Application of compound 6-BIO in the preparation of HIV latent infection virus activator and in the preparation of drugs for eradicating the virus

Abstract

The present invention provides the use of compound 6-BIO in preparing an agent for activating latent HIV infection and in preparing a drug for eradicating the virus. Compound 6-BIO can significantly activate latent HIV-1 in peripheral blood and has an activating effect on latent HIV viruses. Compound 6-BIO can be combined with antiviral therapeutic drugs to kill newly generated HIV viruses or combined with immunotherapy or cell therapy to kill latent cells, thereby being used to eliminate latent HIV viruses and achieve the purpose of curing latent HIV-1 infection. The compound has important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to the use of compound 6-BIO in preparing an HIV latent infection virus activator and in preparing a drug for eradicating the virus. Background Art

[0002] Although highly active antiretroviral therapy (HAART) can effectively control human immunodeficiency virus type 1 (HIV-1) replication in patients with acquired immunodeficiency syndrome (AIDS), it is unable to eradicate latent infection, a major challenge in current AIDS treatment. Transcriptional inhibition of HIV-1 proviral DNA integrated into the human genome is crucial for maintaining HIV-1 latency. Several immunotherapies or gene therapies have been proposed for latent HIV-1 infection, and some strategies are currently in clinical trials. These include the "activation-killing" approach, which involves reactivating latent HIV-1 using epigenetic inhibitors or certain signaling pathway activators ("activators"), followed by combined antiretroviral therapy (ART), immunotherapy, or cell therapy to eliminate newly generated virus and activated latent cells ("killers"), ultimately achieving latent viral clearance. Several HIV-1 latency-activating compounds, such as the epigenetic inhibitor SAHA (also known as Vorinostat), have shown promising effects at the cellular level, but clinical trials have not yielded the expected significant results. It is necessary to extensively screen for highly effective and low-toxic HIV-1 latent reactivating compounds.

[0003] 6-BIO, (2ˊZ,3ˊE)-6-bromoindirubin-3ˊ-oxime, English name: 6-BIO (6-Bromoindirubin-3-acuteoxime), molecular formula: C 16 H 10 BrN3O2, a derivative of the purple indirubin dye isolated from Tyrian purple indirubins, is a specific inhibitor of glycogen synthase kinase-3 (GSK-3) and can activate the WNT canonical signaling pathway (Meijer, L., et al., GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol, 2003. 10(12): p. 1255-66). This signaling pathway is associated with the development of cancer. Currently, there are no reports indicating that 6-BIO can be used to treat AIDS. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention aims to provide the use of compound 6-BIO in the preparation of an HIV latent infection virus activator and in the preparation of a drug for eradicating the virus.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] On the one hand, the compound 6-BIO is used in the preparation of an HIV latent infection virus activator.

[0007] To address the problem that current antiretroviral therapy (ART) can effectively control the replication of human immunodeficiency virus type 1 (HIV-1) in AIDS patients but cannot eradicate latently infected viruses, the present invention utilizes compound 6-BIO as an HIV latent virus activator to activate latent HIV viruses (such as HIV-1), thereby completely curing the virus.

[0008] In the present invention, the compound 6-BIO has the following structure:

[0009]

[0010] Preferably, the HIV latent infection virus activator is a CD4 + Drugs for latent HIV-1 in T cells.

[0011] In the present invention, the CD4 + Using T cell lines as a cell model, it was found that compound 6-BIO could activate latent HIV-1.

[0012] Preferably, the HIV latent infection virus activator is a drug that promotes the transcription of HIV-1 proviral DNA.

[0013] In the present invention, the compound 6-BIO can promote the transcription of HIV-1 proviral DNA, thereby activating the latent virus.

[0014] In another aspect, the present invention provides use of compound 6-BIO in the preparation of a drug for eradicating latent HIV infection.

[0015] Preferably, the drug for eradicating latent HIV infection virus includes compound 6-BIO as an HIV latent virus activator, and also includes any one or a combination of at least two of a killing agent, an immunotherapeutic agent, or a cell therapy agent that kills HIV virus (e.g., activates newly generated HIV virus).

[0016] The compound 6-BIO of the present invention can be combined with antiviral drugs to kill newly generated HIV viruses or combined with immunotherapy or cell therapy to kill latent cells, thereby achieving a radical cure for AIDS.

[0017] In the present invention, compound 6-BIO is used to activate latent HIV virus, and is combined with a killer agent that kills newly generated HIV virus or a cell or immunotherapy agent that kills latent cells to achieve the purpose of eradicating latent HIV infection virus, thereby achieving a complete cure of latent HIV infection.

[0018] In the present invention, the HIV-killing agent is preferably an antiretroviral therapeutic agent.

[0019] Preferably, the antiretroviral therapeutic agent is, but is not limited to, any one of a reverse transcriptase inhibitor, a proteolytic enzyme inhibitor or an integrase inhibitor, or a combination of at least two thereof.

[0020] Preferably, the drug for eradicating latent HIV infection is a drug for activating latent HIV-1 in peripheral blood cells of HIV-1 infected individuals being treated with antiretroviral drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] In the present invention, compound 6-BIO can significantly activate latent HIV-1 in peripheral blood and has an activating effect on latent HIV viruses. It can be combined with antiviral therapeutic drugs to kill newly generated HIV viruses or combined with immunotherapy or cell therapy to kill latent cells, and is used to eliminate latent HIV viruses to achieve the purpose of curing latent AIDS infection. It has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 HIV-1 latently infected CD4 + The expression level of HIV-1 gag mRNA in T cell ACH2 under the action of compound 6-BIO;

[0024] Figure 2 HIV-1 latently infected CD4 + The supernatant of T cell ACH2 was collected under the action of compound 6-BIO, and the newly produced HIV-1 was further titrated in TZMB1 cells to detect the activation of latent HIV-1;

[0025] Figure 3 Schematic diagram of the experimental process and reaction process for compound 6-BIO to activate latent HIV-1 in peripheral blood cells of patients treated with antiretroviral drugs;

[0026] Figure 4 This is a graph showing the HIV-1 gag mRNA expression levels of 8 patient samples after 6-BIO treatment in Example 2;

[0027] Figure 5This is a graph showing the HIV-1tat-rev mRNA expression levels in 8 patient samples after 6-BIO treatment in Example 2;

[0028] Figure 6 The CD4 + Results of HIV-1 protein Env expression on the surface of T cells;

[0029] Figure 7 This is the result diagram of luciferase expression level in Example 3;

[0030] Figure 8 This is the result diagram of HIV-1 gag mRNA expression level in Example 3. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0032] The experimental materials used in the following examples are as follows:

[0033] 1. Compound 6-BIO: 10 mM compound 6-BIO was dissolved in DMSO and stored at -80°C.

[0034] 2. Reagents and antibodies: RPMI-1640 medium (GIBCO); fetal bovine serum (FBS) (Millipore); human lymphocyte separation medium (Ficoll) (GE); dimethyl sulfoxide (DMSO) (Sigma); phytohemagglutinin-P (PHA-P) (Sigma); Cell Counting Kit-8 (Biyuntian); Trizol (Invitrogen); Thunderbird SYBR qPCR Mix (Toyobo); ReverTra qPCR RT Master Mix gDNA remove (Toyobo); anti-CD3 PerCP-Cy5.5 (eBioscience), anti-CD4 PE-cy7 (BD Biosciences); anti-HIV-1 Env (N6 bnAbs) antibody; PE Streptavidin (BD Biosciences).

[0035] Example 1

[0036] Compound 6-BIO was tested for HIV-1 latent activation using the following method:

[0037] (1) Latent activation assay using HIV-1 latently infected T cells (ACH2). Cells were cultured in RPMI-1640 / 10%-FBS at 4×10 5 Cells / well were seeded in a 24-well cell culture plate and the compound to be tested was added. After 24 hours, the cells were collected and lysed with Trizol, RNA was extracted, and reverse transcribed into cDNA using ReverTra qPCR RT Master Mix gDNA remover. HIV-1 Gag was amplified on an ABI Real-Time PCR instrument using Thunderbird SYBR qPCR Mix. The amount of virus in the supernatant of ACH2 cells was titrated using TZM-bl cells to detect the activation of latent HIV-1.

[0038] (2) CD4 in HIV-1 infected individuals +T cell viral latent activation assay: HIV-1 patients undergoing ART treatment and with undetectable plasma HIV-1 viral load were recruited from Zhengzhou Sixth People's Hospital. Informed consent was obtained and medical ethics approval was obtained. Peripheral blood was drawn, anticoagulated with EDTA, and peripheral blood lymphocytes (PBMCs) were isolated using lymphocyte separation medium (Kim, M., et al., A primary CD4(+) T cell model of HIV-1 latency established after activation through the T cell receptor and subsequent return to quiescence. Nat Protoc, 2014. 9(12): p. 2755-70). PBMC cells were cultured in RPMI-1640 / 10% FBS and the test compounds were added. After 5 days, the cells were stained with Trypan Blue and the viable cells were counted. The cells were then collected, lysed with Trizol, and RNA was extracted. The RNA was reverse-transcribed into cDNA using ReverTra qPCR RT Master Mix gDNA remover, and the HIV-1 Gag and Tat-rev genes were amplified on an ABI Real-Time PCR instrument using Thunderbird SYBR qPCR Mix (Ren, XX, et al., HIV-1 Nef-associated factor 1 enhances viral production by interacting with CRM1 topromote nuclear export of unspliced ​​HIV-1 gag mRNA. J Biol Chem, 2016). The primer sequences are as follows: Gag forward primer: 5′-GTGTGGAAAATCTCTAGCAGTGG-3′, reverse primer: 5′-CGCTCTCGCACCCATCTC-3′; Tat-rev, forward primer: 5′-ATGGCAGGAAGAAGCGGAG-3′, reverse primer: 5′-ATTCCTTCGGGCCTGTCG-3′; GAPDH forward primer: 5′-ATCCCATCACCATCTTCCAGG-3′, reverse primer 5′-CCTTCTCCATGGTGGTGAAGAC-3′; At the same time, cells were collected and analyzed by flow cytometry for CD4 +The expression of HIV-1 Env protein on the surface of T cells was assessed by collecting cells, adding biotin-coupled HIV-1 gp120 broad-spectrum antibody N6 to identify HIV-1 envelope protein (Env) expressed on the surface of latent cells, and then enriching them with PE-coupled avidin. Finally, PerCP-Cy5.5-coupled anti-human CD3 antibody and PE-cy7-coupled anti-human CD4 antibody were added, and CD3 was detected by flow cytometry. + CD4 + The expression of HIV-1 protein Env on the surface of double-positive T cells detects the activation of latent HIV-1.

[0039] The above test results are as follows:

[0040] 1.6-BIO can activate latent HIV-1 in CD4+ T cells

[0041] HIV-1 latently infected CD4 + The number of T cells is too low, per 1 million CD4 + The isolation of a latently infected cell from a T cell (Kim, M., et al., A primary CD4(+) T cell model of HIV-1 latency established after activation through the T cell receptor and subsequent return to quiescence. Nat Protoc, 2014. 9(12): p. 2755-70) has increased the difficulty of studying HIV-1 latent infection. Currently, studies on the molecular mechanisms of HIV-1 latent infection and the development of therapeutic approaches primarily utilize latently infected cell models.

[0042] The present invention utilizes HIV-1 latently infected CD4 + The T cell ACH2 was studied. Different concentrations of 6-BIO were co-cultured with ACH2 cells for 24 hours. The cells and supernatant were collected and the activation of latent HIV-1 was detected by quantifying the production of HIV-1 gag mRNA. Figure 1 As shown, it can be seen that compared with the control without drug addition (Medium), under the action of compound 6-BIO, the expression level of HIV-1 gag mRNA was significantly upregulated, indicating that compound 6-BIO can significantly activate latent HIV-1.

[0043] The supernatant of the above 6-BIO and ACH2 cell co-culture was collected and further infected with TZMB1 cells to titrate the infectivity of the newly generated virus. The newly generated HIV-1 in the ACH2 supernatant was detected by quantitative infection of TZMB1 cells. The results are shown in Figure 2. Figure 2 As shown, it can be seen that the culture supernatant of ACH2 cells treated with compound 6-BIO can significantly enhance the luciferase activity in TZMB1 cells, indicating that compound 6-BIO can significantly activate latent HIV-1.

[0044] Example 2

[0045] 6-BIO activates latent HIV-1 in peripheral blood cells of patients on antiretroviral therapy (ART)

[0046] To verify the effect of 6-BIO on the activation of latent HIV-1, HIV-1 infected individuals on antiretroviral therapy (ART) whose plasma viral load was undetectable were recruited; peripheral blood was drawn and peripheral blood mononuclear cells (PBMCs) were isolated. After PBMCs were treated with compound 6-BIO (1 μM) for 5 days, cells were collected and PHA-P (5 μg / mL) was used as a drug control. The treatment process and reaction process in this experiment are shown in the figure below. Figure 3 Shown: HIV-1 latent reactivation was detected by quantifying HIV-1 gag and tat-rev mRNA levels; and CD3 was analyzed by flow cytometry. + CD4 + The expression of HIV-1 protein Env on the surface of T cells is used to analyze the activation of latent HIV-1. That is, after the patient's peripheral blood mononuclear cells (PBMCs) are treated with compound 6-BIO for 5 days, the cells are collected, and biotin-coupled HIV-1gp120 broad-spectrum antibody N6 is added to recognize HIV-1 envelope protein (Env) expressed on the surface of latent cells. Then, PE-coupled avidin is used for enrichment, and finally, PerCP-Cy5.5-coupled anti-human CD3 antibody and PE-cy7-coupled anti-human CD4 antibody are added. CD3 is detected by flow cytometry. + CD4 + The expression of HIV-1 protein Env on the surface of double-positive T cells was used to detect the activation of latent HIV-1. After 6-BIO treatment, the expression levels of gag and tat-rev mRNA in 8 patient samples were as follows: Figure 4 and Figure 5 As shown, the levels of gag and tat-rev mRNA were significantly increased.

[0047] After 6-BIO treatment, CD4 + The expression of HIV-1 protein Env on the surface of T cells is as follows Figure 6 As shown, CD4 + The percentage of Env-positive T cells increased significantly, indicating that 6-BIO could activate resting CD4 T cells in PBMCs. +Latent HIV-1 in T cells.

[0048] Example 3

[0049] Verification of compound 6-BIO promoting the transcription of HIV-1 proviral DNA by the following method:

[0050] HIV-Luc / VSV-G infection of Jurkat CD4 + T, Infected cells were treated with different concentrations of 6-BIO for 24 h, and the cells were collected. The viral infection was analyzed by luciferase expression, and HIV-1 gag mRNA expression was detected.

[0051] The expression results of luciferase were as follows Figure 7 As shown, the mRNA expression level of HIV-1 gag is as Figure 8 As shown, according to Figure 7 and Figure 8 The results showed that compound 6-BIO could significantly promote the transcription of HIV-1.

[0052] In summary, the present invention uses HIV-1 latent cell lines and PBMCs isolated from HIV-1 infected patients treated with ART to confirm that compound 6-BIO can activate CD4 + HIV-1 is latent in T cells, suggesting that compound 6-BIO can be used as an activator to efficiently activate latent HIV-1 and can be combined with antiviral therapy, immunotherapy or cell therapy to eliminate latent HIV-1.

[0053] The applicant declares that the present invention is illustrative of the above-described embodiments, but the present invention is not limited to these embodiments. This does not mean that the present invention must rely on these embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for the raw materials used, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. The use of compound 6-BIO in the preparation of HIV latent infection virus activator, wherein the HIV latent infection virus activator is to activate HIV-1 latently infected CD4 + Drugs for latent HIV-1 in T cells; The compound 6-BIO has the following structure:

2. The use according to claim 1, characterized in that The HIV latent infection virus activator is a drug that promotes the transcription of HIV-1 proviral DNA.

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