Application of compound Y500-3645 as c-Myc protein degradation agent in preparation of drugs for resisting multiple myeloma

Abstract

The invention relates to application of a compound Y500-3645 as a c-Myc protein degradation agent in preparation of a drug for resisting multiple myeloma, and belongs to the technical field of multiple myeloma. The pharmaceutical composition for treating multiple myeloma provided by the invention takes a compound shown as a formula Y500-3645 or pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, and is supplemented with a pharmaceutically acceptable carrier. In multiple myeloma, the compound Y500-3645 inhibits the expression of c-Myc by promoting the degradation of c-Myc protein, regulates the expression levels of mRNA and protein of related genes, and promotes the death of myeloma cells. As a drug for resisting multiple myeloma, a new thought is provided for research and development of the drug for resisting multiple myeloma. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of multiple myeloma, and particularly relates to the application of a compound Y500-3645 in the preparation of a drug for treating multiple myeloma by promoting the degradation of c-Myc protein, inhibiting the expression of c-Myc, and regulating the mRNA and protein expression levels of related genes. Background Art

[0002] Cancer, characterized by uncontrolled cell growth, remains a major global health challenge and one of the leading causes of global mortality. Despite significant progress in our understanding of its molecular mechanisms, the incidence of cancer is still rising due to population aging and lifestyle changes. Current treatment methods, including surgery, chemotherapy, and radiotherapy, aim to eliminate cancer cells; however, they are often accompanied by significant side effects and limited efficacy. Small molecule targeted therapy, as a promising alternative, offers specific treatment while reducing systemic toxicity. However, there is an urgent need to develop more targeted therapies. Multiple Myeloma (MM) is a malignant hematological tumor caused by abnormal proliferation of plasma cells, with high genetic heterogeneity and complex molecular pathological mechanisms. Although drugs such as proteasome inhibitors (e.g., Bortezomib), immunomodulators (e.g., Lenalidomide), and monoclonal antibodies (e.g., Daratumumab) have significantly improved the survival of MM patients, drug resistance and recurrence remain the main challenges in treatment. Therefore, it is particularly important to develop new targeted drugs for MM.

[0003] c-Myc is a key transcription factor that regulates cell proliferation, differentiation, and metabolism, and is abnormally activated in various tumors, especially playing an important role in the occurrence and development of MM. Research shows that overexpression of c-Myc promotes the survival, metabolic reprogramming, and drug resistance of myeloma cells, and is closely related to poor prognosis. Therefore, drugs that target c-Myc to promote its degradation may provide new strategies for the treatment of MM.

[0004] Currently, there are few direct small molecule inhibitors targeting c-Myc, mainly because it lacks a classical drug-binding pocket. Traditional c-Myc targeting strategies mainly include: (1) inhibiting c-Myc transcription: by interfering with the transcriptional regulation of the c-Myc gene. For example, the small molecule BET inhibitor JQ1 can indirectly reduce the level of c-Myc mRNA, but its effect is non-specific and prone to side effects. (2) blocking c-Myc / Max dimerization: such as the small molecule inhibitor 10074-G5, which can interfere with the interaction between c-Myc and Max and prevent it from regulating the expression of downstream genes. However, the in vivo efficacy of such inhibitors is weak and it is difficult to be used as an effective clinical candidate drug. (3) promoting c-Myc degradation: the stability of c-Myc protein is regulated by the ubiquitin-proteasome system, and E3 ubiquitin ligase (such as FBW7) can mediate its degradation. Therefore, promoting c-Myc degradation based on strategies such as PROTAC (Proteolysis Targeting Chimeras) is an emerging direction in recent years. Summary of the Invention

[0005] An object of the present invention is to provide the use of compound Y500-3645 in the preparation of a drug for treating multiple myeloma on the basis of the prior art.

[0006] Another object of the present invention is to provide the use of compound Y500-3645 in the preparation of a drug for promoting the degradation of c-Myc protein.

[0007] A third object of the present invention is to provide the use of compound Y500-3645 in the preparation of a drug for promoting the degradation of c-Myc protein, inhibiting the expression of c-Myc, and regulating the mRNA and protein expression levels of related genes.

[0008] The technical solution of the present invention is as follows:

[0009] The use of the compound represented by formula Y500-3645 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating multiple myeloma,

[0010]

[0011] The compound represented by formula Y502-3645 or a pharmaceutically acceptable salt thereof, when used in the preparation of a drug for treating multiple myeloma, the drug can be made into a liquid preparation, a solid preparation or a semi-solid preparation. Further, based on the technical solution provided by the present invention, the drug can be made into an injection, a tablet, a capsule or an oral liquid. Pharmaceutically acceptable excipients play a key role in the entire development process of formulation technology. In many formulations, excipients account for the majority, so to a large extent, the properties of the excipients determine the properties of the formulation. Excellent excipients can enhance the stability of the main drug and extend the shelf life of the medicament; can regulate the release rate of the main drug in vivo and in vitro; can change the absorption of the drug in vivo and increase the bioavailability. The specific type of excipient is not limited.

[0012] In a preferred embodiment, the present invention provides a pharmaceutical composition for the preparation of an anti-multiple myeloma drug, which uses the compound represented by formula Y500-3645 mentioned in the present invention or a pharmaceutically acceptable salt thereof as the active ingredient or the main active ingredient, supplemented with a pharmaceutically acceptable carrier. Further, the pharmaceutical composition can be made into a liquid preparation, a solid preparation or a semi-solid preparation. Even further, the pharmaceutical composition can be made into an injection, a tablet, a capsule or an oral liquid. Pharmaceutically acceptable excipients play a key role in the entire development process of formulation technology. In many formulations, excipients account for the majority, so to a large extent, the properties of the excipients determine the properties of the formulation. Excellent excipients can enhance the stability of the main drug and extend the shelf life of the medicament; can regulate the release rate of the main drug in vivo and in vitro; can change the absorption of the drug in vivo and increase the bioavailability. The specific type of excipient is not limited.

[0013] Use of the compound represented by formula Y500-3645 or a pharmaceutically acceptable salt thereof in the preparation of a drug for promoting c-Myc protein degradation

[0014]

[0015] The compound represented by formula Y502-3645 or a pharmaceutically acceptable salt thereof, when used in the preparation of a drug for promoting c-Myc protein degradation, the drug can be made into a liquid preparation, a solid preparation or a semi-solid preparation. Further, based on the technical solution provided by the present invention, the drug can be made into an injection, a tablet, a capsule or an oral liquid. Pharmaceutically acceptable excipients play a key role in the entire development process of formulation technology. In many formulations, excipients account for the majority, so to a large extent, the properties of the excipients determine the properties of the formulation. Excellent excipients can enhance the stability of the main drug and extend the shelf life of the medicament; can regulate the release rate of the main drug in vivo and in vitro; can change the absorption of the drug in vivo and increase the bioavailability. The specific type of excipient is not limited.

[0016] In a preferred embodiment, the present invention provides a pharmaceutical composition for promoting the degradation of c-Myc protein, wherein the compound represented by formula Y500-3645 mentioned in the present invention or a pharmaceutically acceptable salt thereof is used as the active ingredient or the main active ingredient, supplemented with a pharmaceutically acceptable carrier. Further, the pharmaceutical composition can be formulated into a liquid preparation, a solid preparation or a semi-solid preparation. Furthermore, the pharmaceutical composition can be formulated into an injection, a tablet, a capsule or an oral liquid.

[0017] Use of the compound represented by formula Y500-3645 or a pharmaceutically acceptable salt thereof in the preparation of a drug for promoting the degradation of c-Myc protein, inhibiting the expression of c-Myc, and regulating the mRNA and protein expression levels of related genes.

[0018]

[0019] When the compound represented by formula Y502-3645 or a pharmaceutically acceptable salt thereof is used in the preparation of a drug for promoting the degradation of c-Myc protein, inhibiting the expression of c-Myc, and regulating the mRNA and protein expression levels of related genes, the drug can be formulated into a liquid preparation, a solid preparation or a semi-solid preparation. Further, based on the technical solution provided by the present invention, the drug can be formulated into an injection, a tablet, a capsule or an oral liquid. Pharmaceutically acceptable excipients play a key role in the development process of the entire formulation technology. In many formulations, excipients account for the majority, so to a large extent, the properties of the excipients determine the properties of the formulation. Excellent excipients can enhance the stability of the main drug and extend the shelf life of the pharmaceutical; they can regulate the release rate of the main drug in vivo and in vitro; they can change the absorption of the drug in the body and increase the bioavailability. The specific types of excipients are not limited.

[0020] In a preferred embodiment, the present invention provides a pharmaceutical composition for promoting the degradation of c-Myc protein, inhibiting the expression of c-Myc, and regulating the mRNA and protein expression levels of related genes, which uses the compound represented by formula Y500-3645 mentioned in the present invention or a pharmaceutically acceptable salt thereof as the active ingredient or the main active ingredient, supplemented with a pharmaceutically acceptable carrier. Further, the pharmaceutical composition can be formulated into a liquid preparation, a solid preparation or a semi-solid preparation. Furthermore, the pharmaceutical composition can be formulated into an injection, a tablet, a capsule or an oral liquid.

[0021] The present invention uses the CCK8 assay to measure cell viability and demonstrates that compound Y500-3645 has cytotoxic effects on multiple myeloma RPMI8226. The q-PCR assay is used to show that Y500-3645 can reduce the expression level of c-Myc mRNA in multiple myeloma RPMI8226. The Western Blot assay indicates that Y500-3645 can decrease the expression level of c-Myc protein in multiple myeloma RPMI 8226. Further, cells are treated with MG132, CHX, and the drug simultaneously, suggesting that Y500-3645 may promote c-Myc protein degradation through the proteasome pathway. Western blot analysis is used to detect the expression levels of p-c-Myc Thr58, p-c-Myc Ser62, p-GSK3-β, and GSK3-β, indicating that Y500-3645 may promote c-Myc degradation through GSK3-β-mediated phosphorylation.

[0022] The advantages of the technical solution of the present invention are as follows:

[0023] The present invention confirms the important role of compound Y500-3645 in anti-multiple myeloma. Experiments show that compound Y500-3645 has anti-multiple myeloma activity, can target c-Myc, promote c-Myc protein degradation, inhibit c-Myc expression, and regulate the mRNA and protein expression levels of related genes, providing new ideas for the research and development of anti-multiple myeloma drugs. Brief Description of the Drawings

[0024] Figure 1 It is for Y500-3645 to have anti-multiple myeloma activity;

[0025] Figure 2 It is for Y500-3645 to be able to specifically reduce the expression level of c-Myc mRNA;

[0026] Figure 3 It is for Y500-3645 to be able to reduce the expression level of c-Myc protein and affect the degradation process of c-Myc through the proteasome;

[0027] Figure 4 It is for Y500-3645 to be able to accelerate the degradation rate of c-Myc protein;

[0028] Figure 5 It is for Y500-3645 to possibly promote c-Myc degradation through GSK3-β-mediated phosphorylation. Detailed Description of the Invention

[0029] To better understand the technical solution of the present invention, the present invention is further described by the following embodiments, but these embodiments do not constitute any limitation to the present invention. Specific Embodiments

[0031] 1. Detect the anti-tumor activity of Y500-3645 by CCK-8 assay

[0032] Experimental materials:

[0033] (1) Cells

[0034] The cell lines were from the cell bank of the School of Medicine, Anhui University of Science and Technology. The human cell line RPMI 8226 was cultured in RPMI-1640 medium (KeyGEN, Shanghai, China) supplemented with 10% fetal bovine serum (FBS; ZetaLife, USA). All cells were maintained in a humidified atmosphere of 37°C and 5% CO2. The cell lines were identified by short tandem repeat (STR) analysis and mycoplasma contamination was regularly detected.

[0035] (2) CCK-8 kit

[0036] Experimental procedures:

[0037] (1) Cell counting: Suspended cells were directly collected and centrifuged at 1000 rpm for minutes; the supernatant was aspirated and discarded, and an appropriate amount of medium was added to resuspend the cells. The cell concentration was calculated using the cell counting method;

[0038] (2) Cell seeding: 5000 cells per well, 100 μL volume, and 3 replicates were set for each group. Calculate the required cell volume and the volume of complete medium to be supplemented. After thoroughly pipetting and mixing, the cells were inoculated into a 96-well plate and cultured in a constant temperature cell incubator;

[0039] (3) CCK-8 detection: 48 hours after cell administration, 10 μL of CCK-8 reagent was added to each well, and the cells were cultured for another 2 hours. Subsequently, the absorbance value of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and a growth curve was plotted.

[0040] Experimental results:

[0041] The study found that the IC50 value of Y500-3645 against the RPMI-8226 cell line was 12.17 μM, indicating that Y500-3645 had significant cytotoxicity against this typical MM cell line ( Figure 1 ).

[0042] 2. Y500-3645 inhibits the mRNA expression level of c-Myc.

[0043] Experimental procedures

[0044] (1) Extract the RNA of RPMI 8226 cells after the action of Y500-3645 for 48 h: Collect the cells, add an appropriate amount of PBS to wash the cells three times, then add an appropriate amount of Trizol and centrifuge at 12,000 r / min. Aspirate the upper chloroform layer and add isopropanol. Let it stand in the refrigerator at 4 °C for 20 minutes and then centrifuge at 12,000 r / min. After washing three times with 70% ethanol, measure the purity and concentration.

[0045] (2) Use a reverse transcription kit to reverse transcribe the RNA into a cDNA template and dilute it to 10 times the original concentration with deionized water for later use.

[0046] (3) Transfer the prepared qPCR reaction system to an eight-well strip tube to ensure that the volume of each well is the same. Then, tighten the tube cap to prevent the reaction solution from leaking during centrifugation. Place the eight-well strip tube in a palm centrifuge for a short centrifugation to ensure that the reaction system is fully and evenly mixed. After centrifugation, gently tap the bottom of the tube to concentrate the liquid in the tube, and ensure that the bubbles that may be generated during the pipetting process are moved to the tube mouth and removed by centrifuging again.

[0047] (4) After the PCR reaction is completed, collect the CT values of each sample. Use appropriate statistical methods and controls to analyze these data to calculate and compare the relative expression levels of the target gene in different samples.

[0048] Experimental results:

[0049] We studied the effect of Y500-3645 on the expression of c-Myc gene in RPMI 8226, including the control group (without drug treatment), 5 μM of Y500-3645, 10 μM of Y500-3645, and 15 μM of Y500-3645. The results showed that Y500-3645 inhibited the c-Myc mRNA level in a concentration-dependent manner in the RPMI 8226 multiple myeloma cell line ( Figure 2 ).

[0050] 3. Y500-3645 downregulates the protein expression level

[0051] Western blot experiment

[0052] Experimental steps:

[0053] (1) Cell protein extraction: Centrifuge to collect the cells, wash them 3 times with PBS buffer, and discard the supernatant. Prepare a protein lysate according to the ratio of RIPA:SPMF:phosphatase inhibitor = 100:1:2, add it to the cells, use a turbo oscillator to shake for 10 s, and lyse on ice for 30 min, shaking for 10 s every 10 min. Pre-cool a 4 °C centrifuge, after lysing on ice, centrifuge at 12,000 r / min at 4 °C for 20 min, and collect the supernatant.

[0054] (2) Measuring protein concentration by BCA method: According to the kit instructions, prepare the BCA working solution, with solution A:solution B = 50:1. Add 2 μl of protein supernatant, 18 μl of PBS buffer, and 200 μl of BCA working solution to a 96-well plate, incubate in the dark for 30 min, measure the absorbance at 462 nm, and calculate the protein concentration. After mixing the protein supernatant with the loading buffer and boiling for 15 min, store at -20 °C.

[0055] (3) Prepare 10% separating gel and 5% stacking gel.

[0056] (4) Protein loading: Take out the protein, load it after boiling.

[0057] (5) Electrophoresis: Electrophorese at 80 V until the Marker boundary is obvious, then adjust to 120 V and electrophorese until the bottom of the separating gel.

[0058] (6) Wet transfer: According to the separation distance of the Marker and the molecular weight of the target protein, cut a PVDF membrane of appropriate size, mark it for standby. Place the SDS-PAGE gel in the electrotransfer solution, moisten the sandwich clamp, place the gel, place the PVDF membrane, drive out the air bubbles, fix it, add the electrotransfer solution to the electrotransfer tank, and perform electrotransfer at 100 V on ice, with the time set according to the molecular weight. Blocking: After electrotransfer, take out the PVDF membrane, place it in TBST containing 5% skim milk powder, and gently shake on a shaker for 2 h.

[0059] (7) Membrane washing: After blocking, take out the PVDF membrane, place it in TBST, and quickly shake on a shaker for 5 min × 3 times.

[0060] (8) Primary antibody incubation: Blot the PVDF membrane dry, place it in the primary antibody, and incubate overnight on a shaker at 4 °C.

[0061] (9) Membrane washing: After primary antibody incubation, wash with TBST for 5 min × 3 times.

[0062] (10) Secondary antibody incubation: Place the PVDF membrane in the secondary antibody, gently shake on a shaker, and incubate at room temperature for 1 h. Membrane washing: Take out the PVDF membrane and wash with TBST for 5 min × 3 times.

[0063] (11) ECL development: Prepare the ECL developing solution and develop with the instrument.

[0064] Experimental results:

[0065] Western blot analysis showed that Y500-3645 effectively downregulated the expression of c-Myc in a concentration-dependent manner in the RPMI 8226 cell line, and the degradation of c-Myc was reversed by MG132 after treatment with Y500-3645, indicating that the drug acts on the ubiquitin-proteasome pathway and promotes the degradation of c-Myc (Figure 3 )。The degradation rate of c-Myc was accelerated after treatment with Y500-3645 and CHX Figure 4 )。After treatment with Y500-3645 (5 μM, 10 μM, and 15 μM) for 48 hours, Western blot analysis was performed to detect the expression levels of p-c-Myc Thr58, p-c-Myc Ser62, p-GSK3-β, and GSK3-β. β-actin was used as an internal reference control. The experimental data showed that Y500-3645 could promote the degradation of c-Myc, with enhanced phosphorylation of Thr58 and no significant change in Ser62, indicating that Y500-3645 might promote the degradation of c-Myc through GSK3-β-mediated phosphorylation Figure 5 )。

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a compound represented by formula Y500-3645 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating multiple myeloma, 2. The use according to claim 1, characterized in that: The medicine is prepared into a liquid preparation, a solid preparation or a semisolid preparation.

3. The use according to claim 2, characterized in that: The medicine is prepared into injection, tablet, capsule or oral solution.

4. A pharmaceutical composition for preparing an anti-multiple myeloma agent, comprising the compound represented by formula Y500-3645 or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient or a main active ingredient, and supplemented with a pharmaceutically acceptable carrier.

5. Use of the compound represented by formula Y500-3645 or a pharmaceutically acceptable salt thereof in the preparation of a drug for promoting the degradation of c-Myc protein, 6. The use according to claim 5, characterized in that: The medicine is prepared into a liquid preparation, a solid preparation or a semisolid preparation.

7. The use according to claim 6, characterized in that: The medicine is prepared into injection, tablet, capsule or oral solution.

8. Use of the compound represented by formula Y500-3645 or a pharmaceutically acceptable salt thereof in the preparation of a drug for promoting the degradation of c-Myc protein, inhibiting the expression of c-Myc, and regulating the expression levels of mRNA and protein of related genes, 9. The use according to claim 8, characterized in that: The medicine is prepared into a liquid preparation, a solid preparation or a semisolid preparation.

10. The use according to claim 9, characterized in that: The medicine is prepared into injection, tablet, capsule or oral solution.

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