CN106265679A - Bromine domain protein inhibitor is preparing the purposes that anti-HIV-1 is hidden in medicine - Google Patents

Abstract

The invention belongs to the field of medicine and relates to the application of a bromostructural protein BET inhibitor in anti-HIV-1 latent treatment. Experiments of the present invention show that the chemical drugs have the effect of inducing the activation of HIV-1 latent cells, and will not cause systemic T cell activation, and have a synergistic activation effect when used in combination with protein kinase C agonists or cytokines, and have the effect of anti-reverse infection. The combined use of recording virus drugs can kill activated latently infected cells, thereby accelerating the clearance of latent virus storage, further, the bromodomain protein inhibitor can be used to prepare anti-HIV-1 latent therapeutic drugs, It will provide new intervention approaches and strategies for the complete cure of AIDS.

Description

Use of Bromodomain Protein Inhibitors in the Preparation of Anti-HIV-1 Latent Therapeutic Drugs

technical field

The invention belongs to the field of medicine and relates to the use of bromodomain protein (BET) inhibitors in the preparation of anti-HIV-1 latent therapeutic drugs

Background technique

Research has disclosed that AIDS, Acquired Immunodeficiency Syndrome (AIDS) is an infectious disease that seriously affects human physical and mental health caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV-1) infection of the immune system. According to statistics, in 2012, about 35.3 million people were infected with HIV-1 in the world, of which 2.3 million were newly infected and 1.6 million died. AIDS has become a serious public health and social problem in the world today. At present, the clinical treatment of AIDS is mainly High Active Antiretroviral Treatment (HAART), which can reduce the plasma HIV-1 level below the clinical detection line, significantly improve the quality of life of patients, and enable them to obtain Nearly normal lifespan, but once treatment is discontinued, viral load quickly returns to pre-treatment levels. Studies have shown that an important reason why HIV-1 is difficult to be completely eliminated in the body is the existence of the virus latent library, which is mainly composed of resting CD4+ T cells, and also includes dendritic cells and macrophages. There are two main ways: one is HIV-1 infection of activated cells, most of these cells are killed by the immune system, and a very small number survive and transform into a static memory state, which is the main source of latent pool; the other is HIV-1 Directly infect quiescent cells, and the viral cDNA is successfully integrated into the host genome in the absence of cell activation. Since the integrated provirus in the latent library lacks transcriptional activity, it can escape the attack of the immune system and HAART therapy. Although the number of latently infected cells is small, but the half-life is long, it is not possible to completely eliminate it only by HAART treatment during the individual survival period. Possibly, the virus latent library becomes the main obstacle to eradicate HIV-1 [Finzi, D. et al. Latent infection of CD4+Tcells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nature Med.1999 , 5, 512–517]. In response to this phenomenon, researchers in the field proposed an activation-clearance strategy, that is, an attempt to induce the expression of proviruses in latently infected cells by drugs, combined with HAART therapy to achieve the goal of completely eradicating HIV-1 (Richman et al. The Challenge of Finding a Cure for HIV Infection, Science, 2009, 1304, 323).

At present, several intervention programs to activate latent HIV-1 have entered clinical trials, such as interleukin-2 (NCT00004978, NCT00001535) and interleukin-7 (NCT00105417) based on cytokines; based on histone deacetylase inhibition There are Valproic acid (NCT00289952), Vorinostat (NCT01319383, NCT01365065), Romidepsin (NUT01933594, NUT02092116), Panobinostat (NCT01680094); based on phosphatase and its homologue inhibitors, there are Disulfiram (NCT01286259), 60 The combination of these inducers with HAART therapy has achieved preliminary clinical efficacy, but there are still problems such as low activation efficiency or toxic side effects. Therefore, the development of new drugs and the development of new anti-latency treatment intervention strategies have become an urgent problem in this field. The key issue.

Bromodomain and extra terminal domain (BET) protein inhibitors are another class of factors that can activate latent HIV-1 transcription [Banerjee C, et al. BET bromodomain inhibition as a novel strategy for reactivation of HIV-1. J Leukoc Biol .2012Dec;92(6):1147-54]. The BET protein inhibitor JQ1(S) was originally developed as an anti-tumor drug, and it has been found that JQ1(S) has a good effect on interfering with HIV latency [Li Z, et al. The BET bromodomain inhibitor JQ1activates HIV latency through antagonizing Brd4inhibition of Tat-transactivation. Nucleic Acids Res.2013Jan 7; 41(1):277-87], the drug may become an expected anti-HIV-1 potential drug candidate.

In addition, a variety of BET inhibitors have been in preclinical or clinical trials, such as the compound OTX015 (CAS number: 202590-98-5) in the treatment of midline cancer with rearrangement of the nuclear protein of the testis (NUT) gene. The clinical phase I (NCT02259114), the treatment of glioblastoma multiforme entered the clinical phase I/II (NCT02296476), and the treatment of acute myeloid leukemia entered the clinical phase I/II (NCT01713582, NCT02303782). RVX-208 (CAS No.: 1044870-39-4) has entered clinical phase II for the treatment of atherosclerosis (NCT01058018), dyslipidemia (NCT01423188, NCT01863225), diabetes (NCT01728467), and coronary artery disease (NCT01067820). PFI-1 (CAS number: 1403764-72-6) has also been used in the study of acute leukemia (Picaud, S. et al. PFI-1, a highly selective protein interaction inhibitor, targeting BET Bromodomains. Cancer research 73, 3336- 3346, doi: 10.1158/0008-5472. CAN-12-3292 (2013)). Bromosporine is a broad-spectrum BET inhibitor. So far, there are no reports about OTX015, RVX-208, PFI-1 and Bromosporine having anti-HIV-1 latent therapeutic effect.

Contents of the invention

The purpose of the present invention is to provide new chemical drugs for anti-HIV-1 latent treatment. Chemopharmaceuticals specifically related to bromostructural protein BET inhibitors. In particular, the use of bromostructural protein BET (including Brd2, Brd3, Brd4, BrdT) inhibitors in the preparation of anti-HIV-1 latent therapeutic drugs.

Experiments in the present invention prove that the bromostructural protein inhibitors Brd2 and Brd4 have inhibitory effects on the initiation and extension of HIV-1 transcription, so the inhibitors can promote the expression of latent HIV-1.

In order to confirm that the chemical drug has a reactivation effect on latent HIV-1 in latently infected cells, the present invention is implemented through the following technical scheme.

Main reagent material used in the present invention is as follows:

The HIV-1 latent infection cell model that the present invention selects is A10.6 and C11, and A10.6 is donated by the AIDS Reference Reagent Program Department (NIH AIDS Research and Reference Reagent Program) of the National Institutes of Health and Health in the United States, and is an internationally general HIV -1 latent infection cell model; C11 was constructed by our laboratory, and many articles have been published (Ding, D. et al. Virol.2013.02.022(2013), Qu,X.etal.Zinc finger nuclease: a new approach forexcising HIV-1proviral DNA from infected human T cells.Molecular biology reports 41,5819-5827,doi:10.1007/s11033-014- 3456-3(2014)); the two cell lines are clones established after HIV-1 carrying the green fluorescent protein gene infects human T lymphocytes, and its biological characteristic is that HIV-1 is integrated, However, the viral gene is not expressed (gene silencing); the green fluorescent protein can be observed on living cells, so it is used as a reporter gene, that is, a marker of whether the latently infected cells are activated;

The BET inhibitors used in the present invention are all provided by Selleckchem, are all soluble in DMSO (purchased from SIGMA), OTX015 product number S7360, storage concentration 10mmol/L; RVX-208 product number S7295, storage concentration 50mmol/L; PFI-1 product number S1216, The storage concentration is 10mmol/L; Bromosporine product number S7233, the storage concentration is 50mmol/L;

In the present invention, the anti-HIV-1 latent therapeutic drug activates HIV latently infected cells; the cells are human mononuclear cells, human macrophages, human CD4 T lymphocytes, human mast cells, human dendritic cells, human Follicular dendritic cells, human hematopoietic progenitor cells, human natural killer cells, human neurons or oligodendrocytes.

In the present invention, a pharmaceutical composition for synergistically activating HIV latently infected cells is provided, which consists of bromodomain protein inhibitors OTX015, RVX-208, PFI-1 or Bromosporine and protein kinase C agonists or cytokines respectively; The aforementioned protein kinase C agonist is preferably Prostratin, and the aforementioned cytokine is preferably TNF-a.

In the present invention, a pharmaceutical composition for activating, eliminating or eradicating HIV latent virus is provided, which is composed of bromodomain protein inhibitors OTX015, RVX-208, PFI-1 or Bromosporine and anti-HIV drugs, and the anti-HIV drugs Can be selected from: (1) Nucleoside reverse transcriptase inhibitors: ① Zidovudine zidovudine (AZT or ZDV); ② Didanosine (ddl, Videx); ③ Zalcitabine (ddc); ④ Stavudine (d4T); ⑤ Lamivudine (3TC); ⑥ abacavir (1592U89Ziagen), or (2) non-nucleoside reverse transcriptase inhibitors (NNRTI): ① nevirapine nevirapine; ② delavird; ③ efavirene, or (3) protease inhibitors: ① saguinavir; ② indinavir; ③ ritonavir; ④ nelfinavir; ⑤ amprenavir amprenavir.

More specifically, the use of the bromodomain protein inhibitor of the present invention in the preparation of anti-HIV-1 latent therapeutic drugs is achieved through the following technical scheme:

(1) Dose-effect relationship of BET inhibitors to activate latent HIV-1

In the present invention, the HIV-1 latently infected cell model is treated with different concentrations of BET inhibitors, and after 48 hours, the activation of HIV-1 latently infected cells is analyzed by fluorescent microscope observation and flow cytometry detection of the reporter gene green fluorescent protein Efficiency, to obtain the dose-effect relationship of drug action; the results showed that with the increase of BET inhibitor concentration, the number of cells expressing green fluorescence increased; HIV-1 latently infected cell C11 was treated with OTX015, and the proportion of green fluorescence positive cells was as high as 88.4 %, the proportion of positive cells after treatment with RVX-208 was as high as 85.4%, the proportion of positive cells after treatment with PFI-1 was as high as 94.3%, and the proportion of positive cells after treatment with Bromosporine was as high as 87.7%; HIV-1 latent infection without induction agent treatment Cells, the proportion of fluorescence-positive cells is only 4.09% background activation; the experimental results show that OTX015, RVX-208, PFI-1 and Bromosporine have an activation effect on HIV-1 latently infected cells, and there is a dose-effect relationship;

(2) Time-effect relationship of BET inhibitors to activate latent HIV-1

In the present invention, the HIV-1 latent infection cell model is treated with the optimal concentration of various BET inhibitors respectively, and after 24h, 48h, and 72h after drug treatment, the fluorescent microscope observation and flow cytometry of the reporter gene green fluorescent protein Cytometry detection, analyzing the activation efficiency of HIV-1 latently infected cells, and obtaining the time-effect relationship of drug action; the results showed that, BET inhibitors were treated with HIV-1 latently infected cell models, and the number of green fluorescent positive cells increased over time Gradually increased, and basically reached the best activation effect after 48 hours on C11 cells; the experimental results indicated that the activation of OTX015, RVX-208, PFI-1 and Bromosporine on HIV-1 latently infected cells had a time-effect relationship;

(3) Effects of BET inhibitors on systemic T cell activation

In the present invention, by analyzing the expression levels of cell surface molecules CD25 and CD69 to detect whether BET inhibitors can cause systemic T cell activation, CD4 T cells were isolated from normal human peripheral blood, and different drugs and positive control Prostratin were used to treat cells for 48 hours, and CD25- FITC and CD69-PE staining, flow cytometry analysis of the expression levels of CD25 and CD69, the results showed that compared with Prostratin activated CD25 28.78%, CD6980.9%, BET inhibitor group was almost similar to the blank control group, did not cause The expression levels of CD25 and CD69 are significantly increased; the experimental results of the present invention suggest that OTX015, RVX-208, PFI-1, and Bromosporine will not cause systemic T cell activation, laying the foundation for clinical application;

(4) Synergistic activation of BET inhibitors, protein kinase C (PKC) agonist Prostratin and tumor necrosis factor-α (TNF-α)

Based on the state of each HIV-1 latently infected cell is not exactly the same, so the HIV-1 latent mechanism is different; in order to maximize the activation of HIV-1 latently infected cells and reduce toxicity, in the present invention, BET inhibitors and Prostratin and Detection and analysis of the synergistic activation of TNF-α: after treatment of HIV-1 latently infected cells C1148h with single drug or combined drug, the reporter gene green fluorescent protein was detected by flow cytometry. The results showed that the proportion of positive cells activated by OTX015 and Prostratin It was 77.83%, which was significantly greater than the sum of the activation effects of single drugs OTX015 (10.76%) + Prostratin (27.27%); the proportion of positive cells activated by the combination of OTX015 and TNF-α was 39.77%, which was greater than the sum of the activation effects of single drugs OTX015 (10.76%) )+TNF-α (4.99%); the results of the other three drugs are similar; the experimental results of the present invention suggest that OTX015, RVX-208, PFI-1 and Bromosporine have obvious synergistic activation with Prostratin, and have a weaker activation effect with TNF-α. synergistic activation.

The experimental results of the present invention show that the BET inhibitor compounds OTX015, RVX-208, PFI-1 and Bromosporine have higher induction and activation effects on different HIV-1 latent infection cell models, and will not cause systemic T cell activation. And when used in combination with Prostratin and TNF-α, it has a synergistic induction and activation effect. The experimental results provide a new intervention approach and idea for the induction, activation and final clearance of latent HIV-1.

The invention provides a new application of the bromostructural protein BET inhibitor in anti-HIV-1 latent treatment. Experiments of the present invention show that the compounds have the effect of inducing the activation of HIV-1 latent cells, and will not cause systemic T cell activation, and have a synergistic activation effect when used in combination with protein kinase C agonists or cytokines, and have a synergistic activation effect with anti-reverse transcription Virus drugs can be used in combination to kill activated latently infected cells, thereby accelerating the clearance of latent virus storage. Further, the bromodomain protein inhibitor can be used to prepare anti-HIV-1 latent therapeutic drugs, which will Provide a new intervention strategy for the complete cure of AIDS.

Description of drawings

Figure 1 Dose-effect relationship of BET inhibitors to activate latent HIV-1,

It shows that different concentrations of drugs were used to treat C11 and A10.6 cells for 48 hours, then flow cytometry was performed to analyze the proportion of fluorescent cells.

Figure 2 Time-effect relationship of BET inhibitors to activate latent HIV-1,

It shows the proportion of fluorescent cells analyzed by flow cytometry after the C11 and A10.6 cells were treated with various drugs at the optimal concentration for 24h, 48h, and 72h respectively.

Figure 3 Effects of BET inhibitors on systemic T cell activation,

It shows that CD4 T cells were isolated from normal human peripheral blood, treated with different BET inhibitors and positive control Prostratin for 48 hours respectively, and the expression levels of cell surface molecules CD25 and CD69 were analyzed by flow cytometry.

Figure 4 Synergistic activation of BET inhibitors with Prostratin and TNF-α,

It shows the proportion of fluorescent cells analyzed by flow cytometry after BET inhibitor was administered alone or co-administered with Prostratin and TNF-α for 48 hours.

detailed description

Example 1 BET inhibitor activates the dose-effect relationship experiment of latent HIV-1

C11 and A10.6 were planted in 96-well plates at ² ×104 cells per well, and 100 uL of 1640 medium (Gibco) containing 10% FBS (Gibco) was added to each well, and the ratio of drug to medium was 1:200. Add different concentrations of BET inhibitors; after 48 hours of drug treatment, observe the expression of GFP in the cells under a fluorescence microscope, collect the cells for flow cytometry detection, and analyze the proportion of fluorescent cells;

The results showed that with the increase of BET inhibitor concentration, the number of cells expressing green fluorescence increased; HIV-1 latently infected cell C11 was treated with OTX015, the proportion of green fluorescent positive cells was as high as 88.4%, and the positive cells after RVX-208 treatment The ratio was as high as 85.4%, the ratio of positive cells was as high as 94.3% after being treated with PFI-1, and the ratio of positive cells was 87.7% after being treated with Bromosporine. 2.75% background activation; the results suggest that OTX015, RVX-208, PFI-1 and Bromosporine can activate HIV-1 latently infected cells, and there is a dose-effect relationship.

Example 2 BET inhibitor activates time-effect relationship of latent HIV-1

C11 and A10.6 were planted in 96-well plates at ² ×104 cells per well, and 100 uL of 1640 medium (Gibco) containing 10% FBS (Gibco) was added to each well, and the ratio of drug to medium was 1:200. Different concentrations of BET inhibitors were added. After 24h, 48h, and 72h of drug-treated cells, the expression of GFP in the cells was observed under a fluorescent microscope, and the cells were collected for flow cytometry detection, and the proportion of fluorescent cells was analyzed to obtain the kinetic characteristics of drug-activated latent HIV-1;

The results showed that when BET inhibitors were treated with HIV-1 latent infection cell models, the number of green fluorescent positive cells gradually increased with time, and the best activation effect was basically achieved after 48 hours on C11 cells; the results suggested that OTX015, RVX- 208, PFI-1 and Bromosporine have a time-effect relationship in the activation of HIV-1 latently infected cells.

Example 3 Effects of BET Inhibitors on Systemic T Cell Activation

CD4 T cells were isolated from normal human peripheral blood, planted in 24-well plate at ¹⁰ cells per well, 500uL 1640 medium (Gibco) containing 10% FBS (Gibco) was added to each well, OTX015, RVX-208, RVX-208, The final concentrations of PFI-1, Bromosporine and Prostratin were 1uM, 50uM, 5uM, 2.5uM and 1uM respectively, and the cells were collected after 48 hours of drug treatment. Wash once with 1mL PBS, then resuspend the cells with 100uL PBS, add 1uL of antibodies CD69-FITC and CD25-PE (BD-Biosciences) respectively, incubate on ice for 45min, wash with PBS three times, and then resuspend the cells with 500uL PBS, The expression levels of CD25 and CD69 were analyzed by flow cytometry;

The results showed that compared with Prostratin activated CD25 28.78% and CD69 80.9%, the BET inhibitor group was almost similar to the blank control group, and did not cause a significant increase in the expression levels of CD25 and CD69; the results suggested that OTX015, RVX-208, PFI-1, Bromosporine does not cause systemic T cell activation.

Example 4 Synergistic Activation of BET Inhibitors with Prostratin and TNF-α

2×10 ⁴ C11 cells were planted in a 96-well plate per well, and 100 uL of 1640 medium (Gibco) containing 10% FBS (Gibco) was added to each well, and OTX015 (0.01 uM), RVX-208 (5 uM), PFI-1 (0.5uM), Bromosporine (0.25uM), Prostratin (200nM), TNF-α (10ng/uL) alone or OTX015, RVX-208, PFI-1, Bromosporine, Prostratin and TNF-α in combination. After the cells were treated with drugs for 48 hours, the expression of GFP in the cells was observed under a fluorescence microscope, and the cells were collected for flow cytometry detection, and the proportion of fluorescent cells was analyzed;

The results showed that the ratio of OTX015 combined with Prostratin to activate positive cells was 77.83%, which was significantly greater than the sum of the activation effects of OTX015 (10.76%)+Prostratin (27.27%) alone; the ratio of OTX015 combined with TNF-α to activate positive cells was 39.77% , which is greater than the sum of the activation effects of individual drugs OTX015 (10.76%) + TNF-α (4.99%); the results of the other three drugs are similar; the results suggest that OTX015, RVX-208, PFI-1 and Bromosporine have obvious synergistic activation with Prostratin It has a weak synergistic activation effect with TNF-α.

Claims (8)

1. bromine domain protein inhibitor is preparing the purposes that anti-HIV-1 is hidden in medicine, described bromine domain Protein inhibitor is selected from OTX015, RVX-208, PFI-1 (PF-6405761), Bromosporine chemicals.

2. the purposes as described in claim 1, it is characterised in that described anti-HIV-1 hide medicine activate HIV Latent infected cells.

3. the purposes as described in claim 2, it is characterised in that described cell is people's mononuclearcell, people is huge to bite Cell, people's CD4T lymphocyte, people's mastocyte, people's dendritic cell, people's folliculus sample dendritic cell, artificial blood ancestral Cell, people's natural killer cell, human neure or oligodendroglia.

4. the pharmaceutical composition of a synergistic activation HIV latent infected cells, it is characterised in that by bromine domain protein Inhibitor OTX015, RVX-208, PFI-1 or Bromosporine respectively with protein kinase C agonist or cell because of Son composition.

5. the pharmaceutical composition of the synergistic activation HIV latent infected cells as described in claim 4, it is characterised in that Described protein kinase C agonist is Prostratin.

6. the pharmaceutical composition of the synergistic activation HIV latent infected cells as described in claim 4, it is characterised in that Described cytokine is TNF-a.

7. one kind is activated the pharmaceutical composition removed or eradicate HIV latent virus, it is characterised in that by bromine domain protein White inhibitor OTX015, RVX-208, PFI-1 or Bromosporine form with inverase.

8. activating as described in claim 7 is removed or the pharmaceutical composition of elimination HIV latent virus, it is characterised in that Described inverase is: (1) efabirenz: 1. zidovudine zidovudine (AZT Or ZDV)；2. didanosine didanosine (ddl, Videx)；3. zalcitabine Zalcitabine (ddc)；④ Stavudine Stavudine (d4T)；5. lamivudine Lamivudine (3TC)；6. Abacavir Abacavir (1592U89Ziagen), or (2) non-nucleoside reverse transcriptase inhibitor (NNRTI): 1. nevirapine nevirapine；2. Delavirdine delavird；3. efavirenz efavirene, or (3) protease inhibitor: 1. Saquinavir saguinavir；2. indinavir indinavir；3. ritonavir ritonavir；4. viracept see nelfinaivr nelfinavirr；5. amprenavir amprenavir.