Use of en219 in preparation of drug for preventing and treating aids

Abstract

The use of EN219 in the preparation of a drug for preventing and treating AIDS. The present application confirms that EN219 can promote the function of innate immune cells, disrupt the immune escape strategy of HIV, and inhibit the replication of HIV in immune cells. The present application overcomes, to a certain extent, the drawback of high medication adherence required by traditional AIDS treatment drugs, discovers a new host drug target, and has brand-new application prospects.

Description

Application of EN219 in preparation of drugs for preventing and treating AIDS TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, more specifically, it relates to an application of EN219 in preparation of drugs for preventing and treating AIDS. BACKGROUND

[0002] For AIDS, the existing main therapy is the anti-retroviral therapy (ART) therapy, that is, the commonly said "cocktail therapy". The limitations of the cocktail therapy mainly manifest in the following aspects: 1. effective for early AIDS patients, but little help for middle and late stage patients, because the immune systems of these patients have been irreversibly destroyed by the HIV; 2. various drugs are all aimed at the key proteins in the life cycle of HIV, and are easy to produce drug resistance, and the compliance of patients is high; 3. the drug taking method is complex, and has side effects, since the cocktail therapy is a mixed taking of various drugs, so the size of the side effects depends on what specific drug the patient takes, and one of the side effects is called fat loss, the fat tissues of the face and arms of the patient will be seriously lost, causing great physical damage to the patient; 4. the drug cannot clear the latent HIV virus bank, and after the drug is stopped, even if there is still 0.001% of the virus, the virus will come back; 5. in addition, the different metabolic rates of the combined drugs will lead to complex pharmacodynamics / pharmacokinetics of the drugs, so a large amount of expensive clinical research is needed before the drug is put on the market, resulting in a high cost of the drug development of the cocktail therapy.

[0003] To sum up, AIDS in the clinic still cannot be completely cured, mainly because HIV integrates with the host genome and enters a long dormant state, forming latent infection, which cannot be completely cleared by the anti-retroviral therapy (ART). SUMMARY

[0004] In order to solve the above technical problems, the present application provides an application of EN219 in preparation of drugs for preventing and treating AIDS.

[0005] The present application analyzes the interaction between the viral antisense protein asp and the host protein TBK1 in the HIV-1 infection process through immunological, biochemical and other multidisciplinary research means, inhibits the production of type I IFN, and realizes the mechanism of immune escape and establishment of latent infection. Mechanistically, the combination of asp and 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-based research, we found that the RNF114 inhibitor EN219 counteracts the ubiquitination of TBK1 by RNF114, thereby blocking the immune evasion of HIV-1 and antagonizing the infection, showing a good development prospect for AIDS treatment and prevention drugs.

[0006] Unlike traditional anti-AIDS drugs, the present application cuts in from the perspective of enhancing the ability of immune cells to resist viruses, similar to the "supporting health" strategy of traditional Chinese medicine treatment, aiming at the host E3 ubiquitin ligase (RNF114), starting from the perspective of the host, developing drugs for HIV-manipulated host targets, overcoming the disadvantages of easy mutation and drug resistance of viruses, opening up new ideas for the preparation of drugs for treating or preventing 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 improve the cure rate, which will reduce the patient's medical expenses and the pressure on the state treasury, reduce the economic worries and psychological burden of HIV-infected patients, maintain social stability and promote economic development.

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

[0008] In a second aspect, the present application provides the use of EN219 in the preparation of a drug for preventing and / or treating AIDS.

[0009] Further, 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 crack 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. The present application overcomes the high compliance of traditional AIDS treatment drugs to a certain extent, discovers a new host drug target, and has a completely new application prospect. BRIEF DESCRIPTION OF DRAWINGS

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

[0015] (Figure 1-A shows the activation of the IFN signaling pathway after 12 hours of HIV-1 and HIV-1 Δasp infection in THP-1; Figure 1-B shows the mRNA transcription level of IFN and other inflammatory signal factors after 12 hours of infection, reflecting the effect of EN219 on the body's innate immunity);

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

[0017] Figure 2-A shows the activation of IFN signaling pathway after 12 hours of HIV-1 and HIV-1 Δasp infection of THP-1; Figure 2-B shows the mRNA transcription level of IFN and other inflammatory signaling factors after 12 hours of infection, reflecting the effect of EN219 on the body's innate immunity. DETAILED DESCRIPTION

[0018] The technical solutions and effects of the present application are further described in detail below in conjunction with examples. It can be understood that the specific examples described herein are only used to explain the present application, and not to limit the present application.

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

[0020] Material source: THP-1 and HEK293T (human embryonic kidney cells) were purchased from ATCC (American Type Culture Collection). TZM-bL cells were donated by Professor Xu Jianqing's research group of Fudan University. HEK293T and TZM-bL cells were cultured in DMEM medium with the addition of 10% inactivated fetal bovine serum and 100 u / mL penicillin streptomycin solution. THP-1 was cultured in RPMI-1640 medium with the addition of 10% inactivated fetal bovine serum and 100 u / mL penicillin streptomycin solution. HIV-1 NL4.3-Δenv and VSVG plasmids were donated by Professor Sauter. EN219 was purchased from MCE company (CAS No.: 380351-29-1). WB chemiluminescent substrates (Super Signal West chemiluminescent substrates) were purchased from Vazyme company; primary antibodies 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 entire experiment was divided into three steps, including 1) coating of HIV-1 pseudovirus; 2) HIV infection of THP-1 cells and drug treatment; 3) evaluation of immune function and viral load.

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

[0023] Procedure:

[0024] 1)Pseudovirus packaging

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

[0026] TCID50 method for HIV-1 virus titer: TZM-b1 cells were digested and counted, and plated according to 1 x 10 4 cells per well in 96-well plates. After the cells adhered, virus dilutions were added. Each virus gradient was added in triplicate wells, for a total of 8 gradients. Another 8 wells were used as negative controls and were added with the same volume of medium. After 48 hours, the 96-well plates were removed from the cell incubator, the liquid was aspirated, and washed twice with PBS for 5 minutes each time. Lysis solution was then added, and the plates were shaken on a low-temperature shaker for 30 minutes. The cell liquid from each well was collected into a new 1.5 mL EP tube, centrifuged at 4°C, 16,000 rpm for 15 minutes, and the supernatant was transferred to an enzyme-labeled plate. After being protected from light, the luciferase substrate was added to each well using a dispenser, and the plate was immediately placed in a luminometer and the "Luciferase" program was selected to read the luminescence value of the sample. Finally, the Reed-Muench method was used to calculate the virus titer, and the formula was lgTCID50 = distance ratio x difference between dilution logarithm + logarithm of dilution higher than 50% lesion rate.

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

[0028] The cultured THP-1 cells were plated according to 2 x 10 6 cells per well in a 6-well plate, the DMEM medium (without serum) was replaced and starved for 12 hours, and HIV-1 pseudovirus was added according to MOI = 10 for 12 hours. After the infection was completed, EN219 (50 mmol) was added for 1 hour, and then cell protein and RNA were extracted.

[0029] Collection of protein samples: 200 ul of cell lysis solution (Bi Yun Tian, P0013, with PMSF, cocktail, NaF and sodium metavanadate to inhibit the degradation of phosphorylated proteins) was added, 4°C lysis for 40 minutes, blown and transferred to a new ep tube, 13,200 r, 4°C centrifugation for 15 minutes. 150 ul of supernatant was taken, loading was added, and the sample was boiled at 100°C for 10 minutes.

[0030] Collection of RNA samples:

[0031] a. Add 1 ml TRIZOL (all EP tubes, head washing, etc. are RNAase free), mix well by blowing, and then transfer to a new EP tube.

[0032] b. Add 200 ul chloroform, cover the tube cap, shake vigorously for 15 seconds, and stand at room temperature for 5 minutes. Centrifuge at 12000 r for 15 minutes. The sample will be divided into three layers. Take 400 ul of the upper aqueous phase, add 400 ul of isopropanol, stand at room temperature for 10 minutes, and centrifuge at 10000 r for 10 minutes. 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. Stand at room temperature to dry the RNA precipitate. Add an appropriate amount of RNAase-free water, and dissolve the RNA by sucking and beating several times with a gun head. Detect the RNA concentration and purity.

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

[0036] 3) Immune function and viral load evaluation

[0037] Western blot evaluation of immune signal pathway activation state

[0038] a. Electrophoresis: concentrated gel 80 mV, 30 min; separation gel 110-120 mV, 80 min.

[0039] b. Membrane transfer: cut 4 cm*8 cm PVDF membrane, activate in methanol in advance, and install the membrane transfer clamp in the order of black clamp-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-white clamp, 100 V, 1 KD / min, and set the membrane transfer time.

[0040] c. Blocking: 5% skimmed milk blocking for 1 h, and TBST (regular membrane washing solution) washing for 3 times, 5 min each time.

[0041] d. Incubate the primary antibody: incubate the primary antibody at 4°C overnight, respectively.

[0042] e. TBST washing for 3 times, 5 min each time, incubate the secondary antibody (the secondary antibody is the antibody combined with the primary antibody, which has an enzyme that can react with the substrate in the luminescent solution), 60 min, TBST washing for 3 times, 5 min each time.

[0043] f. Development.

[0044] qPCR assesses cytokine transcription level and viral load

[0045] qPCR, according to the system of 0.2ul upstream and downstream primers + 1ul cDNA + 3.6ul ddH2O + 5ul 2Xsybr, using reagents ChamQ Blue Universal SYBR qPCR Master Mix of Vazyme Company, using instrument Roche 480, exporting data.

[0046] The statistical analysis involved in the present application is carried out by using GraphPad Prism statistical software, t-test is used for comparing two group means, single factor analysis of variance is used for comparing multiple group means, and P<0.05 is considered to have statistical difference.

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

[0048] Example 2 is the same as example 1 except that the infected cells (RAW264.7) are different from example 1 (THP-1), which proves that EN219 has the effect of activating the body's natural immunity to inhibit HIV infection in different source cells.

[0049] The specific embodiments are only an explanation of the present application, and are not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution according to the needs after reading the present 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. Application of RNF114 as a target in preparation of a drug for preventing and / or treating AIDS.

2. Application of EN219 in preparation of a drug for preventing and / or treating AIDS.

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

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