Use of phd3 inhibitors in the prevention and treatment of aids

Abstract

The application discloses application of a PHD3 inhibitor in prevention and treatment of AIDS. Through immunological, biochemical and other multidisciplinary research means, the application analyzes the mechanism that a viral antisense protein ASP inhibits production of type I IFN to realize immune escape and establish latent infection in an HIV-1 infection process. Mechanically, after being expressed into a protein in a cell in the body, the ASP needs to undergo post-translational modification to exert its inhibiting effect, and after hydroxylation modification of a 47-position proline, the ASP can help to realize the ability of inhibiting the body immune response, and the post-translational modification depends on a host proline hydroxylase 3 (PHD3) to realize. Based on the mechanism-based research, it is found that a PHD3 inhibitor Molidustat offsets the hydroxylation of the ASP by PHD3, thereby blocking the immune evasion of HIV-1 and antagonizing infection, and showing a good development prospect of an AIDS treatment and prevention drug.

Description

TECHNICAL FIELD

[0001] The application relates to application of a PHD3 inhibitor in prevention and treatment of AIDS and belongs to the technical field of biological medicine. BACKGROUND

[0002] At present, the main treatment method for AIDS (AIDS) is high-efficiency anti-retrovirus therapy (ART), commonly known as "cocktail therapy". The therapy targets key proteins in the HIV life cycle to inhibit virus replication by using a plurality of anti-virus drugs in combination. In recent years, a plurality of pre-exposure (PrEP) and post-exposure (PEP) blocking drugs for AIDS have been developed in China, such as domestic generic drugs based on emtricitabine, tenofovir and lamivudine, which have improved the prevention and early intervention ability of AIDS to a certain extent. However, whether ART or blocking drugs, there are a series of technical bottlenecks to be solved in clinical application:

[0003] 1. Strong dependence on disease progression: ART is mainly effective for patients in the early stage of infection, and for patients who have entered the middle and late stages, the immune system is often irreversibly damaged by HIV, and the curative effect is limited;

[0004] 2. Virus is easy to produce drug resistance: whether ART or existing domestic blocking drugs, the action target is mostly concentrated in the key links of HIV reverse transcription, integration or protease, and the virus is easy to produce mutations to form drug-resistant strains under long-term selection pressure, resulting in decreased curative effect;

[0005] 3. High requirement for patient compliance and obvious side effects: ART and a plurality of blocking drugs need to be taken regularly, and the treatment scheme is complex, and long-term use may cause a series of side effects, such as fat malnutrition, liver and kidney toxicity, and bone density decrease. Among them, some domestic blocking drugs have the problems of fixed composition ratio and insufficient response to high-risk exposure situations;

[0006] 4. Cannot clear the latent virus library: existing drugs are difficult to completely clear the HIV virus library latent in CD4 + T cells and other host cells, and once the drug is stopped, even if only a trace amount of virus remains in the body, the virus may also be reactivated.

[0007] 5. Complex pharmacokinetics, high development threshold: the plurality of drugs in the cocktail therapy have different metabolic rates and drug efficacy durations, increasing the complexity of the design of the drug administration scheme, and making it difficult to predict the interaction between drugs, which urgently needs to rely on a large number of clinical trials for verification, resulting in long development cycle and high cost. Some domestic blocking drugs still have room for improvement in pharmacokinetic adaptability, oral bioavailability and targeted distribution;

[0008] 6. Limited use range: The current domestic blocking drugs are mainly used for short-term prevention of high-risk HIV groups, and there is still a lack of effective intervention means for early viral clearance or immune recovery support after infection.

[0009] In summary, the current AIDS treatment and prevention field urgently needs to develop innovative treatment strategies with new mechanisms, low drug resistance, broad-spectrum antiviral, easy to use, and can intervene the latent viral reservoir, to further improve the clinical intervention effect and the quality of life of patients. SUMMARY

[0010] The purpose of the present application is: in view of the deficiencies of the existing drugs for treating AIDS, the present application provides the application of PHD3 inhibitor in preventing and treating AIDS.

[0011] In order to achieve the above purpose, the present application provides the application of PHD3 inhibitor in preparing a drug for preventing and treating AIDS.

[0012] Preferably, the PHD3 inhibitor comprises Molidustat, CAS No.: 1154028-82-6.

[0013] Preferably, the drug comprises an effective component and a pharmaceutically acceptable carrier or excipient, and the effective component comprises the PHD3 inhibitor.

[0014] Preferably, the dosage form of the drug comprises injection, tablet, powder, suspension, capsule, pill or syrup.

[0015] Unlike traditional anti-AIDS drugs, the present application starts from the perspective of enhancing the anti-viral ability of immune cells, similar to the traditional Chinese medicine treatment of "supporting health", aiming at PHD3, starting from the perspective of the host, by analyzing the key molecular mechanism of targeting or regulating HIV latency, developing anti-AIDS drug PHD3 inhibitor aiming at HIV manipulated host target, the drug can effectively intervene the latency or activation process of virus in host cells; significantly enhance the host anti-viral immune response, improve the recognition and clearance ability of HIV in the early stage of infection and even in the latent period; reduce the risk of drug resistance and systemic toxicity, improve the safety and compliance of long-term use of patients; have good potential for combined use with existing antiviral drugs, enhance the comprehensive treatment effect without increasing the complexity of pharmacokinetics.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The PHD3 inhibitor Molidustat offsets the hydroxylation of ASP by PHD3, thereby blocking the immune evasion of HIV-1 and antagonizing infection, promoting the function of innate immune cells, and breaking the immune evasion strategy of HIV; the drug to some extent overcomes the drawbacks of high compliance of traditional AIDS treatment drugs and easy mutation and drug resistance of viruses, and provides a new host drug target for preventing and treating AIDS, and presents good prospects for drug development and clinical application. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figures a, b, c and d show that Molidustat promotes the immune function of cells and inhibits the viral load of HIV in MDM cells (human monocyte-derived macrophages) and Jurkat (human CD4+ T cells) in an embodiment of the present application; wherein, figure a shows the activation of the IFN signaling pathway after 12 hours of HIV-1 and HIV-1 ASP P47A (ASP 47 proline mutation, which can cause loss of function of ASP) pseudovirus infection using MDM; figure b shows the mRNA transcription level of IFN and other inflammatory signaling factors and viral load after 24h of infection, reflecting the antiviral effect of Molidustat in macrophages; figure c shows the mRNA transcription level of IFN and other inflammatory signaling factors and viral load after 24 hours of HIV-1 and HIV-1 ASP P47A infection using MDM, reflecting the antiviral effect of Molidustat in T cells; figure d shows the mRNA transcription level of IFN and other inflammatory signaling factors and viral load after 24 hours of HIV-1 and HIV-1 ASP P47A true virus infection using MDM, reflecting the antiviral effect of Molidustat in macrophages;

[0019] Figure 2 Figures a, b and c show that Molidustat promotes the antiviral function of immune cells in humanized mice (hu-PBL mice) in an embodiment of the present application; wherein, figure a shows a mode diagram of HIV-1 and HIV-1 △asp infection using humanized mice and Molidustat treatment; figure b shows the mRNA transcription level of IFN and IFN B and HIV-1 tat in the spleen, liver and rectum of the mice after a certain time of Molidutat treatment, and the cytokine antigen level in the blood, reflecting the influence of Molidustat on the antiviral immune response of the body; figure c shows the viral load (green fluorescence) of HIV in the spleen of the mice after a certain time of Molidutat treatment. DETAILED DESCRIPTION

[0020] The technical solutions and effects of the present application are further described in detail below in conjunction with the embodiments. It can be understood that the specific embodiments described herein are merely intended to explain the present application and not to limit the present application.

[0021] The experimental methods not specified in the following examples are generally carried out according to the conventional conditions or according to the conditions recommended by the manufacturers. The materials, reagents, etc. used in the following examples are commercially available reagents and materials unless otherwise specified.

[0022] Example 1: Molidustat promotes immune function of macrophages and T cells and inhibits viral load of HIV

[0023] Material source: Jurkat cells (human peripheral blood leukemia T cells) and HEK293T cells were purchased from ATCC (American Type Culture Collection). TZM-bL cells were donated by Professor Xu Jianqing's research group of Fudan University. MDMs (human peripheral blood macrophages) were collected from peripheral blood of healthy volunteers recruited by the laboratory and met the human ethics regulations. MDMs and Jurkat cells were cultured with RAPI-1640 medium supplemented with 10% inactivated fetal bovine serum and 100 U / mL penicillin-streptomycin solution. TZM-bL and HEK293T were cultured with DMEM medium supplemented with 10% inactivated fetal bovine serum and 100 U / mL penicillin-streptomycin solution. HIV-1 NL4.3-Δenv and VSVG plasmids and pnL 4-3 infectious clone were donated by Professor Sauter. Molidustat was purchased from MCE company (CAS No.: 1154028-82-6). WB chemiluminescent substrate (Super Signal West chemiluminescent substrates) was purchased from Vazyme company; primary antibodies TBK1, IRF3, P38, P65 and their phosphorylated antibodies were purchased from Cell Signaling Technology company; GAPDH was purchased from Abclonal company.

[0024] Model construction: The entire experiment was divided into three steps, including 1) HIV-1 virus coating; 2) HIV infection and drug treatment; 3) immune function and viral load evaluation.

[0025] Experimental grouping: The experiment was divided into 6 groups, DMSO treatment group, Molidustat treatment group, HIV-1 infection group, HIV-1 ASP P47A infection group, HIV-1 infection + Molidustat treatment group, HIV-1 ASP P47A infection + Molidustat treatment group.

[0026] Implementation process:

[0027] 1) HIV-1 virus and viral packaging

[0028] Pseudovirus packaging: HIV-1 and HIV-1 ASP P47A were transfected in HEK293T cells with VSVG and Δenv or Δenv P47A plasmids (according to VSVG: Δenv / Δenv P47A = 1:2), and harvested at 48 hours post-transfection. The viral supernatant was filtered through a 0.45 μm filter and concentrated using PEG8000 (V900156, VETEC) according to the instructions.

[0029] True virus packaging: HIV-1 and HIV-1 ASP P47A were transfected in HEK293T cells with pnL 4-3 or pnL4-3 ASP P47A plasmids, and harvested at 48 hours post-transfection. The viral supernatant was filtered through a 0.45 μm filter and stored in the BSL-3 (Biological Safety Level 3) laboratory of Fudan University.

[0030] TCID50 method for determining HIV-1 virus titer: TZM-bl cells were digested and counted, and plated at a cell amount of 1 x 10 4 per well of a 96-well plate. After the cells adhered, the virus dilution was added. Each virus gradient had 3 replicate wells, for a total of 8 gradients. Another 8 wells were used as negative controls and the same volume of medium was added. After 48 hours, the 96-well plate was removed from the cell incubator, the liquid was aspirated, and washed twice with PBS for 5 minutes each time. Then, lysis solution was added, and 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 after centrifugation was transferred to an enzyme-labeled plate. After being protected from light, the luciferase substrate was added to each well using a dispensing gun, and immediately placed in an enzyme-labeled instrument to read the luminescence value of the sample by selecting the “Luciferase” program. Finally, the Reed-Muench method was used to calculate the virus titer, with the formula being lgTCID50 = distance ratio x difference between dilution logarithm + logarithm of dilution above 50% lesion rate.

[0031] 2) HIV infection of MDM cells and Jurkat cells and drug treatment

[0032] The cultured cells were plated at a cell amount of 2 x 10 6 per well of a 6-well plate, the DMEM culture medium (without serum) was replaced for 12 hours of starvation treatment, and HIV-1 pseudovirus infection was performed at an MOI of 10 for 12 hours (24 hours for true virus). After the infection was completed, Molidustat (5 μmol) was added for 20 minutes of treatment, and then cell protein and RNA were extracted.

[0033] Collection of protein sample: add 200 μl cell lysate (Bi Yun Tian, P0013, add PMSF, cocktail, NaF and sodium metavanadate to inhibit degradation of phosphorylated proteins), lyse at 4°C for 40 minutes, transfer to a new EP tube by blowing, centrifuge at 13200 r / min for 15 minutes at 4°C. Take 150 μl supernatant, add loading, and boil the sample at 100°C for 10 minutes.

[0034] Collection of RNA sample:

[0035] a. Add 1 ml TRIZOL (all EP tubes, washing heads, etc. are RNAase free thereafter), mix well by blowing and transfer to a new EP tube.

[0036] b. Add 200 μl chloroform, cover the tube cap, shake vigorously for 15 seconds, and stand at room temperature for 5 minutes. Centrifuge at 12000 r / min for 15 minutes, the sample will be divided into three layers, take 400 μl of the upper aqueous phase, add 400 μl of isopropanol, stand at room temperature for 10 minutes, centrifuge at 10000 r / min for 10 minutes, discard the supernatant.

[0037] c. Wash the RNA precipitate with 75% ethanol, centrifuge at 10000 r / min for 5 minutes, discard the supernatant, repeat 3 times.

[0038] d. Dry the RNA precipitate at room temperature, 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.

[0039] 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.

[0040] Evaluation of immune function and viral load

[0041] Western blot evaluation of immune signal pathway activation state

[0042] a. Electrophoresis: concentrated gel 80 mV, 30 min; separation gel 110-120 mV, 80 min.

[0043] b. Membrane transfer: cut 4 cm*8 cm PVDF membrane, activate in methanol in advance, install the membrane transfer clamp in the order of black clamp-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-white clamp, set the membrane transfer time at 100 V and 1 KD / min.

[0044] c. Blocking: block with 5% skimmed milk for 1 h, wash with TBST (regular membrane washing solution) for 3 times, 5 min each time.

[0045] d. Incubation with primary antibody: Incubate with primary antibody overnight at 4°C.

[0046] e. Wash three times with TBST for 5 minutes each time, incubate with secondary antibody (the secondary antibody is an antibody that binds to the primary antibody and carries an enzyme that can react with the substrate in the luminescent solution) for 60 minutes, then wash three times with TBST for 5 minutes each time.

[0047] f. Development.

[0048] qPCR assessment of cytokine transcription levels and viral load

[0049] qPCR was performed using a mixture of 0.2 μl upstream and downstream primers + 1 μl cDNA + 3.6 μl ddH2O + 5 μl 2X sybr. The reagent used was Vazyme's ChamQ Blue Universal SYBR qPCR Master Mix, and the instrument used was a Roche 480. Data were then exported.

[0050] The statistical analysis involved in this invention was performed using GraphPad Prism statistical software. The t-test was used to compare the means of two groups, and the one-way ANOVA was used to compare the means of multiple groups. P < 0.05 was considered to be statistically significant.

[0051] Experimental results are as follows Figure 1 As shown, Figure 1 Molidustat was shown to promote cellular immune function and suppress HIV viral load in MDM cells (human monocyte-derived macrophages) and Jurkat (human CD4+ T cells); among which, Figure 1 a shows the activation of the IFN signaling pathway 12 hours after infection with HIV-1 and HIV-1ASP P47A (ASP 47 proline mutation, which can lead to loss of ASP function) pseudoviruses using MDM; Figure 1 b shows the mRNA transcription levels of IFN and other inflammatory signaling factors and viral load 24 h after infection, reflecting the antiviral effect of Molidustat in macrophages; Figure 1 c. The mRNA transcription levels and viral load of IFNG and other inflammatory signaling factors were measured 24 hours after MDM infection with HIV-1 and HIV-1ASP P47A to reflect the antiviral effect of Molidustat in T cells. Figure 1 d shows the mRNA transcription levels of IFN and other inflammatory signaling factors and viral load 24 hours after infection with HIV-1 and HIV-1ASP P47A evovirus using MDM, reflecting the antiviral effect of Molidustat in macrophages.

[0052] Example 2: Molidustat promotes immune function and inhibits HIV viral load in humanized mice.

[0053] Materials sourced from: C-NKG (severely immunodeficient mice) were purchased from Cyagen Biosciences. PBMCs (human peripheral blood mononuclear cells) were obtained by isolating peripheral blood from recruited volunteers, and human medical ethics were approved. The coating of HIV-1 evovirus was consistent with that in Example 1. HIV-1 P24, Human IFN-γ, and IFN-β antigen ELISA kits were purchased from Enzyme-Linked Biotechnology Co., Ltd. CD45, CD3, CD64, and HIV-1 P24 antibodies were purchased from Abcam Biotechnology Co., Ltd.

[0054] Model construction: The entire experiment was divided into three steps, including 1) construction of humanized mice; 2) HIV infection of humanized mice and drug treatment; 3) assessment of immune function and viral load.

[0055] Experimental groups: The experiment was divided into 4 groups: DMEM treatment group, DMEM + Molidustat treatment group, HIV-1 infection + DMSO treatment group, and HIV-1 infection + Molidustat control group.

[0056] Implementation process:

[0057] 1) Construction of humanized mice

[0058] 5 × 10^5 cells were obtained from the peripheral blood leukocyte layer of healthy donors by Ficoll density gradient centrifugation. 6 Peripheral blood mononuclear cells (PBMCs) were injected intraperitoneally (ip) 200 μL into C-NKG mice using a 1 mL syringe and a 25-gauge needle. Immune cell differentiation was assessed 14 days post-transplantation. PBMCs were obtained by collecting 100 μL of blood via retroorbital sampling, separated by Ficoll density gradient centrifugation, stained with fluorescently labeled anti-human CD45 antibody, and analyzed by flow cytometry to confirm successful model establishment.

[0059] 2) HIV infection of humanized mice and drug treatment

[0060] Mice with stable human leukocyte reconstitution were infected via intraperitoneal injection (ip), with each mouse receiving 5 ng of p24 HIV-1 virus. Mice infected with DMEM served as controls. Ten days after infection, mice were treated with a small molecule drug, molidustat, via intraperitoneal injection (0.5 mg / kg). DMSO was administered intraperitoneally as a control, and the treatment continued for 14 days. The entire infection experiment was conducted in the P3 biosafety laboratory (BSL-3) at Fudan University.

[0061] 3) Assessment of immune function and viral load

[0062] After processing, mice were anesthetized and euthanized according to animal ethics guidelines. Venous blood, spleen, liver, and rectum were collected for subsequent serum separation, organ RNA extraction, and fluorescent immunohistochemistry. RNA sample extraction and qPCR detection were performed as described in Example 1. ELISA detection was performed according to the kit instructions.

[0063] Spleen multiplex immunofluorescence:

[0064] Spleen tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 5 μm. After dewaxing and rehydration, the sections were subjected to antigen retrieval in citrate buffer (pH 6.0) at 95°C for 20 min. They were then blocked with 5% BSA and incubated overnight at 4°C with primary antibodies against human CD3, CD68, and HIV-1P24, followed by incubation with corresponding fluorescently labeled secondary antibodies. Cell nuclei were counterstained with DAPI. After mounting, images were acquired using a confocal fluorescence microscope (Leica TCS SP8). Quantitative fluorescence intensity analysis was performed using ImageJ software (NIH).

[0065] Experimental results are as follows Figure 2 As shown, Figure 2 Molidustat was shown to promote the antiviral function of immune cells in humanized mice (hu-PBL mice); among which, Figure 2 a shows a schematic diagram of humanized mice infected with HIV-1 and HIV-1Δasp and treated with Molidustat; Figure 2 b shows the levels of IFNG, IFNB, and HIV-1tat mRNA transcription in the spleen, liver, and rectum of mice treated with Molidutat for a certain period of time, as well as the levels of cytokine antigens in the blood, reflecting the effect of Molidutat on the body's antiviral immune response. Figure 2 c shows the HIV viral load in the spleen of mice after Molidutat treatment for a certain period of time (shown in green fluorescence). It can be seen from the figure that the viral load is significantly reduced.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of PHD3 inhibitors in the preparation of drugs for treating AIDS, characterized in that, The PHD3 inhibitor is Molidustat, CAS No.: 1154028-82-6.

2. The application as described in claim 1, characterized in that, The drug comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient is Molidustat.

3. The application as described in any one of claims 1 to 2, characterized in that, The dosage forms of the drug include injections, tablets, powders, suspensions, capsules, pills, or syrups.

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