Application of compound I as proteasome inhibitor

Abstract

The invention belongs to the field of biomedical engineering, and particularly provides an application of a compound as a proteasome inhibitor, the compound is lithospermum furan A, the CAS number of the compound is 85022-66-8, and the compound is a natural small molecule compound. Experiments prove that the lithospermum furan A can obviously inhibit the proteasome function and is a novel proteasome inhibitor. The proteasome inhibitor screened by the invention may become a new generation of proteasome inhibitor which is better in effect, stronger in specificity, smaller in side effect and also plays a role in solid tumors; the proteasome inhibitor can be finally used for preparing drugs for inhibiting tumor cell proliferation and / or inducing tumor cell death, and has important drug and medical clinical values.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical engineering, and particularly relates to application of a compound as a proteasome inhibitor. BACKGROUND

[0002] Proteasome is a multi-subunit macromolecular protein complex, which is widely present in eukaryotes and some prokaryotes. The main function of proteasome is to selectively degrade most of the intracellular proteins (80-90%). Therefore, the function of proteasome is crucial for the intracellular balance of proteins, and regulates almost all important life activities. Proteasome is an important part of the ubiquitin-proteasome pathway (UPP). UPP is composed of ubiquitin, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), ubiquitin-ligating enzyme (E3) and proteasome. The proteins to be degraded in cells are marked with polyubiquitin molecules under the action of E1, E2 and E3, and the polyubiquitinated proteins are finally recognized by proteasome and then degraded.

[0003] The sedimentation coefficient of proteasome density gradient centrifugation is 26S, so it is also called 26S proteasome. The 26S proteasome is composed of a catalytic particle (CP) and a regulatory particle (RP). The sedimentation coefficient of CP is 20S, also known as 20S proteasome, and its main function is to hydrolyze proteins. The sedimentation coefficient of RP is 19S, also known as 19S regulatory particle or PA70, and its main function is to recognize proteins with ubiquitination tags. CP is a four-loop barrel structure (α7β7β7α7) composed of seven α subunits and seven β subunits. In eukaryotic cells, the outer two loops are composed of seven different α subunits, and the inner two loops are composed of seven different β subunits. Among them, 3 β subunits are active subunits with protease activity: β1 subunit has caspase-like (C-L) activity, β2 subunit has trypsin-like (T-L) activity, and β5 subunit has chymotrypsin-like (CT-L) activity. RP is located on the outside of CP and is composed of 19 subunits; these subunits can be divided into two categories: ATPase-like subunits Rpt (Rpt1-6) and non-ATPase-like subunits Rpn (Rpn1-13), which form the lid and base of the RP, respectively. The base can be directly combined with the α loop of CP and is composed of 10 protein members, which are Rpt1-6 and Rpn1, Rpn2, Rpn10, Rpn13, respectively; the lid is composed of 9 non-ATPase-like subunits, which are Rpn3, Rpn5-9, Rpn11, Rpn12 and Rpn15, respectively. RP is mainly responsible for recruiting polyubiquitinated protein substrates (Rpn10 and Rpn13), removing ubiquitin molecules (Rpn11), and unfolding, promoting the opening of the intermediate channel in CP, and making the substrate protein enter the CP for degradation.

[0004] Proteasome is closely related to many diseases, such as tumor. In cells, many anti-tumor protein molecules are degraded by ubiquitin-proteasome pathway (UPP). Proteasome inhibitors block UPP by inhibiting proteasome activity, inhibit the degradation of anti-tumor protein molecules, and thus can inhibit the proliferation of various tumor cells and induce tumor cell apoptosis. For example, proteasome inhibitors can inhibit I-κB degradation, I-κB can bind to NF-κB and prevent NF-κB activation, so that the expression of many genes related to cell proliferation regulated by NF-κB is blocked, thereby inhibiting tumor growth and inducing apoptosis. Therefore, proteasome and its inhibitors have become a new target for anti-tumor therapy. According to the chemical structure of the pharmacophore, proteasome inhibitors can be divided into five categories: peptide aldehyde (MG-132), boronic acid peptide (bortezomib), peptide epoxy ketone (epoxomicin, epoxomicin and carfilzomib), peptide vinyl sulfone and β-lactone (lactacystin and its derivatives). Bortezomib (Bortezomib or PS341) is the first proteasome inhibitor approved by the US Food and Drug Administration (FDA) for marketing, and is clinically used for the treatment of multiple myeloma and mantle cell lymphoma. Bortezomib inhibits the activity of 20S proteasome by reversibly binding to the β5 subunit. Bortezomib can effectively delay tumor progression and improve patient survival, and has become a first-line drug for relapsed multiple myeloma. Bortezomib has side effects and is prone to drug resistance, and has poor effect on solid tumors. In order to overcome the above defects, the second generation of proteasome inhibitors has been developed. Carfilzomib is the second proteasome inhibitor approved by the FDA for the treatment of multiple myeloma patients who have received at least 2 drugs (including bortezomib and immunomodulator therapy) before. Carfilzomib is a specific and irreversible proteasome inhibitor, which belongs to the peptide epoxy ketone compound. Carfilzomib can irreversibly covalently bind to the β5 subunit of proteasome and the β5i (LMP7) subunit of immunoproteasome. In 2015, the first oral proteasome inhibitor Ixazomib Citrate (MLN9708) was approved for marketing. Ixazomib Citrate is hydrolyzed in vivo to Ixazomib (MLN2238), which is biologically active, namely Ixazomib; Ixazomib reversibly inhibits the β5 subunit of 20S proteasome. In addition, some proteasome inhibitors are undergoing clinical trials, such as Delanzomib (CEP-18770), Oprozomib (ONX-0912), NPI-0052 (Marizomib).

[0005] As can be seen from the above, the development of proteasome inhibitors is currently a hot research topic internationally. At present, proteasome inhibitors are mainly used to treat hematologic malignancies, and their efficacy against solid tumors is not ideal. They also have drawbacks in the treatment of multiple myeloma, including significant side effects and a high likelihood of drug resistance. Therefore, the identification and discovery of new proteasome inhibitors is of great significance. Summary of the Invention

[0006] Therefore, this invention provides an application of compound one as a proteasome inhibitor, wherein compound one is shikonin A, with CAS number 85022-66-8, and its chemical structural formula is as follows:

[0007]

[0008] In one specific embodiment, the compound is used as a proteasome inhibitor and ultimately for the preparation of a drug that inhibits tumor cell proliferation and / or induces tumor cell death.

[0009] In one specific embodiment, the compound is used as a proteasome inhibitor and ultimately for the preparation of a drug that inhibits the proliferation of human or mammalian tumor cells and / or induces tumor cell death, preferably the tumor cells being multiple myeloma, nasopharyngeal carcinoma, colorectal carcinoma, or lung cancer cells.

[0010] The present invention has at least the following beneficial effects: the proteasome inhibitor drug screened by the present invention may become a new generation of proteasome inhibitor with better efficacy, stronger specificity, fewer side effects, and also effective against solid tumors, and has significant pharmaceutical and clinical value. Attached Figure Description

[0011] Figure 1 This diagram illustrates the screening system for small-molecule proteasome inhibitors using the recombinant engineered protein TMEM8B-a-GFP described in this invention. Figure A shows the working principle, and Figure B shows the working steps.

[0012] Figure 2 Fluorescence microscopy images illustrating that the proteasome inhibitors MG132 and bortezomib PS341 can inhibit the degradation of recombinant engineered proteins N-copGFP TMEM8B-a or N-mScarlet TMEM8B-a in 293FT cells. copGFP is a green fluorescent protein, and mScarlet is a red fluorescent protein.

[0013] Figure 3This image shows the results of screening eight small molecule compounds from a variety of natural products using N-copGFP TMEM8B-a 293FT cells. The compound names are in the upper right corner, among which PS-341 is bortezomib, a specific proteasome inhibitor used as a positive control.

[0014] Figure 4 A and B represent shikonin and thiostreptin, two compounds, which were used to treat cells for 24 hours at concentrations of 10 μM, 5 μM, 1 μM, and 0.2 μM, respectively. The results of Western Blot detection of N-copGFP TMEM8B-a expression are shown in the figure.

[0015] Figure 5 The image shows the results of Western Blot analysis of total ubiquitin protein expression after treating cells with shikonin and thiostreptin at a concentration of 10 μM for 24 h.

[0016] Figure 6 Fluorescence microscopy results of N-copGFP TMEM8B-a 293FT cell screening to verify whether comfrey furan A is a proteasome inhibitor.

[0017] Figure 7 Figure 1 shows the effect of different concentrations of lithospermum erythrorhizon A on the degradation of copGFP TMEM8B-a by the proteasome.

[0018] Figure 8 The figure shows the effect of different concentrations of lithospermum furan A on the expression of total ubiquitin protein in 293FT cells.

[0019] Figure 9 The figure shows the results of the inhibitory effect of lithospermum erythrorhizon A on the proliferation of different cells. Detailed Implementation

[0020] The TMEM8B gene (also known as the NGX6 gene) is a metastasis suppressor gene closely related to nasopharyngeal carcinoma and colorectal cancer, independently cloned by our team. Our previous research demonstrated that the TMEM8B gene-encoded protein TMEM8B-a can be rapidly degraded by the proteasome in cells. Western blotting detected TMEM8B-a protein expression after treatment of stably transfected cells with the proteasome inhibitor MG-132 or bortezomib, with expression levels increasing with increasing MG-132 concentration. Immunofluorescence assays also showed that fluorescence appeared in stably transfected cells after 24 hours of treatment with MG-132 at concentrations above 10 μM, and the fluorescence intensity increased with increasing MG-132 concentration. However, treatment of stably transfected cells with the lysosomal inhibitor chloroquine and the calpain inhibitor calpeptin did not prevent the degradation of TMEM8B-a protein. These results indicate that TMEM8B-a protein is degraded via the proteasome pathway, rather than via calpain or lysosomal pathways. The above content has been published in scientific papers and doctoral dissertations by the inventors of this invention.

[0021] Because TMEM8B-a can be rapidly and completely degraded by the proteasome in cultured cells in vitro, the degradation of TMEM8B-a protein can be prevented by using proteasome-targeting inhibitors, leading to its accumulation in cells. We are the first to propose utilizing this characteristic of rapid proteasome degradation by fusing TMEM8B-a with a fluorescent protein (TMEM8B-a-GFP) as a reporter protein, constructing a cell line stably expressing this fusion protein. We then treated the cells with different small molecule compounds (from a small molecule compound library). When a compound did not target the proteasome and could not inhibit proteasome activity, the TMEM8B-a-GFP fusion protein was rapidly degraded by the proteasome, and no fluorescent signal was detected in the cells. Conversely, when a compound specifically targeted the proteasome and inhibited its activity, the TMEM8B-a-GFP fusion protein could not be degraded by the proteasome but accumulated in the cells, and a fluorescent signal could be detected.

[0022] Therefore, the present invention first provides a recombinant engineered protein, which is a fusion protein, comprising an effector protein and a reporter protein, wherein the effector protein is TMEM8B-a protein, and the reporter protein comprises a fluorescent protein or a luciferase.

[0023] In this invention, fluorescent proteins are a class of proteins with chromophores. They are activated by irradiation with light of a certain wavelength (excitation light) and release energy in the form of light energy. That is, they are excited by short-wavelength light and emit light of a longer wavelength than the excitation wavelength.

[0024] In one specific embodiment, the reporter protein is a fluorescent protein, preferably a green fluorescent protein or a red fluorescent protein.

[0025] In this invention, the fluorescent protein is not limited to green fluorescent protein and red fluorescent protein; for example, blue fluorescent protein, far-infrared fluorescent protein, etc., are all possible. Furthermore, the green fluorescent protein can be wild-type GFP, enhanced eGFP, or other variants such as copGFP. The red fluorescent protein can be wild-type RFP or other variants such as mScarlet.

[0026] The present invention also provides an application of the recombinant engineered protein described above for screening proteasome inhibitors.

[0027] In one specific implementation, cells that stably express the fusion protein are first established, preferably 293 series cells.

[0028] In this invention, the 293 series cells include cells of 293 origin such as 293, 293T, and 293FT.

[0029] This invention also provides a high-throughput screening method for proteasome inhibitors, comprising the following steps:

[0030] Step A: Seed cells stably expressing the fusion protein into a multi-well plate. The fusion protein is a recombinant engineered protein, including TMEM8B-a protein and fluorescent protein.

[0031] Step B: Add the small molecule compound to be tested into the cultured cells;

[0032] Step C: Use a fluorescence microscope or a fluorescence multi-functional microplate reader to detect whether the small molecule compound is a proteasome inhibitor.

[0033] In one specific embodiment, the perforated plate is a 96-well plate or a 348-well plate.

[0034] In one specific implementation, step A is preceded by a step of constructing a stable cell line for expressing the fusion protein. Specifically, this includes first constructing a eukaryotic expression vector TMEM8B-a with a fluorescent protein fused to its N-terminus, and then transfecting it into 293 series cells, thereby constructing a stable cell line for expressing the fusion protein.

[0035] Figure 1 Figure A shows the working principle diagram of the TMEM8B-a-GFP proteasome small molecule inhibitor screening system, and Figure B shows the working steps.

[0036] The screening system built based on this principle can conveniently and quickly screen a large number of compounds in a short time, rapidly and accurately obtaining new small molecule inhibitors targeting the proteasome. The entire high-throughput screening can be completed in just three steps: First, cells stably expressing TMEM8B-a-GFP are seeded into multi-well plates, such as 96-well plates; second, the small molecule compound to be tested is added to the cultured cells; third, the compound is detected using a fluorescence microscope or a fluorescence multi-mode microplate reader. Figure 1 B). The entire process can be completed in living cells without lysing cells or adding additional colorimetric or luminescent reagents.

[0037] We first successfully constructed eukaryotic expression vectors of TMEM8B-a, with green fluorescent protein (copGFP) and red fluorescent protein (mScarlet) fused to their N-termini, respectively, and transfected them into 293FT cells to construct stable expression cell lines of N-copGFP TMEM8B-a 293FT and N-mScarlet TMEM8B-a 293FT. Fluorescence signals were almost undetectable in both cell types without treatment or with DMSO treatment. After treatment with the proteasome inhibitor MG132 (10 μM) and bortezomib PS341 (60 nM) for 24 hours, significant green and red fluorescence signals were detected, with no clear difference in fluorescence intensity between the two. (See [link to documentation]). Figure 2 . Figure 2 Fluorescence micrographs illustrating that the proteasome inhibitors MG132 and bortezomib PS341 can both inhibit the degradation of N-copGFPTMEM8B-a or N-mScarlet TMEM8B-a proteins in 293FT cells; Figure 2 The scale bars in the diagrams are all 50 micrometers. This indicates that TMEM8B-a can successfully transport copGFP or mScarlet to the proteasome for degradation. When proteasome function is inhibited, copGFP or mScarlet can effectively accumulate in the cell and be detected. Unless otherwise specified, the fluorescence acquisition conditions for the fluorescence microscope in this invention are: exposure time 58 ms, sensitivity 800 ISO. Figure 2 The images in the first and third rows show fluorescence field testing, specifically the green and red fluorescence signals detected under a fluorescence microscope; while Figure 2 The images in the second and fourth rows are white field of view, which are comparison images under white field light corresponding to the images in the first and third rows.

[0038] It is important to note that while the proteasome is responsible for degrading the vast majority of intracellular proteins, not any protein fusion with a fluorescent protein can serve as a reporter protein for proteasome activity and for high-throughput screening of small-molecule proteasome inhibitors. Such a protein and its corresponding reporter protein must possess two key characteristics: First, the protein must be rapidly and completely degraded by the proteasome. Many proteins in cells are degraded via the proteasome pathway, but most degradation is controlled. When the protein level falls below a certain level, a feedback mechanism is triggered to prevent further degradation, protecting the protein from complete degradation. For example, some proteins in pathways such as p53, NF-κB, and β-catenin cannot be completely degraded. If protein degradation is incomplete, fusion with a fluorescent protein will produce a strong fluorescent signal, making it unsuitable for screening proteasome inhibitors. Second, the key degradative domains of the protein must not be masked or interfered with by the fluorescent protein, allowing it to successfully transport the fluorescent protein to the proteasome for degradation while undergoing its own degradation. TMEM8B-a perfectly possesses these characteristics, therefore, its fusion with a fluorescent protein can be used as a reporter protein for proteasome activity. The scheme described in this invention also provides an important approach and method for studying proteasome function.

[0039] We selected N-copGFP TMEM8B-a 293FT stably transfected cells as a high-throughput screening system to screen 477 small molecule natural product compounds (MCE, Natural Product Library). Several positive compounds were successfully identified, including Shikonin and Thiostrepton. All compounds were screened at a concentration of 10 μM. We found that Shikonin and Thiostrepton strongly induced the accumulation of TMEM8B-a in cells, and extremely strong fluorescence signals were observed. Figure 3 ).

[0040] To further verify that these compounds can inhibit proteasome activity and induce TMEM8B-a expression, we used Western blotting to detect the induction of N-copGFPTMEM8B-a expression by different concentrations of these compounds. The results showed that N-copGFPTMEM8B-a expression was extremely weak after cell treatment with DMSO as a negative control; only a small amount of polymerized copGFP TMEM8B-a fusion protein was observed at the high molecular weight band. In contrast, both shikonin and thiosericin effectively induced copGFP TMEM8B-a fusion protein expression, and the expression was strong and dose-dependent, meaning that the expression level of copGFP TMEM8B-a fusion protein increased sequentially with increasing cell concentrations of the two drugs. Figure 4To further verify that shikonin and thiosericin target the proteasome, we treated 293FT cells with these compounds and detected the expression of total ubiquitinated protein using Western blotting. The results showed that shikonin and thiosericin significantly promoted the aggregation of polyubiquitinated proteins within the cells, indicating that proteasome function was inhibited. Through these experiments, we preliminarily confirmed that shikonin and thiosericin are highly effective proteasome inhibitors. Figure 5 ).

[0041] Specifically, Figure 3 The image shows the results of screening eight small molecule compounds from various natural products using N-copGFP TMEM8B-a 293FT cells. Five of the small molecule compounds that produced negative results are not proteasome inhibitors: Icaritin (CAS No.: 118525-40-9); Levoleucovorin (CAS No.: 68538-85-2); Sisomicin (CAS No.: 32385-11-8); Octopamine (CAS No.: 104-14-3); and DHEA (CAS No.: 53-43-0). The other three small molecule compounds that produced positive results are all proteasome inhibitors: Shikonin, Thiostrepton, and bortezomib PS341.

[0042] The following are the chemical formulas of Shikonin and Thiostrepton from the small molecule compound library.

[0043]

[0044] Figure 4 Figures A and B represent the results of Western Blot analysis of N-copGFP TMEM8B-a expression, showing the effects of treating cells with shikonin and thiostreptin at concentrations of 10 μM, 5 μM, 1 μM, and 0.2 μM, respectively, for 24 h. GAPDH in the figure is the internal control molecule used for this Western Blot analysis, ensuring consistent loading amounts. "anti-strep" indicates that the antibody used in this Western Blot analysis is a strep antibody, and the detected signal represents the copGFP TMEM8B-a recombinant protein. "poly-modified TMEM8B-a" in the figure represents the polymerized copGFP TMEM8B-a recombinant protein.

[0045] Figure 5The image shows the results of Western blotting analysis of total ubiquitin protein expression after treating cells with shikonin and thiostreptin at a concentration of 10 μM for 24 h. DMSO served as the negative control; poly-Ub, anti-Ub, and mono-Ub represent polyubiquitin signal, antibody detection of ubiquitin, and monoubiquitin signal, respectively; GAPDH served as the internal control.

[0046] In summary, this invention utilizes the rapid degradation of the TMEM8B-a protein by the proteasome and proposes for the first time to fuse TMEM8B-a with a fluorescent protein that serves as a reporter protein, constructing a cell line stably expressing this fusion protein. This serves as a novel high-throughput screening system for small molecule inhibitors targeting the proteasome in live cells. The screening system built based on this principle can conveniently and quickly screen a large number of compounds in a short time, rapidly and accurately obtaining new small molecule inhibitors targeting the proteasome. The entire high-throughput screening can be completed in just three steps, and the entire process can be performed in live cells without cell lysis or the addition of additional chromogenic or luminescent reagents.

[0047] Example 1

[0048] The inventors' team conducted high-throughput screening of more than 6,500 small molecule compounds and found that the vast majority of them were negative and did not belong to the proteasome inhibitors; among these more than 6,500 small molecule compounds, only less than 20 were positive and all belong to the proteasome inhibitors.

[0049] The inventors' team screened fewer than twenty positive small molecule compounds, including shikonin and thiotetracycline, which they had previously listed in their patent applications; and six compounds with structures related to shikonin, including deoxyshikonin, biotin-esterified shikonin, and esterified derivatives of the secondary hydroxyl group of shikonin (specifically including 4-bromobutyrate shikonin ester, biotin-shikonin, β,β-dimethylacryloylshikonin, and acetylshikonin), which they had previously claimed protection for in their patent applications. The team also included six compounds listed in this batch of patent applications, including Shikonofuran A (CAS No.: 85022-66-8). Because the six compounds listed in this batch of patent applications do not share common chemical structures, they are not suitable for protection claims in the same patent application due to the requirement of unity of composition. Therefore, the applicant has filed separate patent applications for these six positive compounds, which are proteasome inhibitors.

[0050] This invention uses N-copGFP TMEM8B-a 293FT stably transfected cells as a high-throughput screening system to perform high-throughput screening on a large number of small molecule compounds (including the MCE natural product library, etc.), and successfully screened positive compounds, including Shikonofuran A (CAS No.: 85022-66-8), whose structural formula is as follows:

[0051]

[0052] The high-throughput screening of small molecule compounds in this invention specifically includes the following steps:

[0053] I. Cell Seeding. Take copGFP-NGX6A 293FT cells, also known as N-copGFP TMEM8B-a 293FT cells, digest and count them, and dilute the cells to 5.833 × 10⁻⁶. 5 At a concentration of / ml, seed 90 μL into each well of a 96-well plate using a pipette. Add the drug after 24 hours of culture. The seeding density of 293FT cells is 1.641 × 10⁻⁶. 5 / cm 2 It will be fully grown in 24 hours.

[0054] II. Adding various drugs to be tested. For example, the MCE natural compound library contains 477 natural small molecule compounds at 10 mM. The drug dilution process is as follows: Take a 96-well plate, use a multi-channel pipette, add 99 μL of serum-free DMEM to each well, and then add 1 μL of 10 mM small molecule compound. At this point, the drug concentration in each well of the 96-well plate is 100 μM. Then, use a multi-channel pipette to add 10 μL of 100 μM drug to a 96-well plate seeded with copGFP-NGX6A293FT cells. At this point, the final drug concentration is 10 μM.

[0055] III. Result Detection. After adding the drug, continue culturing for 24 hours, observe the results using a fluorescence microscope, and take photographs.

[0056] Figure 6 This image shows the results of screening N-copGFP TMEM8B-a 293FT cells to verify whether lithospermum erythrorhizon A is a proteasome inhibitor. The vast majority of compounds in the library are non-proteasome inhibitors; treatment of N-copGFP TMEM8B-a 293FT cells with these compounds resulted in almost no green fluorescence. For example... Figure 3 The present invention contains lcaritin and Levoleucovorin. Figure 6 The lithospermum erythrorhizon A shown can effectively inhibit the proteasome, causing the cell to emit green fluorescence. This demonstrates that this compound can significantly inhibit proteasome function and is a novel proteasome inhibitor.

[0057] Example 2

[0058] To further verify that Shikonofuran A (CAS No.: 85022-66-8) can inhibit proteasome activity and induce TMEM8B-a expression, we used Western blotting to detect the induction of copGFP TMEM8B-a expression by different concentrations of Shikonofuran A. The results showed that Shikonofuran A effectively induced the expression of the copGFP TMEM8B-a fusion protein, and the expression was strong and dose-dependent; that is, the expression level of the copGFP TMEM8B-a fusion protein increased sequentially with increasing drug concentration. Figure 7 To further verify that the target of shikonin A is the proteasome, we treated 293FT cells with different concentrations of shikonin A and detected the expression of total ubiquitinated proteins using Western blotting. The results showed that shikonin A significantly induced and promoted the expression of ubiquitinated proteins in cells, with both monoubiquitin and polyubiquitin proteins showing significantly increased expression levels in a dose-dependent manner, indicating that proteasome function was inhibited. Through these experiments, we preliminarily confirmed that shikonin A is a highly effective proteasome inhibitor. Figure 8 ). Figure 7 Figure 1 shows the effect of different concentrations of lithospermum erythrorhizon A on the degradation of copGFP TMEM8B-a by the proteasome. Figure 8 The figure shows the effect of different concentrations of lithospermum furan A on the expression of total ubiquitin protein in 293FT cells. Figure 7 and 8 In this context, SFA stands for Lithospermum erythrorhizon A.

[0059] Example 3

[0060] To verify whether shikonin A can inhibit cell proliferation by inhibiting proteasome function, we treated different cell types with shikonin A, including 8226, HCC827, H441, BEAS2B, H1299, and A549. The inhibitory effect of shikonin A on the proliferation of these cell types was detected using a CCK-8 assay. The experimental procedure was as follows: 5000 viable cells were seeded into 96-well plates, and shikonin A was added after 24 hours, with cell treatment lasting 48 hours. Then, 10 μL of WST-8 reagent from the CCK-8 kit was added, and the mixture was reacted at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader. The results showed that shikonin A significantly inhibited the growth and proliferation of 8226, HCC827, H441, BEAS2B, H1299, and A549 cells. Figure 9 ).in, Figure 9This figure shows the inhibitory effect of zirconia furan A on the proliferation of cells 8226, HCC827, H441, BEAS2B, H1299, and A549. The vertical axis represents the absorbance value measured at 450 nm using a microplate reader, and the horizontal axis represents the effect of treatment with the negative control DMSO and the treatment with zirconia furan A.

[0061] CCK-8 Assay Principle: The Cell Counting Kit-8 (CCK-8) is a rapid, highly sensitive, and non-radioactive colorimetric assay kit based on WST-8, widely used for cell proliferation and cytotoxicity detection. The CCK-8 solution can be directly added to cell samples without the need for pre-mixing of various components. In the presence of electron coupling reagents, the WST-8 reagent in the CCK-8 kit is reduced by certain dehydrogenases in mitochondria to produce orange-yellow formazan. The more and faster the cell proliferation, the darker the color; conversely, the greater the cytotoxicity, the slower the cell proliferation, or even cell death, the lighter the color. For the same number of cells, the intensity of the color (the amount of formazan produced) is linearly related to the number of cells.

[0062] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions and substitutions can be made without departing from the inventive concept, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. The application of a compound as a proteasome inhibitor, characterized in that, The first compound is lithospermum furan A, with CAS number 85022-66-8, and its chemical structural formula is as follows:

2. The application according to claim 1, characterized in that, The compound is used as a proteasome inhibitor and is ultimately used to prepare drugs that inhibit tumor cell proliferation and / or induce tumor cell death.

3. The application according to claim 2, characterized in that, The compound is used as a proteasome inhibitor and is ultimately used to prepare a drug that inhibits the proliferation of human or mammalian tumor cells and / or induces tumor cell death, preferably the tumor cells being multiple myeloma, nasopharyngeal carcinoma, colorectal carcinoma, or lung cancer cells.

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