Application of EN219 in the preparation of drugs for preventing and treating AIDS

Abstract

This application relates to the field of biomedicine and discloses the use of EN219 in the preparation of drugs for preventing and treating AIDS. This application has confirmed that EN219 can promote the functions of innate immune cells, break through the immune escape strategy of HIV, and can inhibit the replication of HIV in immune cells. To a certain extent, it overcomes the drawback of high compliance of traditional AIDS treatment drugs, discovers a new host drug target, and has a brand-new application prospect.

Description

Technical Field

[0001] This application relates to the field of biomedicine. More specifically, it relates to the use of EN219 in the preparation of drugs for the prevention and treatment of AIDS. Background Art

[0002] For AIDS, the existing main treatment is antiretroviral therapy (ART), namely the so-called "cocktail therapy". The limitations of the cocktail therapy are mainly manifested in: 1. It is effective for early-stage AIDS patients, but it is of little help to mid- and late-stage patients because the immune systems of these patients have been irreversibly damaged by the HIV virus; 2. Multiple drugs are all targeted at key proteins in the HIV life cycle, and drug resistance is likely to occur, and the compliance of patients is relatively high; 3. The drug-taking method is complex and has side effects. Since the cocktail therapy is a mixture of various drugs, the size of the side effects depends on what specific drugs the patient is taking. One of the side effects is called fat loss, and the fat tissues on the patient's face and arms will be severely lost, causing great physical damage to the patient; 4. The drugs cannot clear the latent HIV virus reservoir. Even if only 0.001% of the virus remains after stopping the drug, the virus will make a comeback; 5. In addition, the different metabolic rates of the combination drugs will lead to complex pharmacodynamic / pharmacokinetic relationships of the drugs. Therefore, a large number of expensive clinical studies are required before the drugs are marketed, resulting in a high R & D cost for the drugs of the cocktail therapy.

[0003] To sum up, AIDS still cannot be completely cured clinically mainly because HIV integrates with the host genome and enters a long-term dormant state, forming latent infection and cannot be completely cleared by antiretroviral therapy (ART). Summary of the Invention

[0004] To solve the above technical problems, this application provides the use of EN219 in the preparation of drugs for the prevention and treatment of AIDS.

[0005] Through multidisciplinary research means such as immunology and biochemistry, the present invention analyzes the interaction between the viral antisense protein asp and the host protein TBK1 during the HIV-1 infection process, inhibits the production of type I IFN, and realizes the mechanism of immune escape to establish latent infection. Mechanistically, the binding of asp to the E3 ubiquitin ligase RNF114 promotes the recruitment of RNF114 to TBK1 and inhibits the phosphorylation of TBK1 by ubiquitinating TBK1, thereby affecting the activation of type I IFN. Based on the mechanism research, we found that the RNF114 inhibitor EN219 counteracts the ubiquitination of TBK1 by RNF114, thereby blocking the immune escape of HIV-1 and antagonizing infection, showing good prospects for the development of drugs for the treatment and prevention of AIDS.

[0006] Different from traditional anti-AIDS drugs, the present invention starts from the perspective of enhancing the antiviral ability of immune cells, similar to the "strengthening the healthy qi" strategy of traditional Chinese medicine treatment. It targets the host E3 ubiquitin ligase (RNF114), develops drugs from the perspective of the host, and targets the host targets manipulated by HIV, overcoming the disadvantages of easy mutation and drug resistance of the virus, and opening up new ideas for the preparation of drugs for the treatment or prevention of AIDS. If the drug development based on EN219 can be clinically verified and approved for marketing, it will enhance the international competitiveness of Chinese pharmaceutical companies and promote the healthy and vigorous development of China's pharmaceutical industry. More importantly, the development of new drugs will increase the cure rate, thereby reducing the medical expenses of patients and the pressure on the national finance, reducing the economic concerns and psychological burdens of HIV-infected patients, and maintaining social stability and promoting economic development.

[0007] In the first aspect, the present application provides the use of RNF114 as a target in the preparation of drugs for the prevention and / or treatment of AIDS.

[0008] In the second aspect, the present application provides the use of EN219 in the preparation of drugs for the prevention and / or treatment of AIDS.

[0009] Furthermore, the EN219 blocks the immune evasion of HIV-1 and antagonizes infection by counteracting the ubiquitination of TBK1 by RNF114.

[0010] In summary, the present application has the following beneficial effects:

[0011] 1. The EN219 of the present application can promote the function of innate immune cells and break the immune evasion strategy of HIV;

[0012] 2. The EN219 of the present application can inhibit the replication of HIV in immune cells;

[0013] 3. To a certain extent, the present application overcomes the disadvantage of high compliance of traditional AIDS treatment drugs, discovers a new host drug target, and has a brand-new application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : EN219 in THP-1 promotes the immune function of macrophages and inhibits the viral load of HIV;

[0015] ( Figure 1 -A shows the activation of the IFN signaling pathway after THP-1 is infected with HIV-1 and HIV-1△asp for 12 hours; Figure 1 -B shows the mRNA transcription levels of IFN and other inflammatory signaling factors after 12 hours of infection, reflecting the effect of EN219 on the innate immunity of the body);

[0016] Figure 2: EN219 promotes the immune function of macrophages and inhibits the viral load of HIV in RAW264.7;

[0017] ( Figure 2 -A shows the activation of the IFN signaling pathway after THP-1 cells were infected with HIV-1 and HIV-1Δasp for 12 hours; Figure 2 -B shows the mRNA transcription levels of IFN and other inflammatory signaling factors after 12 hours of infection, reflecting the effect of EN219 on the innate immunity of the body). Specific implementation manner

[0018] The technical solutions and effects of this application are further described in detail below in conjunction with the embodiments. It can be understood that the specific embodiments described herein are only used to explain the invention, rather than limiting the invention.

[0019] Example 1: EN219 promotes the immune function of macrophages and inhibits the viral load of HIV in THP-1 (human monocytes)

[0020] Source of materials: THP-1 and HEK293T (human embryonic kidney cells) were purchased from ATCC (American Type Culture Collection). TZM-bL cells were donated by the research group of Professor Xu Jianqing of Fudan University. HEK293T and TZM-bL cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 100 u / mL penicillin-streptomycin solution. THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 100 u / mL penicillin-streptomycin solution. HIV-1NL4.3-Δenv and VSVG plasmids were donated by Professor Sauter. EN219 was purchased from MCE Company (CAS No.: 380351-29-1). WB chemiluminescent substrate (SuperSignal West chemiluminescent substrates) was purchased from Vazyme Company; primary antibodies against STING, TBK1, P65, IRF3 and their phosphorylated antibodies were purchased from Cell Signaling Technology Company; GAPDH was purchased from Abclonal Company.

[0021] Model construction: The whole experiment was divided into three steps, including 1) coating of HIV-1 pseudovirus; 2) infection of THP-1 cells with HIV and drug treatment; 3) evaluation of immune function and viral load.

[0022] Experimental grouping: The experiment was divided into 6 groups, namely DMSO treatment group, EN219 treatment group, HIV-1 infection group, HIV-1Δasp infection group, HIV-1 infection + EN219 treatment group, and HIV-1Δasp infection + EN219 treatment group.

[0023] Implementation process:

[0024] 1) Coating of HIV-1 pseudovirus

[0025] HIV-1 and HIV-1Δasp were transfected with VSVG and Δenv or Δasp plasmids (VSVG:Δenv / Δasp = 1:2) in HEK293T cells, and the virus was harvested 48 hours after transfection. After the virus supernatant was filtered through a 0.45 μm filter, it was concentrated using PEG8000 (V900156, VETEC) according to the instructions.

[0026] Determination of HIV-1 virus titer by TCID50 method: Digest TZM-bl cells, perform cell counting, and seed the cells at a density of 1×10 4 per well in a 96-well plate. After the cells adhered, add the virus dilution. Add 3 replicates for each virus gradient, with a total of 8 gradients. Another 8 wells were used as negative control groups, and the same volume of medium was added. After 48 hours, take out the 96-well plate from the cell culture incubator, aspirate the liquid, wash it 2 times with PBS for 5 minutes each time, then add the lysis buffer, and shake it on a low-temperature shaker for 30 minutes. Collect the cell liquid in each well into a new 1.5 mL EP tube, centrifuge at 4°C, 16,000 rpm for 15 minutes, transfer the centrifuged supernatant to an enzyme-linked immunosorbent assay (ELISA) plate, and after shielding from light, use a multichannel pipette to add luciferase substrate to each well, then immediately place it in an ELISA reader and select the "Luciferase" program to read the luminescence value of the sample. Finally, the Reed-Muench method was used to calculate the virus titer, and the calculation formula was lgTCID50 = distance ratio × difference between dilution logarithms + logarithm of the dilution with a lesion rate higher than 50%.

[0027] 2) HIV infection of THP-1 cells and drug treatment

[0028] Seed the cultured THP-1 cells at a density of 2×10 6 per well in a 6-well plate, replace the DMEM culture medium (serum-free) and perform starvation treatment for 12 hours, and infect with HIV-1 pseudovirus at an MOI of 10 for 12 hours. After the infection is completed, add EN219 (50 mmol) and treat for 1 hour, then extract cell proteins and RNA.

[0029] Collection of protein samples: Add 200 μl of cell lysate (Beyotime, P0013, add PMSF, cocktail, NaF and sodium orthovanadate to inhibit the degradation of phosphorylated proteins), lyse at 4 °C for 40 minutes, pipette and transfer to a new EP tube, centrifuge at 13200 r, 4 °C for 15 minutes. Take 150 μl of the supernatant, add loading buffer, boil the sample at 100 °C for 10 minutes.

[0030] Collection of RNA samples:

[0031] a. Add 1 ml of TRIzol (all subsequent EP tubes, pipettes, etc. are RNAase free), pipette and mix well, then transfer to a new EP tube.

[0032] b. Add 200 μl of chloroform, cover the tube cap, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 12000 r for 15 minutes, and the sample will be divided into three layers. Take 400 μl of the upper aqueous phase, add 400 μl of isopropanol, let stand at room temperature for 10 minutes, centrifuge at 10000 r for 10 minutes, and discard the supernatant.

[0033] c. Wash the RNA precipitate with 75% ethanol, centrifuge at 10000 r for 5 minutes, discard the supernatant, and repeat 3 times.

[0034] d. Let the RNA precipitate dry at room temperature, add an appropriate amount of RNAase-free water, pipette a few times with a pipette tip to dissolve the RNA, and detect the RNA concentration and purity.

[0035] e. Reverse transcription: Use the HiScript IV RT SuperMix for qPCR (R423-01) kit from Vazyme company, and reverse transcribe into cDNA according to the kit instructions.

[0036] 3) Evaluation of immune function and viral load

[0037] Evaluate the activation status of the immune signaling pathway by Western blot

[0038] a. Electrophoresis: Stacking gel at 80 mV for 30 min; Separating gel at 110 - 120 mV for 80 min.

[0039] b. Transfer: Cut a 4 cm * 8 cm PVDF membrane, activate it in methanol in advance, install the electrotransfer clip in the order of black clip - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - white clip, set the transfer time at 100 V, 1 KD / min.

[0040] c. Blocking: Block with 5% skim milk for 1 h, wash three times with TBST (conventional membrane washing solution), 5 min each time.

[0041] d. Incubate with primary antibody: Incubate with primary antibody overnight at 4°C respectively.

[0042] e. Wash three times with TBST, 5 min each time, incubate with secondary antibody (the secondary antibody binds to the primary antibody and has an enzyme that can react with the substrate in the luminescent solution), for 60 min, then wash three times with TBST, 5 min each time.

[0043] f. Develop the image.

[0044] qPCR was used to evaluate the transcriptional levels of cytokines and viral loads

[0045] For qPCR, load the sample according to the system of 0.2 μl of upstream and downstream primers + 1 μl of cDNA + 3.6 μl of ddH2O + 5 μl of 2Xsybr. The reagent used is ChamQ Blue Universal SYBR qPCR Master Mix from Vazyme Company, and the instrument used is Roche 480. Export the data.

[0046] The statistical analysis involved in the present invention was performed using GraphPad Prism statistical software. The t-test was used for comparison of means between two groups, and one-way ANOVA was used for comparison of means among multiple groups. P < 0.05 was considered to have statistical significance.

[0047] Example 2: EN219 promotes the immune function of macrophages and inhibits the viral load of HIV in RAW264.7

[0048] Except that the infected cells (RAW264.7) in Example 2 are different from those in Example 1 (THP-1), other treatment methods are the same as those in Example 1, which proves that EN219 has the effect of activating the innate immunity of the body and inhibiting HIV infection in cells from different sources.

[0049] This specific embodiment is only an explanation of the present application and does not limit the present application. Those skilled in the art can make modifications without creative contributions to this embodiment as needed after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. Use of EN219 in the preparation of drugs for preventing and / or treating AIDS.

2. The use according to claim 1, wherein EN219 blocks the immune escape of HIV-1 and antagonizes infection by counteracting the ubiquitination of TBK1 by RNF114.

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