Application of disulfiram in preparation of KSHV infection resisting medicine and KSHV related lymphoma resisting medicine

Abstract

The invention provides an application of disulfiram in preparation of KSHV infection resisting medicines and KSHV related lymphoma resisting medicines, and belongs to the technical field of biological medicines. Research finds that disulfiram can inhibit splitting replication of Kaposi sarcoma related herpes viruses and inhibit generation of Kaposi sarcoma related herpes virus particles; a xenotransplantation mouse tumor experiment proves that the disulfiram can induce death of a large number of PEL cells in the incubation period of the KSHV and inhibit growth of PEL tumors, so that the obstacle that the KSHV escapes from immune surveillance of host cells in PEL treatment is overcome, and a new solution is provided for treatment of Kaposi sarcoma-related herpes viruses and treatment of KSHV-related lymphoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of disulfiram in the preparation of anti-KSHV infection drugs and anti-KSHV related lymphoma drugs. Background Art

[0002] Primary effusion lymphoma (PEL) is a rare HIV-related non-Hodgkin lymphoma, accounting for about 4% of all HIV-related non-Hodgkin lymphomas, and is prone to occur in body cavities such as the pleura, pericardium, and peritoneum. Currently, conventional drugs for PEL are ineffective, and the prognosis of patients is very poor. There is currently no consensus or established standard treatment recommendations. There are few drugs based on targeted specific treatments for this type of tumor. PEL is generally associated with Kaposi sarcoma-associated herpesvirus (KSHV) infection.

[0003] The life cycle of KSHV includes two distinct phases, the latent phase and the lytic phase, and plays a crucial role in the pathogenesis. After infecting host cells, KSHV usually enters a latent state characterized by minimal viral gene expression. This restrictive expression strategy helps KSHV evade the immune surveillance of host cells, which is currently considered to be an obstacle to the elimination of KSHV-related tumors. Under the influence of external stimuli such as hypoxia, as well as physiological and pathological factors such as oxidative stress, inflammation, bacterial and viral co-infection (such as papillomavirus, HIV, HSV-1, HSV-2, etc.), and chemical inducers (such as sodium butyrate, valproic acid, etc.), KSHV can transition from the latent phase to the lytic phase. This process is called lytic replication or reactivation of KSHV. Once reactivated, KSHV cascades express viral lytic genes, resulting in the production and release of a large number of viral particles, which is essential for the development and spread of tumors.

[0004] At present, the main treatments for lymphoma are chemotherapy, radiotherapy, immunotherapy, targeted therapy and hematopoietic stem cell transplantation, etc. However, the main treatment for most lymphoma patients is drugs. Common drugs such as rituximab have been widely used in the treatment of lymphoma. Rituximab is a human-mouse chimeric antibody targeting the CD20 antigen on the surface of B lymphocytes. It contains mouse protein structure and other components of the drug, which leads to large adverse reactions after use. Another clinical drug for lymphoma patients, rituximab, is an antibody-drug conjugate composed of a monoclonal antibody targeting CD30 protein and a microtubule disruptor coupled by a protease-sensitive cross-linker. However, the drug can cause neutropenia, peripheral sensory neuropathy, fatigue, nausea, anemia, upper respiratory tract infection, diarrhea, fever, rash, thrombocytopenia, cough, and vomiting. The capsule drug has adverse symptoms such as toxic and side effects that cannot be ignored, and the effect of single treatment is not significant enough. Other drugs such as bortezomib, lenalidomide, dexamethasone, temsirolimus, etc. usually require combined treatment, and their effectiveness and tolerability still need to be solved. The effectiveness and tolerability of current tumor-targeted drugs related to KSHV still need to be improved. Finding a highly effective, specific and low-toxic drug is still the top priority for current disease treatment. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide an application of disulfiram in the preparation of anti-KSHV infection drugs and anti-KSHV related lymphoma drugs. Studies have found that disulfiram can effectively improve the occurrence process of lymphoma and significantly slow down its onset rate. In a xenograft mouse tumor model, it significantly inhibits the occurrence and development of KSHV-related lymphoma, and the drug has no effect on the appearance and development of various organs of mice.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The first object of the present invention is to provide the use of disulfiram and its pharmaceutically acceptable salts in the preparation of a drug for treating Kaposi's sarcoma-associated herpes virus infection.

[0007] Furthermore, the disulfiram inhibits the lytic replication of Kaposi's sarcoma-associated herpes virus.

[0008] Furthermore, disulfiram inhibits the reactivation of Kaposi's sarcoma-associated herpes virus by triggering the innate immune response.

[0009] Furthermore, the disulfiram inhibits the production of Kaposi's sarcoma-associated herpes virus particles.

[0010] The second object of the present invention is to provide the use of disulfiram and its pharmaceutically acceptable salts in the preparation of drugs for treating KSHV-related lymphoma.

[0011] Furthermore, the KSHV-related lymphoma is primary effusion lymphoma.

[0012] Furthermore, the disulfiram induces cell death of Kaposi's sarcoma-associated herpesvirus latent primary effusion lymphoma cells.

[0013] Furthermore, disulfiram inhibits the proliferation and survival of primary effusion lymphoma cells by activating the innate immune response.

[0014] The third object of the present invention is to provide an anti-Kaposi's sarcoma-associated herpes virus pharmaceutical composition, the active ingredient of which includes disulfiram.

[0015] Furthermore, pharmaceutically acceptable excipients are also included.

[0016] The fourth object of the present invention is to provide a pharmaceutical composition for treating KSHV-related lymphoma, the active ingredient of which includes disulfiram.

[0017] Furthermore, pharmaceutically acceptable excipients are also included.

[0018] The pharmaceutically acceptable salts of the above compounds refer to compounds having the same parent core structure as the compound itself, which can generate molecules with the same or similar activity as the original compound through hydrolysis in the body, achieving the same or similar therapeutic effects. Pharmaceutically acceptable salts of compounds can be synthesized from parent compounds by conventional chemical methods. Generally speaking, these salts can be prepared by reacting the free base and acid of the compound in water or an organic solvent or a mixture of the two; usually, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are used.

[0019] Acid addition salts can be prepared by various acids (inorganic and organic). Examples of acid addition salts include salts made from acids, wherein the "inorganic acid" includes, but is not limited to, hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid. The "organic acid" includes, but is not limited to, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, phenylacetic acid, salicylic acid, 2- (4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

[0020] The term "effective amount" or "therapeutically effective amount" refers to an amount of the compound of the present invention sufficient to achieve the intended application (including but not limited to the treatment of diseases as defined below). The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and the disease condition being treated, such as the weight and age of the subject, the severity of the disease condition, and the mode of administration, which can be easily determined by a person of ordinary skill in the art. The specific dosage will vary depending on the specific compound selected, the dosage regimen followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system carried.

[0021] The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the drug, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0022] The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.

[0023] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention found that disulfiram can induce KSHV latent PEL cell death and inhibit the growth of PEL tumors, thereby overcoming the obstacle of KSHV escaping host cell immune surveillance during PEL treatment. It not only does not induce KSHV lytic replication, but also effectively inhibits the NaB-induced lytic replication program. The advantage of DSF in treating PEL is that it does not increase the spread of KSHV and tumor cells, providing a new solution for the treatment of KSHV and KSHV-related lymphoma.

[0025] (2) The present invention demonstrates that disulfiram can effectively improve the progression of lymphoma and significantly slow down its onset. In a xenograft mouse tumor model, it significantly inhibits the occurrence and development of KSHV-related lymphoma. Furthermore, the drug has no effect on the appearance and development of the various organs of the mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Proliferation curves of DG75, BJAB, BJAB-kshv and PEL (BC3, BCBL1 and BCP1) cells after treatment with 0 (DMSO), 0.025, 0.05 and 0.1 μM DSF for 1, 2 and 3 days; Figure 2 The percentage of dead cells in DG75, BJAB, BJAB-kshv and PEL cells treated with 0 (DMSO), 0.025, 0.05 and 0.1 μM DSF for 1, 2 and 3 days; Figure 3Cell viability of DG75, BJAB, BJAB-kshv and PEL (BC3, BCBL1 and BCP1) cells after 72 h of DSF treatment; Figure 4 This is the IC50 curve of different cells treated with DSF; Figure 5 The figure shows the results of apoptosis detection of DG75, BJAB, BJAB-kshv and PEL cells using Annexin V and PI staining; Figure 6 Western blot analysis of DG75, BJAB, BJAB-kshv and PEL cells treated with 0 (DMSO), 0.025, 0.05 and 0.1 μM DSF for 72 h; Figure 7 The results of RT-qPCR analysis of the mRNA levels of KSHV lytic genes such as RTA, PAN RNA, ORFK8, ORF65, ORF59 and ORF57 in BCBL1 cells treated with DMSO, 0.1 μM DSF, 0.5 mM sodium butyrate (NaB) or both for 72 h; Figure 8 Western blots were used to detect the protein level of ORFK8 in BCBL1 cells treated with DMSO, 0.1 μM DSF, 0.5 mM sodium butyrate (NaB), or both for 72 h. The scale bar is 20 μm. Fig. 9 To detect the ORFK8 protein level in BCBL1 cells treated with DMSO, 0.1 μM DSF, 0.5 mM sodium butyrate (NaB), or both for 72 h by immunofluorescence, the scale bar is 20 μm; Fig.10 The supernatant of BCBL1 cells treated with DMSO, 0.1 μM DSF, 0.5 mM sodium butyrate (NaB), or DMSO, 0.1 μM DSF, and 0.5 mM NaB for 96 h was used to quantify the produced KSHV virus particles by qPCR; *, p<0.05, **, p<0.01, **, p<0.001, ns, not statistically significant; Fig.11 Real-time imaging of PEL tumors in mice treated with vehicle or DSF 8 and 15 days after inoculation with BCBL1-Luc cells; Fig.12 Quantification of the luminescence signal of PEL tumors in group G, expressed as ROI (p / s / (μW / cm 2)). *, p<0.05, **, p<0.01, **, p<0.001, ns, no significant difference compared with cells treated with 0 (DMSO) μM DSF; Fig.13 iSLK-BAC16-RGB cells were treated with DMSO, 1 μM DSF, 1 μg / mL doxycycline (Dox) plus 1 mM NaB, or both for 48 h, and GFP and RFP images of the cells were captured under a fluorescence microscope; Fig.14 Figure 1: GFP-positive results of flow cytometry analysis after treatment with DMSO, 1 μM DSF, 1 μg / mL Dox + 1 mM NaB or both for 48 h; Fig.15 The results of RT-qPCR analysis of the mRNA levels of KSHV lytic genes such as RTA, PAN RNA, ORFK8, ORF65, ORF59 and ORF57 in iSLK-BAC16-RGB cells treated with DMSO, 0.1 μM DSF, 0.5 mM sodium butyrate (NaB) or both for 72 h; Fig.16 Western blots were used to detect the protein level of ORFK8 in iSLK-BAC16-RGB cells treated with DMSO, 0.1 μM DSF, 0.5 mM sodium butyrate (NaB), or both for 72 h. The scale bar is 20 μm. Fig.17 The results of qPCR detection of KSHV virus particle concentrations produced in the supernatant of iSLK-BAC16-RGB treated with DMSO, 1 μM DSF, 1 μg / mL Dox + 1 mM NaB, or both for 96 h; Fig.18 Fluorescence microscopy images of HEK293T cells infected with KSHV virions prepared from the supernatant of iSLK-BAC16-RGB cells treated with DMSO, 1 μM DSF, 1 μg / mL Dox + 1 mM NaB, or both for 96 h, 48 h after infection; Fig.19 HEK293T cells were infected with KSHV virus particles prepared from the supernatant of iSLK-BAC16-RGB cells treated with DMSO, 1 μM DSF, 1 μg / mL Dox + 1 mM NaB or both for 96 h. Flow cytometry was used to detect RFP-positive cells 48 h after infection. In the figure, * *, p < 0.001, ns, not statistically significant; Fig. 20The figure shows the results of Western blot analysis of BJAB, BJAB-kshv, BC3 and BCBL1 cells treated with 0 (DMSO), 0.025, 0.05 and 0.1 μM DSF for 24 h; Fig.21 The results of RT-qPCR analysis of IFIT1, IFIT3, ISG15, IRF7 and IFNβ mRNA levels in BJAB cells treated with 0 (DMSO), 0.025, 0.05, 0.1, and 1 μM DSF for 24 h; Fig. 22 The results of RT-qPCR analysis of IFIT1, IFIT3, ISG15, IRF7 and IFNβ mRNA levels in BJAB-KSHV cells treated with 0 (DMSO), 0.025, 0.05, 0.1, 1 μM DSF for 24 h; Fig.23 The results of RT-qPCR analysis of IFIT1, IFIT3, ISG15, IRF7 and IFNβ mRNA levels in BC3 cells treated with 0 (DMSO), 0.025, 0.05, 0.1, and 1 μM DSF for 24 h; Fig.24 The results of RT-qPCR analysis of IFIT1, IFIT3, ISG15, IRF7 and IFNβ mRNA levels in hBCBL1 cells treated with 0 (DMSO), 0.025, 0.05, 0.1, and 1 μM DSF for 24 hBCBL1 cells; Fig.25 Western blotting analysis results of BCBL1 and BC3 wild-type and TBK1 knockout cells treated with 0 (DMSO) or 1 μM DSF for 24 hours; Fig.26 RT-qPCR analysis results of isg (including IFIT1, IFIT3, IRF7, IFNβ and ISG15) mRNA levels in BCBL1 and BC3 wild-type or TBK1 knockout cells treated with 0 (DMSO) or 1 μM DSF for 24 hours; Figure 27a-27b The results of RT-qPCR analysis of the mRNA levels of KSHV lytic genes such as RTA, PAN RNA, ORFK8, ORF65, ORF59 and ORF57; Fig.28 Western blotting analysis results of BCBL1, BC3 wild-type and TBK1 knockout cells treated with 0 (DMSO), 1 μM DSF, 0.5 mM NaB or both for 3 days; Fig.29 The results of RT-qPCR detection of KSHV virus particles in the supernatant of BCBL1 cells, BC3 wild-type and TBK1 knockout cells treated with 0 (DMSO), 0.1 μM DSF, 0.5 mM NaB or both for 3 days; Fig.30a The proliferation curves of BCBL1, BC3 wild-type and TBK1 knockout cells under the action of 0 (DMSO) or 0.1 μM DSF for 3 days; Fig.30b Proliferation curves of BC3 and bcbl1 cells treated with 0.1 μM DSF, 0.2 μM GSK8612, or both for 3 consecutive days; Fig.31 Annexin V and PI staining were used to detect apoptotic cells in BCBL1 cells, BC3 wild-type cells, and TBK1 knockout cells after treatment with 0 (DMSO) or 0.1 μM DSF for 3 days. Fig.32 Western blotting analysis results of BCBL1 or BC3 wild-type and TBK1 knockout cells treated with 0 (DMSO) or 0.1 μM DSF for 3 days; Fig.33 Live imaging of mice implanted with BCBL1-Luc wild-type or TBK1 knockout cells treated with vehicle or the indicated concentrations of DSF for 11, 17, and 22 days; Fig.34 Quantification of luminescent signals of PEL tumors in each group; Fig.35 is the daily weight gain of mice; Fig.36 The volume of ascites of mice on day 22 after inoculation; Fig.37 This is a photo of the mouse spleen on day 22 after inoculation; Fig.38 The length of the mouse spleen on the 22nd day after inoculation; Fig.39 The weight of the spleen of mice on day 22 after inoculation. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0028] The disulfiram (DSF) used in the present invention has the molecular formula: 10 H 20 N 2 S 4 Molecular weight: 297, CAS: 97-77-8. It has been proven that it can inhibit the activity of aldehyde dehydrogenase (ALDH), thereby increasing the acetaldehyde concentration in the blood of alcoholics, causing uncomfortable symptoms such as flushing, headache, nausea, and vomiting, thereby causing alcoholics to give up drinking and achieve the purpose of quitting drinking. It is an FDA-approved drug for the treatment of alcohol dependence.

[0029] The human B cell lymphoma cells used in the present invention: BJAB, DG75; human lymphoma cells: BC3, BCP1, BCBL-1, are from Shantou University, and the virus experiments involved in the present invention were completed in the P2 laboratory of Shanghai Jiaotong University.

[0030] Cell culture: The PEL cells were taken out from liquid nitrogen, quickly dissolved in a 37°C water bath, and added to a centrifuge tube. Next, 9 ml of RPMI1640 medium containing 10% FBS was added to the centrifuge tube. After mixing, the tube was centrifuged at 500 rpm for 5 minutes. The supernatant was discarded, and 1 ml of the corresponding medium was added to resuspend the cells. The cells were transferred to a 10 cm culture dish, and RPMI1640 medium containing 10% FBS was added. After mixing, the cells were cultured in a 37°C, 5% carbon dioxide cell culture incubator. The medium was changed after 24 hours.

[0031] PEL cells were cultured in RPMI1640 medium containing 10% FBS and 1% double antibody in a 37°C, 5% carbon dioxide incubator.

[0032] Observe the cells under a microscope and select cells in good growth condition (the number of viable cells determined by trypan blue staining is more than 95%). Cell subculture can be performed when the cell density reaches 80-90%.

[0033] Western Blot: After the PEL cells were treated with DSF for 72 h, the culture medium was centrifuged to obtain the cell pellet, protein samples were prepared, and protein immunoblotting was performed, and the development images were saved.

[0034] Fluorescence quantitative PCR: Total RNA was extracted by chloroform extraction and isopropanol precipitation. Reverse transcription was performed according to the instructions of the cDNA synthesis kit. SYBR Green was used to perform quantitative polymerase chain reaction on cDNA, and all quantitative polymerase chain reactions were repeated three times. The expression of related genes was normalized with the internal reference β-actin to obtain the 2-ΔΔCT value.

[0035] Apoptosis detection: The 12-well plate cells used for cell proliferation assay were taken out after 72 hours, and the cells were centrifuged at 1500 rpm, then washed once with PBS, and then double-stained with DAPI and Annexin V for 15 minutes at room temperature. Flow cytometry (Thermo Fisher, Attune NXT) showed cell apoptosis, and FlowJoV10 analyzed it. No staining of Annexin V and DAPI indicated cell survival, staining of both Annexin V and DAPI indicated early cell apoptosis, staining of both Annexin V and DAPI indicated late cell apoptosis, and staining of both Annexin V and DAPI indicated cell death.

[0036] Example 1 Study on the effect of DSF on the proliferation of PEL cells.

[0037] Five types of PEL cells, namely BJAB, DG75, BCBL1, BC3 and BCP1, were inoculated into 12-well cell culture plates with 2 mL of culture medium per well and 2×10 cells per well. 5 After DSF powder was dissolved in DMSO (dimethyl sulfoxide), it was added to a 12-well plate to a final concentration of 0, 0.025, 0.05, and 0.1 µM. The plate was cultured in a cell culture incubator. At the 72nd hour of culture, trypan blue staining was used and a hemocytometer was used to count the number of live and dead cells.

[0038] This example evaluates the physiological effects of DSF on multiple PEL cell lines, including BCBL1, BC3, and BCP1 cells. To provide a comparative reference, the KSHV-negative Burkitt lymphoma cell lines BJAB and DG75 were included.

[0039] like Figure 1 As shown, DSF effectively inhibited the proliferation of KSHV-positive B lymphoma cells, including BJAB cells latently infected with KSHV (BJAB-KSHV) and three PEL cell lines, showing higher sensitivity than BJAB and DG75 cells.

[0040] like Figure 2 As shown, DSF treatment resulted in a significant increase in the number of dead cells in BJAB-KSHV and PEL cells compared with DG75 and BJAB cells.

[0041] like Figure 3 and Figure 4As shown, the 50% inhibitory concentration (IC50) of DSF for BJAB-KSHV and PEL cells ranged from 0.04 μM to 0.06 μM, and the 50% inhibitory concentrations for BJAB and DG75 cells were 0.09 μM and 0.08 μM, respectively.

[0042] like Figure 5 and Figure 6 As shown, DSF also induced apoptosis in BJAB-KSHV and PEL cells in a dose-dependent manner, as evidenced by increased levels of cleaved caspase 3 (c-caspase 3). In contrast, KSHV-negative BJAB and DG75 cells showed greater resistance to DSF-induced apoptosis. Example 2 Study on the effect of DSF on KSHV lytic replication.

[0043] PEL cells were inoculated into 12-well cell culture plates with 2 mL of culture medium per well and 2×10 5 Sodium butyrate (an activator of KSHV lytic replication) powder was dissolved in DMSO (dimethyl sulfoxide) and added to a 12-well plate to a final concentration of 0.5 µM. DSF powder was dissolved in DMSO (dimethyl sulfoxide) and added to a 12-well plate to a final concentration of 0.1 µM. The plates were cultured in a cell culture incubator. At 72 hours of culture, the culture medium was centrifuged to obtain the cell precipitate. A portion of the culture medium was used to prepare protein samples for protein immunoblotting, and a portion of the culture medium was used to prepare RNA samples for fluorescent quantitative PCR.

[0044] Sodium butyrate (NaB) is a well-established KSHV lytic reactivation inducer that acts by inhibiting cellular deacetylases, leading to the accumulation and activation of KSHV-encoded lytic proteins, including RTA, a key regulator of KSHV reactivation. NaB induced a 20- to 1000-fold increase in the expression of KSHV lytic genes (including RTA, Pan RNA, ORFK8, ORF65, and ORF59) in BCBL1, BC3, and BCP1 cells, whereas DSF significantly inhibited this induction (e.g., Figure 7 In addition, NaB significantly increased ORFK8 protein expression, an effect that was largely reversed by DSF treatment in all three PEL cell lines (as shown in Figure 8 Immunofluorescence analysis further confirmed that DSF reduced the expression of ORFK8 protein in NaB-induced BCBL1 cells (as shown in Fig. 9 In line with these results, DSF also reduced the production of KSHV progeny virions induced by NaB in the supernatants of BCBL1, BC3, and BCP1 cells (as shown in Fig.10Together, these results suggest that DSF inhibits NaB-induced lytic reactivation of KSHV in PEL cells.

[0045] Example 3 Xenograft mouse tumor experiments.

[0046] The cultured Luc-BCBL1 cells were inoculated into the peritoneal cavity of NOD SCID mice, with 2×10 5 cells, and were randomly divided into 5 control groups and 5 DSF groups after injection. The first part of the experiment was carried out. The day of injection was recorded as Day 0. Starting from Day 1, the DSF group was injected once a day with an injection dose of 50 mg / kg. The inhibitor was dissolved in 5% DMSO + 50% PEG300 + 45% saline and was prepared and used immediately. The control group was injected with the same dose of 5% DMSO + 50% PEG300 + 45% saline. The body weight was weighed once every two days to observe the health status of the mice. On Day 8 and Day 15, live imaging was performed respectively. Each mouse was intraperitoneally injected with 50 mg / kg of D-luciferin. The growth process of the mouse abdominal tumor and the intensity of the fluorescence signal were photographed in the live imaging instrument. After the shooting was completed, the tumor fluorescence signal picture was saved and the fluorescence signal difference was analyzed. All mice were killed, and the ascites and heart, liver, spleen, lung and kidney were collected by dissection, and the organ photos were taken. The appearance of the organs was observed and the organs were removed and immersed in 10% formalin.

[0047] In this example, the effect of DSF on the survival of PEL in vivo was evaluated. A xenograft mouse model was established by inoculating BCBL1 cells stably expressing firefly luciferase (BCBL1-luc) into NOD-SCID mice. Treatment with DSF or vehicle was initiated by intraperitoneal injection on day 1 after implantation and continued daily. Notably, on day 8 after inoculation, 5 of 5 mice (100%) developed PEL, while in the DSF-treated group, 2 of 5 mice (40%) developed PEL (e.g. Fig.11 In addition, the bioluminescence signal intensity of DSF-treated mice on day 15 after inoculation was significantly lower than that of the control group (as shown in Fig.11 and Fig.12 The results showed that DSF effectively inhibited the occurrence and development of PEL in vivo.

[0048] Example 4 DSF inhibits KSHV reactivation in iSLK-BAC16-RGB cells.

[0049] To further demonstrate the inhibitory effect of DSF on KSHV lytic replication, the present invention uses iSLK- BAC16-RGB cells to study the inhibition of KSHV lytic replication by DSF. iSLK is a derivative of SLK cells that expresses doxycycline-inducible RTA encoded by KSHV. RTA is a major regulator of KSHV lytic reactivation by promoting the cascade expression of viral lytic genes. BAC16-RGB is a recombinant KSHV strain that expresses monomeric red fluorescent protein 1 (mRFP1) under the control of the elongation factor 1α (EF1α) promoter and is constitutively active in living cells. In addition, it expresses enhanced green fluorescent protein (EGFP) under the control of the KSHV lytic PAN RNA promoter. This dual fluorescence system is able to track KSHV latent and lytic phases.

[0050] like Fig.13 As shown, fluorescence microscopy showed that all iSLK-BAC16-RGB cells expressed mRFP1, while only a few cells expressed EGFP without induction, indicating that KSHV remained in tight latency in this system. DSF treatment did not significantly change the expression of mRFP1 and EGFP. However, simultaneous treatment with NaB and doxycycline (Dox) induced EGFP expression, and DSF treatment significantly reduced EGFP expression.

[0051] like Fig.14 As shown, flow cytometry analysis was used to further confirm that DSF reduced the proportion of EGFP-positive cells from approximately 56.5% to 37.2%. Under non-inducing conditions, less than 0.09% of cells expressed EGFP regardless of DSF treatment.

[0052] We then investigated whether DSF could regulate the transcriptional program of KSHV lytic genes. Fig.15 As shown in Figure 2, under non-inducing conditions, DSF treatment increased the transcription levels of KSHV lytic genes RTA, ORF59, and ORF57, while decreasing the expression of ORFK8, ORF65, and PAN RNA transcripts. This is consistent with the observations of EGFP expression and KSHV ORFK8 protein levels (Figure 2 Fig.16 The dual treatment of NaB and Dox significantly increased the transcription of KSHV lytic genes by up to 80,000 times, and DSF significantly reduced this effect (as shown in Fig.15 In addition, the induction of KSHV ORFK8 protein by NaB and Dox was consistently attenuated by DSF (as shown in Fig.15 and Fig.16 as shown).

[0053] To further determine whether DSF reduces the production of infectious KSHV virions, we first used qPCR to quantify the copy number of viral genomes released into the supernatant. Although viral genomes were barely detectable under normal conditions, the production of NaB- and Dox-induced KSHV viral progeny in the supernatant of iSLK-BAC16-RGB cells was significantly reduced after DSF treatment, regardless of DSF treatment (e.g., Fig.17 In addition, culture supernatants of iSLK-RGB-BAC16 cells were collected on day 4 after NaB and Dox induction and used to infect HEK293T cells, which are highly susceptible to KSHV infection. Cells expressing mRFP1 were tested to determine the relative titer of KSHV infectious virions (as shown in Fig.18 DSF treatment reduced the production of infectious virions by 5.3-fold (as shown in Fig.19 as shown).

[0054] Taken together, these results indicate that DSF effectively inhibits KSHV lytic replication in both PEL and endothelial cells.

[0055] Example 5 Studies on DSF-triggered innate immune responses.

[0056] Preparation of TBK1 knockout cell lines.

[0057] A single guide RNA (sgRNA) targeting TBK1 was designed using CRISPR tools (CRISPR / Cas9 system) (http: / / crispor.tefor.net) and cloned into the pLentiCRISPR-V2 vector. The sgTBK1 expression vector was then packaged into lentivirus and used to infect BC3 and BCP1 cells. 24 hours after infection, 2 μg / mL puromycin was used for 4 days of selection to eliminate uninfected cells. Surviving cells were then diluted into single cells and re-seeded into 96-well plates for clonal expansion.

[0058] To reveal whether DSF inhibits PEL progression by activating TBK1, 1x10 7 BCBL1-Luc wild-type or TBK1 knockout cells were injected intraperitoneally into individual NOD / SCID mice. One day after inoculation, mice received 50 or 25 mg / kg DSF or the corresponding vehicle. To visualize PEL tumors, D-luciferin (PerkinElmer, 122799) was injected intraperitoneally at a final concentration of 50 mg / kg and incubated for 8 minutes.

[0059] Examples 1-3 Studies have shown that DSF can effectively modulate the immune system of cancer cells. Given that DSF effectively prevented the reactivation of KSHV in PEL and endothelial cells, the inventors speculated that DSF may activate antiviral immune responses. TBK1 is a central node protein that plays a key role in antiviral innate immunity. To verify this hypothesis, the inventors evaluated the phosphorylation of TBK1 serine 172 (pTBK-S172) after DSF treatment, which indicates TBK1 activation. DSF significantly activated TBK1 in PEL cells (including BC3 and BCBL1) in a dose-dependent manner ( Fig. 20 As shown). Notably, DSF at a concentration of 0.05 μM was sufficient to induce TBK1 activation in BC3 and BCBL1 cells. In addition, DSF treatment activated TBK1 in BJAB-KSHV only at a concentration of 1 μM, indicating that BJAB-KSH V cells were less responsive than KSHV-positive PEL cells. In addition, TBK1 in BJAB cells was minimally affected by DSF in the concentration range of 0 to 1 μM. To further validate these results, the present invention examined the downstream effectors of TBK1 after DSF treatment. DSF treatment consistently upregulated the mRNA levels of IFNβ and interferon-stimulated genes (ISGs), including IFIT1, IFIT3, ISG15, and IRF7, in KSHV-positive BC3 and BCBL1 cells in a dose-dependent manner, but not in BJAB cells ( Fig.21 , 23 and 24). In addition, 1 μM DSF significantly increased the transcriptional expression of IFNβ and ISGs in BJAB-KSHV cells compared with other concentrations, which is consistent with TBK1 activation ( Fig. 22 Collectively, these findings suggest that DSF preferentially triggers antiviral innate immune pathways by activating TBK1 in KSHV-positive but not KSHV-negative lymphoma cells.

[0060] Example 6 DSF inhibits KSHV reactivation by triggering innate immune responses.

[0061] We investigated whether DSF inhibits KSHV reactivation in PEL cells by activating TBK1. We inhibited TBK1 by genomic disruption or pharmacological inhibition. As expected, TBK1 knockout or treatment with the potent TBK1 inhibitor GSK8612 effectively blocked DSF-induced TBK1 activation in BCBL1 and BC3 cells ( Fig.25 In addition, DSF treatment significantly increased the mRNA levels of IFNβ and ISGs, including IFIT1, IFIT3, ISG15, and IRF7, in BCBL1 and BC3 cells ( Fig.26TBK1 knockout or GSK8612 treatment significantly attenuated this increase ( Fig.26 Furthermore, TBK1 deficiency significantly enhanced NaB-induced KSHV reactivation, as evidenced by increased expression of KSHV lytic genes and increased viral progeny production ( Figure 27a-Figure 29 ). These results highlight the critical role of TBK1 in preventing KSHV lytic replication latency. Consistently, TBK1 knockdown or treatment with GSK8612 significantly reversed the DSF inhibition of NaB-induced transcription of KSHV lytic genes in BCBL1 and BC3 cells, including RTA, Pan RNA, ORFK8, ORF65, ORF59, and ORF57 ( Fig.27a and Figure 27b In addition, TBK1 knockout or GSK8612 treatment reversed the inhibitory effect of DSF on NaB-induced ORFK8 protein expression ( Fig.28 In addition, TBK1 knockdown or GSK8612 treatment also reduced NaB-induced KSHV viral progeny production after DSF treatment in BCBL1 and BC3 cells ( Fig.29 Collectively, these findings suggest that DSF inhibits lytic reactivation of KSHV in PEL cells by activating the innate immune response.

[0062] Example 7 DSF inhibits the proliferation, survival and progression of PEL by activating TBK1.

[0063] TBK1 knockout or GSK8612 treatment was used to verify that DSF inhibited the proliferation and survival of PEL by activating the innate immune response.

[0064] like Fig.30a and Fig.30b As shown, inhibition of TBK1 by knockdown and GSK8612 treatment attenuated the inhibitory effect of DSF on the proliferation of BCBL1 and BC3 cells. In addition, TBK1 knockdown or GSK8612 treatment significantly reduced the percentage of apoptotic cells and c-caspase3 protein levels induced by DSF in BCBL1 and BC3 cells ( Fig.31 and Fig.32 These results strongly suggest that DSF inhibits PEL proliferation and survival by mediating TBK1 activation.

[0065] Then, the in vivo dependence of DSF on TBK1 in inhibiting PEL cell survival was evaluated in a mouse model. To this end, a BCBL1-Luc cell line with stable TBK1 knockout was constructed (BCBL1-Luck-sgTBK1), and the bioluminescent signal of BCBL1-Luc wild-type cells was verified to be comparable to that of BCBL1-Luck-sgTBK1 cells, thus enabling the evaluation of the dependence of DSF on TBK1 in regulating PEL growth in vivo. Wild-type and TBK1 knockout BCBL1-Luc cells were transplanted into NOD-SCID mice, and vehicle or DSF treatment was started on the next day ( Fig.33 Consistently, all five mice (100%) in the vehicle control group developed PEL on day 22 post-inoculation, whereas three of five mice (60%) in the DSF-treated group developed PEL ( Fig.34 In contrast, all five mice (100%) harboring BCBL1-Luc-sgTBK1 cells developed PEL on day 22, even after DSF treatment ( Fig.34 In addition, DSF treatment resulted in a significant decrease in bioluminescence signal intensity compared with the control group, and TBK1 knockout reversed this effect ( Fig.34 The development of PEL often results in abnormal weight gain, in part due to the formation of ascites. DSF treatment reduced the weight gain observed in the control group, and TBK1 knockout reversed this effect ( Fig.35 Furthermore, DSF treatment significantly reduced the amount of ascites and spleen enlargement caused by PEL, and TBK1 deficiency abolished these effects ( Figure 36-Figure 39 ). Peritoneal and retroperitoneal solid tumor metastases were smaller after DSF treatment, but TBK1 knockout reversed this effect. Neither TBK1 knockout nor DSF treatment caused significant changes in organs such as the heart, liver, spleen, lung, or kidney, indicating that DSF is safe at these concentrations. Taken together, these results indicate that TBK1 mediates the inhibitory effects of DSF on PEL survival in vivo and in vitro. TBK1 deficiency abolished the inhibitory effects of DSF on PEL.

[0066] For matters not mentioned above, the prior art applies.

[0067] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make modifications or supplements to the described specific embodiments or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above implementations based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of disulfiram and its pharmaceutically acceptable salts in the preparation of drugs for treating Kaposi's sarcoma-associated herpes virus infection.

2. The use according to claim 1, characterized in that The disulfiram inhibits the lytic replication of Kaposi's sarcoma-associated herpes virus; The disulfiram inhibits the reactivation of Kaposi's sarcoma-associated herpes virus by triggering the innate immune response.

3. The use according to claim 1, characterized in that The disulfiram inhibits the production of Kaposi's sarcoma-associated herpes virus particles.

4. Use of disulfiram and its pharmaceutically acceptable salts in the preparation of drugs for treating KSHV-related lymphoma.

5. The use according to claim 4, characterized in that The KSHV-related lymphoma is primary effusion lymphoma.

6. The use according to claim 4, characterized in that The disulfiram induces cell death in Kaposi's sarcoma-associated herpesvirus latent primary effusion lymphoma cells; The disulfiram inhibits the proliferation and survival of primary effusion lymphoma cells by activating the innate immune response.

7. An anti-Kaposi's sarcoma-associated herpes virus pharmaceutical composition, characterized in that: Its active ingredient includes disulfiram.

8. The anti-Kaposi's sarcoma-associated herpes virus drug according to claim 7, characterized in that: Pharmaceutically acceptable excipients are also included.

9. A pharmaceutical composition for treating KSHV-related lymphoma, characterized in that: Its active ingredient includes disulfiram.

10. The anti-Kaposi's sarcoma-associated herpes virus drug according to claim 9, characterized in that: Pharmaceutically acceptable excipients are also included.

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