Multiple myeloma cells were seeded into 24-well culture plates at a density of 20,000 cells per well, with 1 mL of cell suspension per well, and the cells were cultured overnight.

[0065] The following experimental wells were set up: wells containing only normal cells without drug treatment (negative control group), and wells containing experimental cells with drug intervention (drug treatment group).

[0066] After 24 hours of drug treatment, the cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, the cells were collected, resuspended in PBS, and centrifuged again at 300g for 5 minutes to discard the supernatant and collect the cells.

[0067] The above test results are as follows Figure 3 As shown, Figure 3 A shows the cell viability of six multiple myeloma cell lines and five non-multiple myeloma cell lines after 72 hours of treatment with gradient concentrations of cucurbitacin E, as determined by the CCK-8 assay. Data are expressed as mean ± standard deviation of three independent experiments. The results indicate that cucurbitacin E can efficiently and selectively kill multiple myeloma cells, while exhibiting significantly lower toxicity to non-multiple myeloma cells.

[0068] Figure 3 F represents flow cytometry analysis of cucurbitacin E-induced cell death in primary bone marrow mononuclear cells from three multiple myeloma patients. Prior to flow cytometry analysis, cells were stained with CD138 antibody and 7-AAD. A 7-AAD-positive group was designated as the dead cell population. Results showed that CD138-positive myeloma cells from patient samples exhibited significantly higher sensitivity to cucurbitacin E treatment compared to CD138-negative myeloma cells; while CD138-negative cells showed virtually no response to cucurbitacin E-induced cytotoxicity.

[0069] The above results indicate that cucurbitacin E has selective anti-multiple myeloma activity, while producing only very low toxicity to other cancer cell lines and primary non-malignant bone marrow cells.

[0070] Figure 3 B and Figure 3 C represents the cell cycle analysis of multiple myeloma cell lines treated with different concentrations of cucurbitacin E, with cell cycle distribution detected by flow cytometry. Figure 3B presents representative data from three independent experiments using the H929 cell line. Figure 3 The C-bar chart presents the detection results for three multiple myeloma cell lines. Data are expressed as mean ± standard deviation of three independent experiments. The results indicate that cucurbitacin E-induced cell death is mainly achieved through cell cycle interference. In three independent multiple myeloma cell lines, cucurbitacin E treatment induced cell accumulation in the G2 / M phase in a concentration-dependent manner, indicating cell cycle arrest at the G2 / M phase.

[0071] Figure 3 D and Figure 3 E represents the apoptosis of multiple myeloma cell lines treated with different concentrations of cucurbitacin E for 24 hours, detected by flow cytometry. Figure 3 D shows a representative flow cytometry plot demonstrating cucurbitacin E-induced apoptosis in H929 cells. (Columnar) Figure 3 Figure E shows experimental data for three types of multiple myeloma cells. The data in the figure are the mean ± standard deviation of three independent experiments. Significance was determined using one-way ANOVA and Dunnett's post-hoc test: ns indicates no statistical significance; * indicates p ≤ 0.05; ** indicates p ≤ 0.01; *** indicates p ≤ 0.001.

[0072] Apoptosis experiments confirmed that cucurbitacin E promotes apoptosis in a dose-dependent manner, which was verified by annexin V staining.

[0073] In summary, these results indicate that cucurbitacin E first eliminates multiple myeloma cells by inducing G2 / M phase arrest, subsequently triggering apoptosis. The data suggest that cucurbitacin E exacerbates endoplasmic reticulum stress in multiple myeloma cells, ultimately leading to G2 / M phase cell cycle arrest and subsequent apoptosis.

[0074] Example 4: Cucurbitacin E inhibits the growth of multiple myeloma in vivo. Reference Figure 4 A. A subcutaneous xenograft model was established using NOD-SCID mice. Specifically, 14 six-week-old female NOD-SCID mice (purchased from Beijing Vital River Biotechnology Co., Ltd.) were randomly divided into two groups (n=7 per group). Each mouse was subcutaneously injected with 100 μl of Matrigel (a product of Biosharp Biotech). This solution contained 100 μl of 2.5 × 10⁻⁶ ppm ... 6 H929 cells were administered PBS buffer. When tumors were palpable, they were treated with either cucurbitacin E (0.25 mg / kg, every other day) or a solvent control. Tumor diameter was measured every two days using calipers, and body weight was recorded. Tumor volume was calculated using the formula V = length × width. 2Animals were euthanized when the largest tumor diameter reached 1.5 cm or signs of distress were observed, calculated as ×0.5. All experiments were approved by the Animal Care and Use Committee and were conducted in strict accordance with laboratory animal care and use guidelines.

[0075] The results are as follows Figure 4 B~ Figure 4 As shown in E, Figure 4 B represents the tumor size of the experimental group and the control group at the end of the experiment. Figure 4 C represents tumor growth in NOD-SCID mice subcutaneously injected with H929 cells after treatment with PBS and cucurbitacin E, respectively. Error bars represent mean ± standard deviation; ***p≤0.001 (one-way ANOVA and Dunnett's post-hoc test were used). Figure 4 D represents the tumor weight measured at the end of the experiment; the error bar represents the mean ± standard deviation; ***p≤0.001 (using one-way ANOVA and Dunnett's post-hoc test). Figure 4 E represents weight data measured every two days; error bars represent mean ± standard deviation; ns: one-way ANOVA and Dunnett's post-hoc test showed no statistical significance.

[0076] The results show that cucurbitacin E significantly inhibited the growth of multiple myeloma tumors in vivo. Compared with the control group, the tumor volume of mice treated with cucurbitacin E was significantly reduced. Furthermore, cucurbitacin E administration did not significantly affect the body weight of mice throughout the experiment, providing evidence that cucurbitacin E has no significant toxicity. These data indicate that cucurbitacin E can effectively inhibit the growth of multiple myeloma in vivo.

[0077] In summary, this invention first establishes a high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress. This method is based on the IRE1 / XBP1 fluorescence reporter system, which can simultaneously detect XBP1 splicing activation and cell viability, enabling rapid and unbiased screening of multiple compounds with reliable results.

[0078] Multiple myeloma cells exhibit high basal endoplasmic reticulum stress due to high immunoglobulin synthesis. Endoplasmic reticulum protein homeostasis is crucial for maintaining the survival of multiple myeloma cells. This invention is the first to demonstrate that cucurbitacin E can exert anti-multiple myeloma activity by activating endoplasmic reticulum stress in multiple myeloma cells. Figure 5 As shown, taking the H929 cell line as an example, cucurbitacin E triggers XBP1 splicing in the human multiple myeloma cell line H929 in a concentration-dependent manner. Figure 5 At the mRNA level, cucurbitacin E significantly upregulated the transcriptional expression of DDIT3 and GRP78 with increasing concentration. Figure 5 B. Figure 5C) Furthermore, immunoblotting analysis confirmed that cucurbitacin E significantly enhanced the phosphorylation levels of IRE1α and eIF2α, and upregulated the protein expression of GRP78 and DDIT3 in a concentration-dependent manner, collectively indicating that the endoplasmic reticulum stress pathway was strongly activated. Figure 5 D).

[0079] Furthermore, cucurbitacin E can efficiently and selectively kill multiple myeloma cells, while exhibiting significantly lower toxicity to non-multiple myeloma cells. Figure 5 E), and cucurbitacin E promotes apoptosis in a dose-dependent manner (E ...). Figure 5 F), and effectively interferes with the cell cycle (F). Figure 5 G), compared with non-multiple myeloma cells (CD138 negative), multiple myeloma cells from patient specimens (CD138 positive) showed significantly higher sensitivity to cucurbitacin E treatment; while non-multiple myeloma cells were essentially unresponsive to cucurbitacin E-induced cytotoxicity. Figure 5 The above results indicate that cucurbitacin E has selective anti-multiple myeloma activity, while producing only very low toxicity to non-multiple myeloma cells.

[0080] Finally, animal experiments showed that cucurbitacin E can effectively inhibit the growth of multiple myeloma in vivo. Figure 5 I~ Figure 5 L).

[0081] Based on the above conclusions, the cucurbitacin E of the present invention can achieve selective tumor treatment, avoid the side effects caused by the broad-spectrum toxicity of traditional drugs, improve treatment safety, and has good drug development potential and clinical translation value.

[0082] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of small molecule compounds that activate endoplasmic reticulum stress in the preparation of drugs for treating multiple myeloma, characterized in that: The small molecule compound that activates endoplasmic reticulum stress is cucurbitacin E, with the structural formula shown in Formula I. Formula I.

2. The use of the small molecule compound that activates endoplasmic reticulum stress as described in claim 1 in the preparation of a medicament for treating multiple myeloma, characterized in that: Cucurbitacin E triggers endoplasmic reticulum stress in human multiple myeloma cells in a concentration-dependent manner.

3. The use of the small molecule compound that activates endoplasmic reticulum stress as described in claim 2 in the preparation of a drug for treating multiple myeloma, characterized in that: The human multiple myeloma cells include, but are not limited to, the H929 cell line, the RPMI-8226 cell line, and the multiple myeloma .1S cell line.

4. The use of the small molecule compound that activates endoplasmic reticulum stress as described in claim 2 in the preparation of a medicament for treating multiple myeloma, characterized in that: Cucurbitacin E enhances the phosphorylation levels of IRE1α and eIF2α in human multiple myeloma cells.

5. The use of the small molecule compound that activates endoplasmic reticulum stress as described in claim 3 in the preparation of a medicament for treating multiple myeloma, characterized in that: Cucurbitacin E upregulates the protein expression of GRP78 and DDIT3 in human multiple myeloma cells in a concentration-dependent manner.

6. The use of the small molecule compound that activates endoplasmic reticulum stress as described in any one of claims 1 to 5 in the preparation of a medicament for treating multiple myeloma, characterized in that: Cucurbitacin E has specific anti-multiple myeloma activity.

7. A drug for treating multiple myeloma, characterized in that: The drug targets endoplasmic reticulum stress signaling molecules, triggering endoplasmic reticulum stress in cells, and contains cucurbitacin E and other pharmaceutically acceptable excipients.

8. A high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress, characterized in that: The filtering method is based on the IRE1 / XBP1 signaling pathway, constructing a high-throughput filtering platform for the IRE1 / XBP1 reporting system, including: The XBP1 reporter plasmid containing a partially alternatively spliced ​​fragment of the XBP1 gene and a mNeonGreen fluorescent fragment was transfected into human multiple myeloma cells via lentiviral transfection. Stable expression lines were selected using puromycin to obtain fluorescent reporter cell lines; Fluorescent reporter cell lines were treated overnight, and a library of small molecule compounds to be detected was added. After culturing for 24 hours, the fluorescence intensity and cell viability of human multiple myeloma cells were detected by flow cytometry. Small molecule compounds with a fluorescence intensity ratio > 2.0 and a cell viability ratio < 0.4 were screened to obtain small molecule compounds that activate endoplasmic reticulum stress.

9. The high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress as described in claim 8, characterized in that: The human multiple myeloma cells were selected from H929 cells.

10. The high-throughput screening method for small molecule compounds that activate endoplasmic reticulum stress as described in claim 8, characterized in that: The XBP1 reporter plasmid containing the mNeonGreen fluorescent fragment contains a partially alternatively spliced ​​fragment of the XBP1 gene. This alternatively spliced ​​fragment is specifically excised when endoplasmic reticulum stress induces IRE1RNase activity activation, triggering a translational frameshift and thereby achieving the expression of the fluorescent protein mNeonGreen.

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