CN113712963B

CN113712963B - Application of BET inhibitor BMS-986158 in preparation of anti-AIDS drug

Info

Publication number: CN113712963B
Application number: CN202111202564.XA
Authority: CN
Inventors: 黄旭升; 郑永唐; 田仁荣; 罗荣华; 杨柳萌; 马梦迪
Original assignee: Kunming Institute of Zoology of CAS
Current assignee: Kunming Institute of Zoology of CAS
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2022-09-23
Anticipated expiration: 2041-10-15
Also published as: CN113712963A

Abstract

The invention relates to application of a BET inhibitor BMS-986158, in particular to application in preparing anti-HIV-1 medicines. The research shows that: the BET inhibitor BMS-986158 can remarkably activate the expression of HIV-1 in the J-Lat, OM10.1 and ACH2 cells of HIV-1 latent cell lines; exhibit synergistic effects when used in combination with other types of latent activators; and the activation effect is not influenced by the anti-HIV drugs, and the activity of the anti-HIV drugs can be further increased. BMS-9861658 has low toxicity and high latent activated HIV-1 activity, and can enhance the antiviral effect of antiviral drugs in vitro. Therefore, the medicine can be used for preparing anti-HIV-1 latent medicines, is combined with anti-HIV medicines for treating AIDS, and provides a new intervention way and strategy for completely curing the AIDS.

Description

Application of BET inhibitor BMS-986158 in preparation of anti-AIDS drugs

Technical Field

The invention relates to the technical field of biomedicine, and provides a new application of a BET inhibitor BMS-986158, in particular to an application in preparing anti-AIDS drugs.

Background

Although current anti-HIV drugs can well inhibit the replication of viruses, can reduce the plasma HIV-1 level below a clinical detection line, obviously improves the life quality of patients and enables the patients to obtain a life close to normal, patients must take medicines for a long time, and once the medicines are stopped, the viruses can rebound rapidly and can quickly return to the viral load of the level before treatment. Studies have shown that one important reason why HIV-1 is difficult to completely clear in vivo is the presence of viral latency depots. The viral latency pool is mainly composed of static CD4 ⁺ T cell composition, dendritic cell, macrophage, etc. and has two main ways: firstly, HIV-1 infects activated cells, most of which are killed by immune system, and the like, and few of which survive and transform into a static memory state, which is a main source of a latent pool; secondly, HIV-1 directly infects resting cells, and the virus cDNA is successfully integrated into the host genome under the condition that the cells are not activated. Since proviruses integrated in the latent pool lack transcriptional activity and thus escape the immune system and attack by anti-HIV drugs, the number of latently infected cells is small but the half-life is long, complete clearance by HAART treatment alone during the lifetime of an individual is not possible, and the viral latent pool is a major obstacle to the eradication of HIV-1. The long-term administration has three problems, namely compliance, drug resistance and burden of patients caused by side effects. "activation and killing" is a strategy for curing HIV-1, which aims to activate HIV-1 virus in a latent state by some drugs (latent activators) while continuing antiviral therapy, and the cell surface containing the latent HIV-1 expresses the proteins of the virus, so that the virus is recognized by the immune system and cleared by the immune system, thereby achieving the purpose of clearing the latent virus bank. Finding a latent activator with high potency and low toxicity is an essential link in this strategy.

Latent activators that have been discovered to date can be largely classified into the following general categories: 1) epigenetic modulators: such as histone deacetylase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors, etc.; 2) transcription initiation promoters: primarily protein kinase C agonists; 3) transcription elongation promoter: such as BET (Bromodomain and Extra-terminal domain) inhibitors, P-TEFb activators and the like; 4) cytokines: such as IL-2, IL-6, etc.; 5) other latent activators, e.g. disulfiram, Toll-like receptor agonists 7, etc ^[1] . Although many HDACi and protein kinase C are currently in clinical trials, the effect is not satisfactory, and no drug is currently approved for clinical use.

The BET family protein is a protein with epigenetic regulation and transcription regulation functions, and a bromodomain of the protein can be combined with acetylated histone. In addition, it can be combined with other proteins such as Positive transcription elongation factor (Positive Transcript)ion amplification Factor b, P-TEFb) forms a protein complex, and promotes the phosphorylation of RNA polymerase II by P-TEFb to increase the transcription activity of RNA polymerase II and activate the expression of downstream genes. There are 4 protein members of this family, BRD1, BRD2, BRD4 and BRDT, respectively, of which BRD4 was the most studied. The Bromostructural Ectodomain (BET) is closely related to the transcription of HIV-1. There are, of course, many BET inhibitors that exhibit the effect of inducing activation of latent HIV-1 cells. The patent No. 2015103662060 discloses that the bromodomain protein inhibitor is selected from OTX015, RVX-208, PFI-1(PF-6405761), Bromosporine chemical drug in inducing HIV-1 latent cell activation, and does not cause systemic T cell activation, and has synergistic activation effect when combined with protein kinase C agonist or cytokine, and can kill activated latent infected cells when combined with antiretroviral drug. The research shows that: RVX-208, PFI-1, JQ1, etc. all reactivate latent HIV-1 cells. JQ1 Effect on BRD4, IC in cell-free assays ₅₀ 77nM/33nM, binds to all bromodomains of the BET family. JQ1 promotes the recruitment and binding of HIV Tat protein and P-TEFb in HIV LTR, promotes HIV transcription, but does not reduce viral repertoire. Research also shows that a novel BET inhibitor, apabetalone, can not only induce the transcription of latent virus pools, but also induce the preferential death of latent cells, and is more beneficial to the elimination of HIV. However, in published data, the activation effect of BET inhibitors on latent HIV in J-Lat cells is different. EC of OTX015 ₅₀ 1.95 to 4.34 times lower than JQ1 ^[2] The activation effects of RVX-208, PFI-1, UMB-136 and Apabetalone are weaker and are not as good as JQ1 ^[3-5] . Therefore, there is an urgent need to find compounds that induce transcription from latent viral pools without affecting the anti-HIV efficacy.

BMS-986158 is a BET inhibitor developed by Geril, USA, for the treatment of solid tumors, binds well to the bromodomain of BRD 4. Is still in clinical research stage ^[6] . The BET inhibitor BMS-986158 is a small molecular compound with the relative molecular mass of 495.62 and the molecular formula C ₃₀ H ₃₃ N ₅ O ₂ The structural formula is as follows:

the inventor researches and discovers that: BMS-986158 has low toxicity and high latent activity for activating HIV-1, and the activity is not affected by anti-HIV drug, and can enhance the antiviral effect of the anti-HIV drug in vitro. At present, no research reports that the compound can be used for activating latent viruses in HIV infection and treating HIV-1 are available.

Reference documents:

1.Battistini,A.,Sgarbanti,M.,2014.HIV-1latency:an update of molecular mechanisms and therapeutic strategies.Viruses.6,1715-1758.https://doi.org/10.3390/v6041715.

2.Lu,P.,et al.,The BET inhibitor OTX015 reactivates latent HIV-1through P-TEFb.Sci Rep,2016.6:p.24100.

3.Lu,P.,et al.,BET inhibitors RVX-208and PFI-1reactivate HIV-1from latency.Sci Rep,2017.7(1):p.16646.

4.Huang,H.,et al.,A Novel Bromodomain Inhibitor Reverses HIV-1Latency through Specific Binding with BRD4 to Promote Tat and P-TEFb Association.Front Microbiol,2017.8: p.1035.

5 Zhang,X.X.,et al.,The BET bromodomain inhibitor apabetalone induces apoptosis of latent HIV-1reservoir cells following viral reactivation.Acta Pharmacol Sin,2018.

6.Sun,Y.,Han,J.,Wang,Z.,Li,X.,Sun,Y.,Hu,Z.,2020.Safety and Efficacy ofBromodomain and Extra-Terminal Inhibitors for the Treatment of Hematological Malignancies and Solid Tumors:A Systematic Study of Clinical Trials.Front Pharmacol.11, 621093.https://doi.org/10.3389/fphar.2020.621093.)。

7.Chou,T.C.2006.'Theoretical basis,experimental design,and computerized simulation of synergism and antagonism in drug combination studies',Pharmacol Rev,58:621-81.

8. flos Pruni Salicinae, radix seu herba Penthori Henryi, radix Et caulis Gnathensi, and radix Asahi Saururi, and its preparation method and application are described in China, CN110183372B [ P ].2020-10-30.

9.Zhang,X.X.,J.Lin,T.Z.Liang,H.Duan,X.H.Tan,B.M.Xi,L.Li,and S.W.Liu. 2018.'The BET bromodomain inhibitor apabetalone induces apoptosis of latent HIV-1reservoir cells following viral reactivation',Acta Pharmacol Sin.

Disclosure of Invention

The invention aims to protect the new application of a BET inhibitor BMS-986158, namely the application of BMS-9861658 in preparing anti-HIV-1 latent therapeutic drugs. BMS-986158 has the potential of clearing HIV-1 latent virus library in the aspect of high-efficiency activation of HIV-1 latent virus library, and the potential medicinal value of the BMS-986158 in anti-HIV-1 treatment is disclosed.

The invention also protects the application of BMS-9861658 in preparing anti-HIV-1 latent treatment medicines by combining with histone deacetylase inhibitors and PKC agonists. The research shows that: BMS-9861658 showed a synergistic effect when used in combination with other types of latent activators; the activation effect is not influenced by anti-HIV drugs, and the activity of the anti-HIV drugs can be increased

Further, the histone deacetylase inhibitor is SAHA, and the PKC agonist is Prostratin.

The invention also protects the application of the BMS-9861658 and anti-HIV therapy drug combination in preparing anti-HIV-1 drugs.

Further, the anti-HIV therapy medicine comprises: zidovudine (AZT), Nevirapine (Nevirapine) NVP, indinavir Indinavir (IDV), efavirenz Efavirenz (EFV), Ratergevir Raltegravir (RAL) and Darunavir (DRV).

The invention discloses a high-efficiency latent activation activity of a BET inhibitor BMS-986158 for the first time, and the BET inhibitor BMS-986158 is suggested to be a candidate drug of an HIV-1 therapeutic drug. The content mainly comprises the following steps: 1) low toxicity: toxicity experiments on PBMC showed CC ₅₀ >100 mu M; 2) high activity: expression of HIV-1, EC, in J-Lat cells, the HIV-1 latent cell line ₅₀ 30.82 nM; 3) Broad activation activity: for HIV-1 latent cell line OM10.1 and HI in ACH2V-1 expression and HIV-1 activation activity in peripheral blood mononuclear cells isolated from HIV-1 patients chronically treated with ART. In conclusion, BMS-986158 can significantly activate the expression of latent HIV-1 in HIV-1 latent Cell lines or in Peripheral Blood Mononuclear Cells (PBMC) of AIDS patients receiving HAART treatment for a long period of time, and the activation effect is not affected by antiviral drugs, and the effect of anti-HIV drugs can be enhanced. Moreover, the research finds that: BMS-9861658 in combination with histone deacetylase inhibitors, PKC agonists, enhances activation of the HIV-1 latent virus pool. The compound can be used as a latent activator for activating and killing HIV-1, and is combined with anti-HIV medicines for treating AIDS.

Drawings and description of the drawings

FIG. 1 is a graph of the activation effect of the BET inhibitor BMS-986158 on latent HIV-1, wherein FIG. 1A is the percentage of GFP positive cells of J-Lat cells in different concentrations BMS-986158, JQ1 and DMSO-treated groups as measured by flow cytometry; FIG. 1B is a graph showing the percentage of GFP positive cells after treating J-Lat cells with BMS-986158(100nM), JQ1 (1. mu.M) for different periods of time by flow cytometry; FIGS. 1C and 1D show the expression level of HIV-1p24 antigen in the culture supernatant after different concentrations of BMS-986158 or JQ1 treated ACH2 cells and OM10.1 cells for 48 hours by ELISA method, respectively.

FIG. 2 shows the MTT method for detecting the toxicity of BMS-9861581 on PBMC or J-Lat cells after 2 days of treatment.

FIG. 3 shows the effect of BMS-986158 and JQ1 on T cell immune activation, wherein BMS-986158(100nM) or JQ1 (1. mu.M) was treated for 48 hours in PBMC, collected and tested for CD4 using flow cytometry in FIGS. 3A, 3B and 3C, respectively ⁺ Changes in expression of cell surface receptors CD25, CD69, HLA-DR in T cells, BMS-986158(100nM) or JQ1 (1. mu.M) in FIG. 3D, FIG. 3E, FIG. 3F, respectively, PBMC were treated for 48 hours, and the cells were harvested and tested for CD8 using flow cytometry ⁺ Changes in expression of cell surface receptors CD25, CD69, and HLA-DR in T cells.

FIG. 4 is a graph of the effect of BMS-986158 and JQ1 on HIV-1 co-receptor expression in PBMCs. FIGS. 4A and 4B show the expression changes of cell surface receptors CXCR4 and CCR5 detected by flow cytometry after PBMCs are treated with BMS-986158(100nM) or JQ1(1 μ M) for 48 hours and the cells are harvested.

FIG. 5 shows the effect of BMS-986158 on BRD4 expression and activation, and the expression of BRD4 and phosphorylated BRD4 proteins was detected by WB assay after J-Lat cells were treated with different concentrations of BMS-986158(20, 100, 500nM) for 24 hours. JQ1 (1. mu.M) was used as a control compound.

FIG. 6 shows the effect of BMS-986158 on cyclin T1 and CDK9 expression. FIG. 6A shows that the protein extracted from J-Lat cells was collected 24 hours after treatment with BMS-986158(20, 100, 500nM) at various concentrations, and WB assay was performed to detect the expression of cyclin T1 and CDK9 proteins. JQ1 (1. mu.M) was used as a control compound. Fig. 6B, 6C and 6D are the results of statistical analysis of the protein expression in fig. 6A.

FIG. 7 is a graph of the effect of BMS-986158 on the expression of phosphorylated CDK 9. FIG. 7A different concentrations of BMS-986158(20, 100, 500nM) were used to treat J-Lat cells for 24 hours before cell extract protein was collected and WB assay was used to detect expression of phosphorylated CDK9 protein. JQ1 (1. mu.M) was used as a control compound. FIGS. 7B and 7C are the results of statistical analysis of the protein expression in FIG. 7A.

FIG. 8 is a graph of the effect of BMS-986158 and JQ1 on CDK9 and the recruitment of RNA polymerase II on the HIV-1 LTR. Wherein FIG. 8A, FIG. 8B shows the relative enrichment of CDK9(A) and RNA polymerase II (B) binding on the HIV-1LTR as measured by ChIP assay with 1% formaldehyde added to crosslink DNA and protein 6 hours after BMS-986158(100nM) or JQ1(1 μ M) treatment of J-Lat cells, respectively.

FIG. 9 shows the effect of latent activation in combination with BMS-986158 and SAHA, Prostratin, JQ 1. After 48 hours of treatment of J-Lat cells with BMS-986158(10nM) alone or in combination with SAHA (600nM), Prostratin (10. mu.M), JQ1 (1. mu.M), respectively, the cells were harvested and the percentage of activated J-Lat cells was determined using flow cytometry.

FIG. 10 Effect of anti-HIV drugs on the latent activation effects of BMS-986158 and JQ 1. Wherein FIG. 10A, FIG. 10B shows the percentage of activated J-Lat cells detected by flow cytometry after treating J-Lat cells with BMS-986158(100nM) or JQ1(1 μ M) alone or in combination with AZT, NVP, and IDV, respectively, for 48 hours.

FIG. 11 is a graph of the effect of BMS-986158 on the antiviral efficacy of anti-HIV drugs. FIG. 11A shows the ELISA method for detecting the expression level of HIV-1p24 antigen in the culture supernatant of HIV-1 infected C8166 cells treated with BMS-986158 or anti-HIV drug alone/in combination for 72 hours; FIG. 11B shows the expression level of HIV-1p24 antigen in the culture supernatant after 72 hours of single/combined treatment of HIV-1 infected C8166 cells with YHP-1 or anti-HIV agent by ELISA;

FIG. 12 shows RT-qPCR assay for HIV-1RNA expression in cells of BMS-986158(100nM), JQ1 (1. mu.M) and DMSO treated PBMC isolated from AIDS patients 24 hours later;

FIG. 13 is a graph showing the effect of BMS-986158 and JQ1 on the activation of latent viruses in PBMCs isolated from HIV-1 infected subjects. After PBMC isolated from peripheral blood of AIDS patients chronically receiving ART treatment were treated with BMS-986158(100nM) or JQ1 (1. mu.M) for 24 hours, intracellular RNA was extracted and expression of cell-associated HIV-1RNA was examined using reverse transcription-quantitative PCR.

FIG. 14 shows the resting CD4 isolated from BMS-986158 and JQ1 in latently infected macaques (North Pingtian monkeys) ⁺ Activation Effect of latent Virus in T cells, wherein FIG. 14A FIG. 14B is BMS-986158(100nM) or JQ1 (1. mu.M) treatment of resting CD4 isolated from peripheral blood of SIV latently infected Macaca mulatta or HIV-1 latently infected Macaca pekinensis, respectively ⁺ After 24 hours of T-cell culture, intracellular RNA was extracted and cell-associated SIV/HIV-1RNA expression was detected using reverse transcription-quantitative PCR.

Detailed Description

Example 1 BMS-986158 activation of latent HIV-1 expression

Activation Effect of BMS-986158 on latent HIV-1 in three HIV-1 latent cell lines (J-Lat, ACH2 and OM10.1) the three cell lines J-Lat, ACH2 and OM10.1 were human T lymphocyte cell lines in RPMI-1640 medium plus 10% FBS.

In the activation experiment of J-Lat cells, 2X 10 cells were used ⁵ J-Lat cells were plated in 48-well cell culture plates, 5-fold dilutions of BMS-986158(500, 100, 20, 4, 0.8nM) were added to each well, using JQ1 as a positive drug control, and 5-fold dilutions were also set (5000, 0.8nM),1000. 200, 40, 8nM) while DMSO controls were set up. 5% CO at 37 ℃ ₂ After 48 hours in a humidified incubator, cells from each well were collected and analyzed by flow cytometry for the percentage of GFP-positive cells in each well. The collected cells were flow-tested 24, 48 and 72 hours after BMS-986158100 nM and JQ 11. mu.M treatment of J-Lat cells, respectively. Since changes in GFP expression in J-Lat cells could not be quantified by fluorescence microscopy, the activation effect of compounds on J-Lat cells was quantified by flow cytometry using the percentage of GFP positive cells and the EC for the activation effect of compounds was calculated ₅₀ (concentration of 50％maximal effect,EC ₅₀ ) The value is obtained.

Flow assay results showed that the percentage of GFP-positive cells increased significantly after BMS-986158 treatment, this change increased with increasing concentration (fig. 1A) and with increasing time (fig. 1B), and the positive control compound JQ1 also showed the same trend. EC of JQ1 compared to the positive control compound JQ1 ₅₀ Is 18.28 times of BMS-986158, and as can be seen from the dose-response curve and the time curve, the concentration of BMS-986158 is 1/10 of JQ1, the effect is still stronger than that of JQ1, which indicates that the latent activation activity of BMS-986158 is much higher than that of JQ 1.

In the activation experiment of ACH2 cell and OM10.1 cell, two kinds of cells were treated at 4X 10 ⁴ Cells were plated in 96-well cell culture plates per well, different concentrations of BMS-986158(100, 20, 4, 0.8, 0.16nM) were added to different wells, and different concentrations (1000, 200, 40, 8, 1.6nM) were also set for the DMSO control using JQ1 as a positive drug control. 5% CO at 37 deg.C ₂ After culturing for 48 hours in a humidified incubator, the culture supernatant was collected, and the content of HIV-1p24 in the supernatant was measured using an HIV-1 antigen-antibody detection kit (Wantai Bio, available from Kunming Jinjiang, Ltd.).

OM10.1 cell and ACH2 cell are latent cell lines carrying live HIV virus, the expression of HIV-1 is at a very low level before being stimulated by latent activator, and the expression of HIV-1 is rapidly increased after being treated by the latent activator. After ACH2 cells and 0M10.1 cells were treated with BMS-986158 for 48 hours, cell culture supernatants were collected and HIV-1 expression was confirmed by measuring the amount of HIV-1p24 in the supernatants. The results show that: in ACH2 cells and OM10.1 cells, compared with a DMSO control group, the content of HIV-1p24 in cell culture supernatant of a BMS-986158 treated group is remarkably increased; after treatment with BMS-986158 at different concentrations, the expression of latent HIV-1 in ACH2 cells and OM10.1 cells increased significantly and in a concentration-dependent manner (fig. 1C), the positive control compound JQ1 also showed similar activity (fig. 1D). It shows that BMS-986158 and JQ1 can promote the expression of latent HIV-1 after treating ACH2 cells and OM10.1 cells. Similar to the results on J-Lat cells, the BMS-986158 concentration is 1/10 of JQ1, and the activation effects on the two cells are similar, which shows that the activation effect of JQ1 on ACH2 cells and OM101.1 cells is also obviously stronger than that of JQ 1.

The results of the experiments show that BMS-986158 can activate the expression of latent HIV-1 in latent infected cell models of three HIV-1 pseudoviruses and live viruses, and the activation effect is obviously stronger than that of JQ 1. BMS-986158 is much more active than other BET inhibitors that have been published for their latent activation activity. And EC of BMS-986158 ₅₀ 17.28 times lower than JQ1, significantly stronger than these publicly published BET inhibitors.

Example 2 BMS-986158 toxicity test on human PBMC and J-Lat cells

Peripheral Blood (PBMC) was obtained by taking 10ml of Peripheral blood from a normal donor and separating the Peripheral blood by density gradient centrifugation [ review by the ethical committee on life sciences of kunming animal institute of china academy of sciences, serial No.: SWYX-2013016]. The isolated PBMC were resuspended in an appropriate volume 1640 (10% FBS) at 5X 10 ⁵ The amount of cells per well was added to 96-well plates containing BMS-986158 at different concentrations (concentrations: 100, 20, 4, 0.8, 0.16, 0.032. mu.M); for J-Lat cells, 4X 10 ⁴ Cell amount per well into 96-well plates containing BMS-986158 at various concentrations (concentration: 200, 40, 8, 1.6, 0.32, 0.064. mu.M) after 2 days of culture in an incubator, 20. mu.l of MTT solution was added to each well, 100. mu.l of supernatant was discarded after 4 hours of culture in the incubator, and 100. mu.l of 12% SDS-5 was added to each well0% DMF solution, followed by overnight incubation in an incubator, next day OD values per well were read using a microplate reader at 570/630nm wavelength, cell viability was calculated, and half the Cytotoxic Concentration (CC) was calculated (concentration of 50% cytotoxicity ₅₀ ). The results show that: BMS-986158 was less cytotoxic to PBMC at 2 days PBMC treatment, CC ₅₀ >100 mu M; has little toxicity to J-Lat cells and CC ₅₀ 43.85 μ M, well above its EC ₅₀ Values (see fig. 2).

Example 3 Effect of BMS-986158 on cells

3.1 Effect of BMS-986158 on T cell activation

BMS-986158 was less toxic to cell lines as well as PBMCs. The existing research shows that: some latent activators cause extensive T cell activation, and continued immune activation is a significant impediment to the therapeutic process of HIV-1, particularly with CD8 ⁺ Cell depletion is closely related (Xing and Siliciano 2013). To further evaluate the effect of BMS-986158 on immune cells, BMS-986158 was tested against CD4, respectively ⁺ And CD8 ⁺ Effect of activation marker (cell surface receptors CD25, CD69 and HLA-DR) expression of T cells. After PBMC were treated with BMS-986158 for 48 hours, cells were harvested, washed with PBS, a premix containing antibodies specific for the three receptors CD25, CD69 and HLA-DR was added, incubated on ice for 30 minutes, washed with large volumes of PBS, after which the cells were resuspended with 300 μ l PBS and the expression of the three cell surface receptors was detected using flow cytometry.

The results showed that, in CD4 ⁺ And CD8 ⁺ In T cells, after BMS-986158 treatment for 48 hours, the expression of CD25, CD69 and HLA-DR had no significant effect (FIG. 3), suggesting that BMS-986158 did not cause extensive cell activation, that BMS-986158 did not promote T cell immune activation to accelerate T cell exhaustion, and that BMS-986158 did not burden the immune system of HIV-1 infected patients.

3.2 Effect of BMS-986158 on the auxiliary receptors required for HIV-1 entry

CXCR4 and CCR5 are co-receptors necessary for HIV-1 entry into cells for X4 tropism and R5 tropism, respectively, and in order to further understand the effect of BMS-986158 on immune cells, the present invention also examined changes in the expression of BMS-986158 on co-receptors necessary for HIV-1 entry. After 48 hours of BMS-986158 treatment of PBMCs, the cells were washed with PBS, then resuspended by adding a premix containing CXCR4 and CCR5 antibodies, incubated on ice for 30 minutes, washed with a large volume of PBS, then resuspended with 300. mu.l PBS, and the expression of both cell surface receptors was examined using flow cytometry. The expression of these two receptors was examined in the present invention and BMS-986158 was found to have no significant effect on the expression of the CCR5 receptor (FIG. 4B), but slightly reduced the expression of CXCR4 (FIG. 4A), which means that latent HIV-1 is activated without increasing the risk of HIV-1 infecting surrounding cells due to upregulation of the co-receptor.

The above experimental results show that: the latent activating activity, cytotoxicity and influence on immune cells of BMS-986158 are researched, and the latent activating agent is confirmed to be a high-efficiency and low-toxicity latent activating agent.

Example 4 mechanism study of BMS-986158 to exert the effect of latent activation

4.1 inhibitory Activity of BMS-986158 on BRD2/4

The inhibitory activity of BMS-986158 on BRD2/4 has not been reported in the prior art. In order to confirm whether BMS-986158 can directly inhibit the activity of BRD2/4, the inhibition of BMS-986158 on the activity of BRD2/4 is detected by using a time-lapse fluorescence energy resonance transfer experiment. As shown in Table 1, BMS-986158 has significant inhibitory effects on both BRD2 and BRD4, and IC is the inhibitory effect on both proteins ₅₀ (concentration of 50％inhibitory,IC ₅₀ ) 1.17nM and 2.75nM, respectively, the control compound JQ1 also showed inhibitory effect on both, IC ₅₀ 30.18nM and 62.71nM, respectively. IC of JQ1 for BRD2/4 inhibitory Activity ₅₀ IC of BMS-986158 ₅₀ This is a factor of 20 higher, similar to the latent activation activity observed on J-Lat cells, suggesting that the latent activation activity of BMS-986158 may be related to its inhibitory activity on BRD 2/4.

Inhibitory Activity of the Compounds of Table 1 on BRD2/4

4.2 Effect of BMS-986158 on the activation of BRD4 and P-TEFb

The strength of the inhibitory activity of BMS-986158 and JQ1 on BRD2/4 is consistent with the strength of the inhibitory activity of the two on J-Lat cells, suggesting that BMS-986158 may exert its latent activation through a similar mechanism as JQ 1. The invention firstly detects the influence of BMS-986158 on the activation of BRD4, namely BMS-986158 treats J-Lat cells for 24 hours, then the cells are collected, PBS is used for washing, total protein in the cells is extracted, and the expression conditions of BRD4 and phosphorylated BRD4 are detected through a WB experiment. As shown in fig. 5, there was no significant change in expression of phosphorylated BRD4 after 24 hours of BMS-986158 treatment, whereas total BRD4 protein expression was increased and the proportion of phosphorylated BRD4 (i.e., activated BRD4) to total BRD4 was decreased, i.e., BMS-986158 inhibited the level of activation of BRD 4.

There are many reports in the literature that BET inhibitors promote the activation of P-TEFb. The invention detects the expression of two subunits CDK9 and cyclin T1 of P-TEFb, collects cells after BMS-986158 treats J-Lat cells for 24 hours, extracts total protein in the cells after PBS washing, and detects the expression of cyclin T1, CDK9 and phosphorylated CDK9 through WB experiments. The experimental results show that after BMS-986158 treatment on J-Lat cells for 24 hours, the expression of cyclin T1 protein in the cells is not obviously changed, and the total CDK9 protein is slightly up-regulated after 500nM BMS-986158 treatment (FIG. 6). The expression of phosphorylated CDK9 increased significantly, and the upregulation was more pronounced at higher concentrations of BMS-986158 (fig. 7). This result indicates that BMS-986158 significantly promoted activation of CDK9, i.e., activation of P-TEFb, suggesting that BMS-986158 promoted transcription of HIV-1 by promoting activation of P-TEFb.

4.3 BMS-986158 recruitment of CDK9 and RNAPII on the HIV-1LTR

Activation of P-TEFb is key to the smooth progression of HIV-1 transcriptional elongation, and recruitment of P-TEFb on the LTR is also required. To confirm whether BMS-986158 promoted recruitment and activation of P-TEFb and RNA polymerase II on the LTR, changes in CDK9 and activated RNAPII binding on the LTR were detected by chromatin co-immunoprecipitation experiments. Treating J-Lat cells for 6 hours by BMS-986158, adding 1% formaldehyde to crosslink DNA and proteins in the cells, adding glycine to stop crosslinking after 10 minutes at room temperature, washing the cells for 2 times by PBS, adding a lysate to crack cell membranes, centrifuging to collect cell nuclei, adding micrococcal nuclease to cut the DNA into fragments of 150-900 bp, extracting purified DNA, adding CDK9 or RNAPII antibodies, incubating overnight at 4 ℃, separating a crosslinked complex bound with the two antibodies by magnetic beads, eluting the crosslinked complex from the magnetic beads, and detecting the content of a target fragment (HIV-1LTR) (namely the amount of the two proteins crosslinked with the target fragment) in the complex by a quantitative PCR experiment.

As shown in fig. 8, CDK9 on HIV-1LTR was significantly increased and, in addition, RNA polymerase binding on LTR was also significantly increased after BMS-986158 treated J-Lat cells for 6 hours, suggesting that BMS-986158 could promote P-TEFb and RNA polymerase recruitment and activation on HIV-1 LTR. These results suggest that BMS-986158 may be activated by inhibiting the activity of BRD4 and activating P-TEFb, promoting the recruitment of Tat to P-TEFb and further promoting the activation of RNA polymerase II on LTR, and finally showing the activity of promoting the transcriptional elongation of HIV-1.

Example 5 Effect of BMS-986158 in combination with other types of latent activators

The activity, toxicity and action mechanism of BMS-986158 were studied in the foregoing, and all these results suggest that BMS-986158 is a latent activator with potential to be clinically applied in the future. According to the results of the current clinical trial, it is difficult to effectively activate the expression of latent HIV-1 in vivo using a single latent activator, similar to the synergistic effect of ART therapy when different latent activators are combined or perhaps combined, and the present invention uses the Zhou's intermediate efficacy model to study the effect of BMS-986158 in combination with three different types of latent activators (histone deacetylase inhibitor SAHA, PKC agonist Prostratin, and BET inhibitor JQ 1). BMS-986158, SAHA, Prostratin and JQ1 are single medicine groups respectively, BMS-986158 is combined with other three compounds respectively to form three combined medicine groups, and a negative control group without medicines is arranged at the same time, and the eight groups are divided into eight groups. The percentage of GFP positive J-Lat cells in each of the 8 groups was determined by flow cytometry 48 hours after treatment of the J-Lat cells with the corresponding compounds in each group.

As shown in FIG. 9, BMS-986158(10nM), SAHA (600nM), Prostratin (10. mu.M) and JQ1 (1. mu.M) alone exhibited 18.23%, 5.05%, 47.20% and 62.63% activation of J-Lat cells, whereas BMS-986158 combined with SAHA exhibited a significant increase in the latent activation effect up to 45.37%, and showed a similar effect to that exhibited by Prostratin, an increase in the latent activation effect up to 93.37%, whereas the increase in the latent activation effect was not significant and only up to 67.7% when combined with the same latent activator, JQ 1. The Zhou's mesogenic model is a model developed by Chou et al for evaluating interactions between compounds ^[7] After analyzing the synergistic effect of BMS-986158 and three latent activators respectively by using a Zhou's mesogenic model, BMS-986158 is found to show a strong synergistic effect with SAHA and Prostratin and show an additive effect when combined with JQ1, which is a common phenomenon in the similar compounds (Table 2). The result shows that BMS-986158 shows a synergistic effect when being combined with different types of latent activators, and in the future treatment process, the optimal concentration can be selected to exert the best latent activation effect, and the side effect brought by the compound can be reduced by properly reducing the dosage of a single medicine on the premise of ensuring the curative effect.

TABLE 2 combination index of combination of latent activators

Example 6 interplay of anti-HIV-1 drugs with the latent reactivation Effect of BMS-986158

"activation and kill" strategy in clinical use, in addition to the use of latent activators to activate the expression of latent HIV-1, patients need continued antiviral therapy to prevent the spread of activated HIV-1.

6.1 Effect of antiviral Agents on the latent activation Effect of BMS-986158

In order to confirm the interaction between BMS-986158 and the antiviral drug, the present invention examined the effect of the antiviral drug on the latent activation effect of BMS-986158.

In the activation experiment of J-Lat cells, 2X 10 cells were used ⁵ J-Lat cells were plated in 48 well cell culture plates per well, BMS-986158(100nM) was added to each well, and J-Lat cells were treated with JQ1 as a positive drug control (1. mu.M), BMS-986158 and JQ1, respectively, in combination with three antiviral drugs, AZT, NVP and IDV. 5% CO at 37 ℃ ₂ After 48 hours in a humidified incubator, cells from each well were collected and analyzed by flow cytometry for the percentage of GFP-positive cells in each well. The results showed that the percentage of GFP-positive cells was not significantly changed in J-Lat cells in cells from the group co-treated with AZT, NVP and IDV with BMS-986158 compared to cells from the group treated with BMS-986158 alone, and similar results were seen in the JQ 1-positive drug control group (fig. 10A, fig. 10B). The results indicate that antiviral drugs do not affect the latent activation effect of BMS-986158, suggesting that BMS-986158 does not affect therapeutic efficacy in future aids virus latency pool clearance strategies due to the persistence of antiviral therapy.

6.2 Effect of BMS-986158 on antiviral drugs

After confirming that the antiviral drug did not affect the latent activation effect of BMS-986158, the present invention further confirmed whether BMS-986158 affected the antiviral effect of the antiviral drug. The three antiviral drugs mentioned above were also used to detect HIV-1 alone or in combination with BMS-986158 _IIIB Inhibitory effect on replication in C8166 cells. YHP-1 was also used as a control experiment. YHP-1 is a BET inhibitor, and a significant latent activation effect was reported in the previous patent ^[8] 。

In the antiviral activity test, HIV-1 was first used _IIIB Virus infects C8166 cells for 3 hours, cells are washed 3 times with PBS to remove free virus, 1X 10 ⁵ C8166 cells were plated in 48-well plates and EFV (0.5 nM), RAL (5nM), DRV (20nM), BMS-986158(10nM) or YHP-1(1 μ M) single drug, BMS-985158 or YHP-1, respectively, was added to each wellA dual drug in combination with three other drugs. 5% CO at 37 ℃ ₂ After 72 hours of incubation in a humidified incubator, cell culture supernatants were collected and tested for expression of HIV-1p24 in the supernatants using ELISA assays.

The results are shown in FIG. 11, and BMS-986158 in combination with anti-HIV agents significantly enhanced the antiviral effects of the three anti-HIV agents, as shown in FIG. 11A, whereas the same compound YHP-1 did not exhibit this effect, as shown in FIG. 11B. When three antiviral drugs are used alone, it can be used for treating HIV-1 _IIIB The replication inhibitory effect was about 10%, while BMS-986158 in combination with them had an HIV-1 inhibitory effect _IIIB The replication inhibition increased to around 90%, which was unexpected to show that BMS-986158 did not only decrease the effect of the antiviral drug, but also significantly enhanced the effect, suggesting that BMS-986158 may have a better effect than thought in clinical applications.

There have been previous reports of the effect of BET inhibitors on the efficacy of antiviral drugs, but the results indicate that the BET inhibitor Apabetalone has no significant effect on the efficacy of anti-HIV drugs ^[9] . The prior art does not report that a BET inhibitor can enhance the antiviral effect of an anti-HIV drug, which means that when the BET inhibitor is clinically applied to anti-HIV treatment, HIV activated by BMS-986158 is more difficult to break through the blockage of the anti-HIV drug on the transmission of the HIV drug, and the elimination of a latent virus bank is facilitated.

Example 7 Effect of BMS-986158 on latent HIV-1 in human and monkey-isolated cells

7.1 latent reactivation Effect of BMS-986158 on human PBMCs

Peripheral blood 10ml was taken from an AIDS patient receiving HAART for a long period of time, and Peripheral Blood Mononuclear Cells (PBMC) were obtained by density gradient centrifugation [ examined and approved by the ethical Committee of Kunming animal research institute of China academy of sciences, No.: SWYX-2013016 ]. After BMS (100nM), JQ1 (1. mu.M) and DMSO (negative control) were directly treated with the isolated PBMC in an appropriate volume 1640 (10% FBS) for 24 hours, intracellular RNA was extracted using Trizol method and quantitative RT-qPCR detection of HIV-1RNA was performed.

The primer and probe sequences are as follows:

the qPCR reaction system was as follows:

the qPCR reaction procedure was as follows:

the results show that after BMS-986158 treatment, the expression of HIV-1 in PBMC of 6 patients out of 10 patients is remarkably increased compared with that of a DMSO treatment group, and the expression amount is 1.4-85.86 times that of a negative control group; after the JQ1 positive control treatment group, HIV-1 expression in PBMCs of 5 of 10 patients is remarkably increased, and the expression amount is 2.61-136.22 times of that of the negative control group (FIG. 12). The above results indicate that BMS-986158 also significantly activates the expression of latent HIV-1 in PBMCs isolated from AIDS patients treated with HAART for a long period of time (HIV virus is in a latent state).

Considering the clinical application scenario of the compound BMS-986158, the present invention examined the activation effect of BMS-986158 on latent HIV-1 in PBMCs isolated from the blood of patients chronically treated with ART in order to more closely approximate the reality of the patient. After 24 hours of PBMC treatment with BMS-986158, total intracellular RNA was extracted and changes in HIV-1RNA expression were detected using reverse transcription-quantitative PCR.

The results are shown in FIG. 13, and show that BMS-986158 can activate the expression of latent HIV-1 in PBMCs of 5 of 10 patients as JQ1, and further verify the latent activation activity of BMS-986158.

7.2 BMS-986158 resting CD4 isolated in vivo against latently infected macaques and North Pingtian climate ⁺ Latent activation Effect of T cells

Thanks to the established HIV-1 latent infection northern flathead monkey model in the early laboratory and the macaque model of SIV latent infection, the present invention attempts to evaluate the effect of BMS-986158 as a latent activator at the animal model level using the models. First, resting CD4 separated from peripheral blood of animal model is collected ⁺ T cells, and detecting the activation effect of BMS-986158 on latent HIV-1/SIV. BMS-986158 treatment of resting CD4 ⁺ After 24 hours of T cells, total RNA was extracted from the cells and changes in HIV-1/SIV RNA expression were detected by reverse transcription-quantitative PCR.

The results are shown in FIG. 14, resting CD4 of 7 HIV-1 latently infected northern flathead monkeys ⁺ BMS-986158 activates 4 resting CD4 in T cells ⁺ Expression of latent HIV-1 in T cells; and resting CD4 in 4 macaques latently infected with SIV ⁺ BMS-986158 was able to activate resting CD4 in 3 individuals in T cells ⁺ Latent SIV expression in T cells. These results indicate that BMS-986158 also significantly activates the expression of latent HIV/SIV in latent cells isolated from peripheral blood of latently infected animal models.

Claims

Use of BMS-9861658 for the manufacture of a medicament against HIV-1 latency.
2. Use according to claim 1, characterized in that: the BMS-9861658 and histone deacetylase inhibitor SAHA and PKC agonist Prostratin are used in the preparation of anti-HIV-1 latent drugs.
3. Use according to claim 1, characterized in that: the application of the BMS-9861658 and anti-HIV drug combination in preparing anti-HIV-1 drugs comprises the following steps: zidovudine, Nevirapine, Indinavir, Efavirenz, Raltegravir and Darunavir.