Application of PHD3 inhibitor in prevention and treatment of AIDS

Abstract

The invention discloses an application of a PHD3 inhibitor in prevention and treatment of AIDS (acquired immune deficiency syndrome). Through multidisciplinary research means of immunology, biochemistry and the like, the situation that virus antisense protein ASP inhibits generation of I-type IFN in the HIV-1 infection process is analyzed, immune escape is achieved, and a latent infection mechanism is established. In the aspect of mechanism, the ASP can play the inhibiting role after transcriptional expression into protein in a body cell through post-translational modification, the 47-site proline of the ASP can be helped to realize the capability of inhibiting the immune response of the body after hydroxylation modification, and the post-translational modification is realized by depending on proline hydroxylase 3 (PHD3) of a host. On the basis of mechanism research, it is found that a PHD3 inhibitor Molidustat counteracts hydroxylation of PHD3 to ASP, so that immune escape of HIV-1 is blocked, infection is antagonized, and good development prospects of AIDS treatment and prevention drugs are shown.

Description

Technical Field

[0001] This invention relates to the application of PHD3 inhibitors in the prevention and treatment of AIDS, and belongs to the field of biomedical technology. Background Technology

[0002] Currently, the main treatment for AIDS is highly active antiretroviral therapy (ART), commonly known as "cocktail therapy." This therapy uses a combination of antiviral drugs to target key proteins in the HIV life cycle to inhibit viral replication. In recent years, China has also developed various pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) drugs, such as domestically produced generic drugs based on emtricitabine, tenofovir, and lamivudine, which have improved AIDS prevention and early intervention capabilities to some extent. However, both ART and PEP drugs face a series of technical bottlenecks that urgently need to be addressed in clinical application.

[0003] 1. High dependence on disease progression: ART is mainly effective for patients in the early stages of infection, while for patients who have entered the middle and late stages, their immune system has often been irreversibly damaged by HIV, and the efficacy is limited.

[0004] 2. Viruses are prone to developing drug resistance: Whether it is ART or existing domestic blocking drugs, their targets are mostly concentrated on key links such as HIV reverse transcription, integration or protease. Under long-term selection pressure, the virus is prone to mutation and form drug-resistant strains, resulting in a decrease in efficacy.

[0005] 3. High patient compliance requirements and significant side effects: ART and various blocking agents require regular, cyclical administration, making treatment regimens complex. Long-term use may lead to a series of side effects, such as lipodystrophy, liver and kidney toxicity, and decreased bone density. Some domestically produced blocking agents also have issues with fixed ingredient ratios and insufficient response to high-risk exposure situations.

[0006] 4. Inability to eliminate latent virus reservoirs: Existing drugs are unable to completely eliminate latent viruses on CD4. + Once medication is stopped, even if only trace amounts of HIV remain in the body, the HIV viral reservoir in host cells such as T cells may trigger a viral rebound.

[0007] 5. Complex pharmacokinetics and high R&D barriers: Multiple drugs in cocktail therapy have different metabolic rates and durations of action, increasing the complexity of dosing regimen design and making drug interactions difficult to predict. This necessitates extensive clinical trials for validation, resulting in long R&D cycles and high costs. Some domestically produced blocking drugs still have room for improvement in pharmacokinetic suitability, oral bioavailability, and target distribution.

[0008] 6. Limited scope of use: Currently, domestically produced post-exposure prophylaxis (PEP) drugs are mainly used for short-term prevention in high-risk groups of HIV, and there is still a lack of effective intervention methods for early viral clearance or immune recovery support after infection.

[0009] In summary, there is an urgent need in the field of HIV / AIDS treatment and prevention to develop innovative treatment strategies with novel mechanisms, low drug resistance, broad-spectrum antiviral activity, ease of use, and the ability to intervene in the latent viral reservoir, in order to further improve the effectiveness of clinical interventions and the quality of life of patients. Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of existing drugs for treating AIDS by providing the application of PHD3 inhibitors in the prevention and treatment of AIDS.

[0011] To achieve the above objectives, the present invention provides the use of PHD3 inhibitors in the preparation of medicaments for the prevention and treatment of AIDS.

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

[0013] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier or excipient, wherein the active ingredient comprises a PHD3 inhibitor.

[0014] Preferably, the dosage form of the drug includes injections, tablets, powders, suspensions, capsules, pills, or syrups.

[0015] Unlike traditional anti-AIDS drugs, this invention approaches the problem from the perspective of enhancing the antiviral capabilities of immune cells, similar to the "strengthening the body's resistance" strategy in traditional Chinese medicine. Targeting PHD3, it takes a host-centric approach, analyzing the key molecular mechanisms that target or regulate HIV latency, and developing PHD3 inhibitors as host targets manipulated by HIV. This drug can effectively intervene in the latency or activation process of the virus within host cells; significantly enhance the host's antiviral immune response, improving the ability to recognize and clear HIV in the early stages of infection and even during the latency period; reduce the risk of drug resistance and systemic toxicity, improving the safety and adherence of long-term patient use; and has good potential for combined use with existing antiviral drugs, enhancing the overall therapeutic effect without increasing pharmacokinetic complexity.

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

[0017] In vitro and in vivo experiments have shown that the PHD3 inhibitor Molidustat counteracts the hydroxylation of ASP by PHD3, thereby blocking HIV-1 immune evasion and antagonizing infection, promoting the function of innate immune cells, and breaking HIV's immune evasion strategy; it also inhibits HIV replication in immune cells. To a certain extent, this drug overcomes the drawbacks of traditional AIDS treatment drugs, such as high adherence and the virus's tendency to mutate and develop drug resistance. This invention provides a new host drug target for the prevention and treatment of AIDS, showing promising prospects for drug development and clinical application. Attached Figure Description

[0018] Figure 1 This invention illustrates how Molidustat promotes cellular immune function and inhibits HIV viral load in MDM cells (human monocyte-derived macrophages) and Jurkat (human CD4+ T cells) in one embodiment of the present invention. Figure a shows the activation of the IFN signaling pathway 12 hours after infection with HIV-1 and HIV-1ASP P47A pseudoviruses (ASP 47-proline mutation, which leads to loss of ASP function). Figure b shows the mRNA transcription levels and viral load of IFN and other inflammatory signaling factors 24 hours after infection, reflecting the antiviral effect of Molidustat in macrophages. Figure c shows the mRNA transcription levels and viral load of IFNG and other inflammatory signaling factors 24 hours after infection with HIV-1 and HIV-1ASP P47A pseudoviruses 24 hours after infection with MDM, reflecting the antiviral effect of Molidustat in T cells. Figure d shows the effect of infection with HIV-1 and HIV-1ASP pseudoviruses using MDM. The mRNA transcription levels of IFN and other inflammatory signaling factors and viral load of P47A evovirus 24 hours later reflected the antiviral effect of Molidustat in macrophages.

[0019] Figure 2 This invention illustrates how Molidustat promotes the antiviral function of immune cells in humanized mice (hu-PBL mice) according to one embodiment of the present invention. Figure a shows a schematic diagram of humanized mice infected with HIV-1 and HIV-1Δasp and treated with Molidustat. Figure b shows the levels of IFNG and IFNB and HIV-1tat mRNA transcription in the spleen, liver, and rectum of mice treated with Molidustat for a certain period, as well as the levels of cytokine antigens in the blood, reflecting the effect of Molidustat on the body's antiviral immune response. Figure c shows the viral load of HIV in the spleen of mice treated with Molidustat for a certain period (shown as green fluorescence). Detailed Implementation

[0020] The technical solutions and effects of this application will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely for explaining the invention and are not intended to limit the invention.

[0021] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

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

[0023] Materials Sources: 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 Jianqing Xu's research group at Fudan University. MDMs (human peripheral blood macrophages) were isolated from peripheral blood collected from healthy volunteers recruited by our laboratory, in accordance with human ethical regulations. Both MDMs and Jurkat cells were cultured in RAPI-1640 medium supplemented with 10% inactivated fetal bovine serum and 100 U / mL penicillin-streptomycin solution. TZM-bL and HEK293T cells were cultured in DMEM medium supplemented with 10% inactivated fetal bovine serum and 100 U / mL penicillin-streptomycin solution. HIV-1NL4.3-Δenv and VSVG plasmids, as well as pnL 4-3 infectious clones, were donated by Professor Sauter. Molidustat was purchased from MCE (CAS No.: 1154028-82-6). The WB chemiluminescent substrates (Super Signal Westchemiluminescent substrates) were purchased from Vazyme; the primary antibodies TBK1, IRF3, P38, P65 and their phosphorylation antibodies were purchased from Cell Signaling Technology; and GAPDH was purchased from Abclonal.

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

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

[0026] Implementation process:

[0027] 1) HIV-1 virus and its coating

[0028] Pseudovirus coating: HIV-1 and HIV-1ASP P47A were transfected into HEK293T cells with VSVG and Δenv or ΔenvP47A plasmids (at a ratio of VSVG:Δenv / Δenv P47A = 1:2). Virus was harvested 48 hours after transfection. The viral supernatant was filtered through a 0.45 μm filter and concentrated using PEG8000 (V900156, VETEC) according to the manufacturer's instructions.

[0029] True virus coating: HIV-1 and HIV-1ASP P47A were transfected into pnL 4-3 or pnL4-3ASPP47A plasmids in HEK293T cells, and the virus was harvested 48 hours after transfection. The viral supernatant was filtered through a 0.45 μm filter and stored in the BSL-3 (Biosafety Level 3) laboratory of Fudan University.

[0030] HIV-1 viral titer determination using the TCID50 method: TZM-bl cells were digested and counted, with 1 × 10⁶ cells per well in a 96-well plate. 4 Cells were seeded into plates. After cell adhesion, viral dilution buffer was added. Each viral gradient was applied in triplicate, for a total of eight gradients. Eight wells served as a negative control group, containing the same volume of culture medium. After 48 hours, the 96-well plate was removed from the cell culture incubator, the liquid was aspirated, and the plate was washed twice with PBS for 5 minutes each time. Lysis buffer was then added, and the plate was shaken on a low-temperature shaker for 30 minutes. The cell lysate from each well was collected into a new 1.5 mL EP tube and centrifuged at 4°C, 16000 rpm, and 15 min. The supernatant was transferred to a microplate, and after protection from light, luciferase substrate was added to each well using a multi-channel pipette. The plate was then immediately placed in a microplate reader, and the luminescence value was read using the "Luciferase" program. Finally, the viral titer was calculated using the Reed-Muench method, with the formula: lgTCID50 = distance ratio × difference between the logarithms of dilutions + logarithm of the dilution with a virulence rate higher than 50%.

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

[0032] The cultured cells were divided into 2 × 10⁶ cells per well in a 6-well plate. 6 Cells were seeded in DMEM medium (serum-free) and starved for 12 h. HIV-1 pseudovirus was added at MOI=10 and infected for 12 h (for real virus, 24 h). After infection, 5 μmol of Molidustat was added and treated for 20 min, followed by extraction of cell proteins and RNA.

[0033] Protein sample collection: Add 200 μl of cell lysis buffer (Beyotime, P0013, with added PMSF, cocktail, NaF, and sodium pervanadate to inhibit phosphorylated protein degradation), lyse at 4°C for 40 minutes, transfer by pipetting to a new EP tube, centrifuge at 13200 rpm at 4°C for 15 minutes. Take 150 μl of supernatant, add loading, and boil at 100°C for 10 minutes.

[0034] RNA sample collection:

[0035] a. Add 1 ml TRIZOL (all subsequent EP tubes, shampoo tubes, etc. should be RNAase-free), mix well by pipetting, and then transfer to a new EP tube.

[0036] b. Add 200 μl of chloroform, cap the tube, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 12000 r / min for 15 minutes. The sample will separate 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 / min for 10 minutes, and discard the supernatant.

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

[0038] d. Allow the RNA precipitate to dry at room temperature, add an appropriate amount of RNase-free water, pipette the solution several times to dissolve the RNA, and then determine the RNA concentration and purity.

[0039] e. Reverse the DNA using the Vazyme HiScript IV RT SuperMix for qPCR (R423-01) kit, following the kit instructions.

[0040] Assessment of immune function and viral load

[0041] Western blot assessment of immune signaling pathway activation status

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

[0043] b. Transfer: Cut a 4cm*8cm PVDF membrane, pre-activate it in methanol, and install the electric transfer clamp in the following order: black clamp-sponge-filter paper-adhesive-PVDF membrane-filter paper-sponge-white clamp. Set the transfer time to 100V and 1KD / min.

[0044] c. Blocking: Block with 5% skim milk for 1 hour, then wash three times with TBST (conventional washing solution) for 5 minutes 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 them, 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. Application of PHD3 inhibitors in the preparation of drugs for the prevention and treatment of AIDS.

2. The application as described in claim 1, characterized in that, The PHD3 inhibitors include Molidustat, CAS No.: 1154028-82-6.

3. 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 includes a PHD3 inhibitor.

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

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