HIV-1 latent infection activator, medicament and use

Abstract

The application relates to an HIV-1 latent infection activator, a medicine and purposes. An HIV-1 latent infection activator comprises ISX-9 and / or ML327. The HIV-1 latent infection activator further comprises an HDAC inhibitor and / or a BET inhibitor. The HDAC inhibitor is quinostat. The BET inhibitor is birabresib or AZD5153. A medicine comprises the HIV-1 latent infection activator and an HIV-1 inhibitor. The HIV-1 inhibitor is a polypeptide EKL1C, and the amino acid sequence is shown in SEQ ID NO:1. The HIV-1 inhibitor comprises one or more of zidovudine, nevirapine or indinavir. The HIV-1 latent infection activator provided by the application can activate latent HIV, and is a new safe and efficient HIV-1 latent infection activator.

Description

Technical Field

[0001] This application relates to the field of small molecule drug technology, specifically to an HIV-1 latent infection activator, drug, and its uses. Background Technology

[0002] Acquired immune deficiency syndrome (AIDS), also known as HIV / AIDS, is a serious infectious disease caused by the human immunodeficiency virus (HIV), posing a significant threat to human health. Due to the HIV virus's extensive mutation and replication capabilities, its ability to establish lifelong infection, and the scale of pandemics, viral strains have diversified considerably over time, presenting immense challenges to prevention and treatment. Currently, the most effective treatment for AIDS is combination antiretroviral therapy (cART). cART can effectively reduce viremia to below the clinical detection limit; however, due to factors such as the latent viral reservoir (LVR), cART cannot completely eradicate HIV-1 from the body.

[0003] Latent infection is a major obstacle to HIV-1 cure. To eliminate the latent HIV reservoir and achieve a functional cure for AIDS, various strategies have been proposed, including the "shock and kill" strategy. The "shock and kill" strategy primarily utilizes various latent infection activators or latency-reversing agents (LRAs) to reactivate latent HIV-1, which is then cleared through antiretroviral therapy and the host's immune function. This strategy is effective when combined with antiviral drugs. The main purpose of LRAs is to specifically reactivate proviral transcription without adversely affecting cellular homeostasis. However, the application of various LRAs currently developed faces many challenges, such as the inability to reduce the latent reservoir, poor diversity, and toxic side effects. Therefore, continuing to develop new, more effective, and safer LRAs, and combining LRAs with antiviral drugs in the "shock and kill" strategy, is an effective treatment approach for achieving a functional cure for AIDS.

[0004] ISX-9 (Isoxazole 9, CAS: 832115-62-5, Molecular Formula: C 11 H 10 N2O2S is a specific neural stem cell differentiation inducer that activates voltage-gated Ca2+. 2+The channel and NMDA receptor promote NeuroD expression and regulate the Notch signaling pathway, exhibiting significant activity in epicardial-derived cell differentiation.137 Currently, no reports have documented its use as an activator of latent HIV-1 infection.

[0005] ML-327 (ML327, CAS: 1883510-31-3, Molecular Formula: C 19 H 18 N4O4 is a small molecule inhibitor of isoxazoline class. Literature reports that it can inhibit tumor cell proliferation by antagonizing MYC transcriptional activity and has demonstrated anti-metastatic effects in preclinical models of breast cancer and colon cancer. Currently, there are no reports on its use as an activator of latent HIV-1 infection. Summary of the Invention

[0006] This application discloses an HIV-1 latent infection activator, a drug, and its uses, exploring new, safe, and effective HIV-1 latent infection activators that can be used in combination with HIV-1 inhibitors to inhibit the replication of activated HIV-1, providing new research and development ideas for the continued development of new and more effective AIDS treatment strategies.

[0007] In one aspect, this application relates to an HIV-1 latent infection activator comprising ISX-9 and / or ML327.

[0008] Furthermore, HIV-1 latent infection activators also include HDAC inhibitors and / or BET inhibitors.

[0009] Furthermore, the HDAC inhibitor is quinolone.

[0010] Furthermore, the BET inhibitor is bilabrecib or AZD5153.

[0011] Secondly, this application relates to a medicine comprising any one of the aforementioned HIV-1 latent infection activators and HIV-1 inhibitors.

[0012] Furthermore, the HIV-1 inhibitor is a polypeptide EKL1C, the amino acid sequence of which is shown in SEQ ID NO:1.

[0013] Furthermore, the HIV-1 inhibitor includes one or more of zidovudine, nevirapine, or indinavir.

[0014] Furthermore, its dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, suppositories, or lyophilized powder injections.

[0015] Thirdly, this application relates to the use of any of the above-mentioned HIV-1 latent infection activators, or any of the above-mentioned drugs, in the preparation of a medicament for the prevention and / or treatment of diseases related to HIV infection.

[0016] Furthermore, the HIV virus is HIV-1.

[0017] This application's HIV-1 latent infection activator can activate latent HIV, and is a novel, safe, and highly effective HIV-1 latent infection activator. Furthermore, when ISX-9 and / or ML327 in this HIV-1 latent infection activator are used in combination with HDAC inhibitors and / or BET inhibitors, the activation effect can be enhanced and a synergistic activation effect can be produced. Moreover, this application's HIV-1 latent infection activator does not induce T cell activation or the release of pro-inflammatory cytokines.

[0018] The drug proposed in this application can inhibit the replication of activated HIV-1 on target cells, providing a new drug combination option for the treatment of AIDS.

[0019] This application can be used in the preparation of drugs for the prevention and / or treatment of diseases related to HIV infection, in conjunction with a "shock and kill" strategy, to increase the diversity of treatment strategies and potential efficacy. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is the result of the first round of screening in Example 1;

[0022] Figure 2 This is the result of the second round of screening in Example 1;

[0023] Figure 3 The results of the second round of screening for compounds in Example 1;

[0024] Figure 4 The activation effect test results of ISX-9 and ML327 in Example 1 are shown.

[0025] Figure 5 The results of the in vitro activation effect test of ISX-9 and ML327 in Example 2;

[0026] Figure 6 The results of ISX-9 and ML327 cytotoxicity assays in Example 3 are shown.

[0027] Figure 7 The results of the combined application analysis of different LRAs in Example 4;

[0028] Figure 8 CD4 of Example 5 + Schematic diagram of the T cell sorting process;

[0029] Figure 9 CD4 of Example 5 + Results of T cell activation detection;

[0030] Figure 10 CD4 of Example 6 + Results of T cell cytokine TNF-α detection;

[0031] Figure 11 CD4 of Example 6 + Results of IFN-γ cytokine detection in T cells;

[0032] Figure 12 CD4 of Example 6 + Results of IL-10 assay in T cells;

[0033] Figure 13 The results of the drug inhibitory activity test in Example 7 are shown. Detailed Implementation

[0034] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and are not intended to limit the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply.

[0035] An HIV-1 latent infection activator comprising ISX-9 and / or ML327. The chemical formula of ISX-9 is C1. 11 H 10 N2O2S, structural formula as follows:

[0036]

[0037] The chemical formula of ML327 is C 19 H 18 N4O4, structural formula as follows:

[0038]

[0039] HIV-1 latent infection activators also include HDAC inhibitors and / or BET inhibitors. The HDAC inhibitor is quinolone. Quinolone's chemical formula is C60. 21 H 26N6O2, structural formula as follows:

[0040]

[0041] BET inhibitors include bisabrecib or AZD5153. The chemical formula of bisabrecib is C2. 25 H 22 C l N5O2S, structural formula as follows:

[0042]

[0043] The chemical formula of AZD5153 is C 36 H 41 N7O6, structural formula as follows:

[0044]

[0045] A drug comprising any one of the above-mentioned HIV-1 latent infection activators and HIV-1 inhibitors. The HIV-1 inhibitor may be a polypeptide EKL1C, with the amino acid sequence shown in SEQ ID NO:1: NVTFLDLEYEMKKLEEAIKKLEESYIDLKELGTYEY. The HIV-1 inhibitor may also be one or more of zidovudine, nevirapine, or indinavir. The drug dosage form may be tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal formulations, suppositories, or lyophilized powder for injection.

[0046] The J-Lat A2 and ACH-2 cell lines were provided by the National Institutes of Health (NIH) AIDS Reagent Program. J-Lat C11 cells were donated by Professor Huanzhang Zhu of the School of Life Sciences, Fudan University. All three cell lines are latent HIV reservoir cell lines. PBMCs were obtained from healthy human donors and provided by the Department of Gastroenterology, Jing'an District Central Hospital, Shanghai (ethics approval number: 2022-029). TZM-Bl cells were provided by the National Institutes of Health (NIH) AIDS Reagent Program.

[0047] Example 1

[0048] I. Screening for novel HIV-1 LRAs using the latently infected cell model J-Lat A2

[0049] Experimental methods:

[0050] (1) Dilute compound ISX-9 / ML327 to a final concentration of 40 μM with PBS in a 96-well round-bottom plate;

[0051] (2) Take 50 μL of the diluted compound and add it to a 96-well cell culture plate, and add an equal volume of 1% penicillin / streptomycin RPMI 1640 medium (purchased from Meilun Biotechnology, China);

[0052] (3) J-Lat A2 cells were divided into 5×10 4 Add the diluted compound at a density of 100 μL / well to bring the final concentration of the compound to 10 μM.

[0053] (4) Pabistat and 10 ng / ml TNF-α were set as positive controls; 0.1% DMSO was set as a negative control;

[0054] (5) After culturing the cells in a 37°C, 5% CO2 incubator for 48 hours, the expression of GFP in J-Lat A2 cells was observed using an inverted fluorescence microscope to determine whether the compound could activate the HIV-1 virus reservoir.

[0055] Results and Analysis:

[0056] like Figure 1 As shown, by using the J-Lat A2 cell line to perform the first round of screening of classic known active libraries, 177 candidate molecules that can reactivate GFP expression in J-Lat A2 cells were obtained in the first round of screening.

[0057] II. Screening for novel HIV-1 LRA using the latently infected cell model ACH2

[0058] Experimental methods:

[0059] (1) Dilute the compounds selected in the first round of screening in 1.5 ml EP tubes with PBS to a final concentration of 40 μM;

[0060] (2) Add 50 μL of the compound at the specified concentration to a 96-well cell culture plate and add an equal volume of 1% penicillin / streptomycin RPMI 1640 medium.

[0061] (3) Pabistat was set as a positive control; 0.1% DMSO was set as a negative control;

[0062] (4) Add 100 μL of 5×10 5 ACH2 cells per mL were added to a cell culture plate and the cells were placed in an incubator at 37°C and 5% CO2 for 48 h.

[0063] (5) Collect 50 μL of cell supernatant and mix it with an equal volume of 5% Triton X-100, and lyse the cells overnight at 4°C;

[0064] (6) Human anti-HIV-1 IgG antibody (HIVIG) was diluted with NaHCO3 (pH 9.6) buffer at 5 μg / mL and coated onto ELISA plates at 50 μL / well overnight at 4°C.

[0065] (7) Wash the ELISA plate 3 times with PBST using an ELISA plate washer, add 150 μL of 5% skim milk to each well and block at 37°C for 2 h;

[0066] (8) Wash the ELISA plate 3 times with PBST, add 40 μL of PBS to each well, then add 10 μL of lysed cell supernatant, and incubate at 37°C for 1 h.

[0067] (9) Wash the ELISA plate three times with PBST using a plate washer, add 50 μL of 3 μg / mL 183-12H-5C primary antibody to each well, and react at 37℃ for 1 h.

[0068] (10) Wash the ELISA plate 3 times with PBST, and dilute the HRP-labeled rabbit anti-mouse IgG secondary antibody with PBS at a ratio of 1:4000, 50 μL / well, and react at 37℃ for 1 h;

[0069] (11) Wash the ELISA plate three times with PBST using an ELISA plate washer, add 50 μL TMB of chromogenic substrate to each well, and react at room temperature for 5–10 min.

[0070] (12) Add 50 μL / well of 1N H2SO4 stop solution to the ELISA plate;

[0071] (13) Use a multi-detection microplate reader to read the absorbance at 450 nm.

[0072] Results and Analysis:

[0073] like Figure 2 and Figure 3 As shown, in the second round of screening, 65 compounds were found to reactivate latent HIV-1 virus, including compounds from different categories such as histone deacetylase inhibitors (HDACi), bromodomains and extra-terminal motif protein inhibitors (BETi), and protein kinase C (PKC) agonists.

[0074] III. ELISA assay to determine the reactivation effect of compounds on ACH2 cells in latently infected cells

[0075] Experimental methods:

[0076] (1) The compounds selected in the second round of screening were serially diluted twice in a 96-well cell culture plate with a final concentration of 40 μM in the first well. Eight dilution gradients were set up, and three replicate wells were set up for each concentration.

[0077] (2) Add an equal volume of 1% penicillin / streptomycin RPMI 1640 medium to each well;

[0078] (3) Pabistat was set as a positive control; 0.1% DMSO was set as a negative control;

[0079] (4) Add 100 μL of 5×10 5 / mLACH2 cells were added to a 96-well plate along with 50 μL of the specified concentration of the compound and incubated for 48 h.

[0080] (5) Collect 50 μL of cell supernatant and mix it with an equal volume of 5% Triton X-100. Incubate overnight at 4°C for lysis. The ELISA detection method is as described above.

[0081] Results and Analysis:

[0082] The selected compounds were then tested for their activation effects on the latently infected cell model ACH2. The results are as follows: Figure 4 As shown, this application identifies two novel HIV-1 latent infection activators, ISX-9 and ML327, that can effectively reactivate latent HIV-1 in ACH2 cells.

[0083] Example 2

[0084] Flow cytometry was used to detect the in vitro activation effect of the compound on HIV-1 LVR.

[0085] Experimental methods:

[0086] (1) Compounds ISX-9 and ML327 were serially diluted in 96-well cell plates with a final concentration of 40 μM in the first well. Three replicate wells were set for each concentration. The 0.1% DMSO treatment group was set as the control group.

[0087] (2) Add 50 μL of RPMI 1640 medium containing 1% penicillin / streptomycin to each well.

[0088] (3) Add 100 μL of 5×10 4 J-Lat A2 and J-Lat C11 cells per mL were incubated with the specified concentration of the compound in 96-well plates for 48 h.

[0089] (4) Using BD LSRFortessaTM The expression of GFP-positive cells was detected by flow cytometry, and the results were analyzed using FlowJo software (v10.0.7).

[0090] Results and Analysis:

[0091] The novel LRAs ISX-9 and ML327 can effectively reactivate the HIV-1 latent reservoir, such as Figure 5 As shown, ISX-9 can induce up to 80% and 40% of GFP-positive cells in J-LatA2 and J-Lat C11 cells, respectively, while ML327 can induce up to 85% and 50% of GFP-positive cells in J-Lat A2 and J-Lat C11 cells, respectively.

[0092] Example 3

[0093] Cytotoxicity assays of compounds ISX-9 and ML327.

[0094] Experimental methods:

[0095] (1) The compound was diluted in a 96-well cell culture plate, with three replicate wells for each concentration. The cell wells were set as the positive group and 1% DMSO as the negative group.

[0096] (2) Add 100 μL of 5×10 5 ACH2, J-Lat A2, and J-Lat C11 cells at a density of cells / mL, 1×10 6 Resting CD4 at a density of 1 / mL + T cells were added to the diluted compound and cultured at 37°C and 5% CO2 for 48 hours.

[0097] (3) Dilute CCK8 reagent 5 times with RPMI 1640 medium containing 1% penicillin / streptomycin, add 50 μL to each well, and continue to incubate at 37℃ for 4-6 h.

[0098] (4) Measure the absorbance at 450 nm using an ELISA reader and calculate the toxicity of different compounds to cells. Calculate cell viability using the formula: [(OD value of experimental group - OD value of negative group) / (OD value of positive group - OD value of negative group)] × 100%. Calculate the half-maximal toxicity concentration (50% cytotoxicity concentration, CC50) of the compounds using Calcusyn software (Biosoft) and plot the curves using GraphPadPrism 8.0.

[0099] Results and Analysis:

[0100] like Figure 6As shown in Figure A, ISX-9 showed CC activity in ACH2, J-Lat A2, and J-Lat C11 cells. 50 The concentrations were 12.2 μM, 130.0 μM, and 33.8 μM, respectively. Figure 6 As shown in B, ML327 is involved in C-cell activity in ACH2, J-Lat A2, and J-Lat C11 cells. 50 The effective concentrations were 8.4 μM, 74.0 μM, and 35.2 μM, respectively. ISX-9 and ML327 exhibited low cytotoxicity in J-Lat A2 and J-Lat C11 cells, and their reactivation efficiency was not affected by drug toxicity.

[0101] Example 4

[0102] Combined application of different LRAs.

[0103] Experimental methods:

[0104] (1) In a 96-well cell culture plate, ISX-9 and ML327 were serially diluted twofold with a final concentration of 10 μM in the first well, quinosat and AZD5153 were serially diluted with a final concentration of 0.1 μM in the first well, and birabreseib was serially diluted with a final concentration of 1 μM in the first well. Eight dilution gradients were set up, and three replicate wells were set up for each concentration.

[0105] (2) The compounds were combined into different types of LRAs: ISX-9-quinolactone, ISX-9-bilabrecib, ISX-9-AZD5153, ML327-quinolactone, ML327-bilabrecib, ML327-AZD5153, and added to 96-well cell culture plates at the final concentration of (1) for two-fold serial dilution. Eight dilution gradients were set up, and three replicate wells were set up for each concentration.

[0106] (3) Add 100 μL of 5×10 4 J-Lat A2 cells per mL were added to 96-well cell culture plates and cultured at 37°C and 5% CO2 for 48 h.

[0107] (4) Using BD LSRFortessa TM The expression of GFP in GFP-positive cells was detected by flow cytometry, and the results were analyzed using FlowJo software (v10.0.7).

[0108] (5) Use Calcusyn software to calculate the combination index (CI) of LRAs and use GraphPad Prism 8.0 to draw a bar chart.

[0109] Results and Analysis:

[0110] This application combines the selected novel LRAs ISX-9 and ML327 with other types of LRAs, including HDACi quinolactone and BETi prabraspirone and AZD5153. The results are as follows... Figure 7 As shown in Table 1, combinations of LRAs with different mechanisms of action have a synergistic effect, and combination therapy can enhance the reactivation of the HIV-1 latent reservoir.

[0111] Table 1

[0112]

[0113] Note: Each sample was made in triplicate, and the experiment was repeated three times. Data from representative experiments are expressed as mean ± SD.

[0114] Example 5

[0115] I. Isolation and Culture of PBMCs

[0116] Experimental methods:

[0117] (1) Prepare 10 mL of fresh human anticoagulated peripheral blood and dilute the sample with PBS at a ratio of 1:1;

[0118] (2) Gently add the human lymphocyte separation solution into a 50mL centrifuge tube, and then carefully add an equal volume of peripheral blood along the side wall using a Pasteur tube. The human lymphocyte separation solution and peripheral blood will form obvious stratification due to their different densities.

[0119] (3) Centrifuge at 2000 rpm for 20 minutes. Note that the acceleration should be set to 9 and the deceleration should be set to no brake.

[0120] (4) After centrifugation, different components will be clearly separated into layers. Carefully aspirate the PBMC cells into a clean 15mL centrifuge tube using a pipette.

[0121] (5) Add 10 mL of PBS, centrifuge at 1600 rpm for 5 min, discard the supernatant, and repeat the washing once;

[0122] (6) Resuspend the cells in RPMI 1640 medium containing 10% FBS and 1% penicillin / streptomycin for subsequent experiments.

[0123] II. Resting CD4 + T cell sorting

[0124] Experimental methods:

[0125] (1) Collect PBMC cells into a 15mL centrifuge tube, centrifuge at 1600rpm for 5min, discard the supernatant, and wash the cells once with PBS.

[0126] (2) Resuspend the cells in 500 μL PBS;

[0127] (3) Dilute CD4-FITC / CD25-APC / CD69-PE / HLA-DR Monoclonal Antibody (LN3), APC-eFluor 780 antibody at a ratio of 1:100, and dilute Fixable Viability Dye eFluor 506-PB antibody at a ratio of 1:1,000, and then stain PBMC cells. Incubate at 4°C in the dark for 30 min.

[0128] (4) After washing the cells twice with 0.5% FBS in PBS, the cell density was adjusted to 10⁻⁶ cells / mL by counting. 7 cells / mL;

[0129] (5) Filter the cells with a flow cytometer to remove cell clumps and place them on ice to await the machine.

[0130] (6) Prepare a collection tube and add 2 ml of 3% penicillin / streptomycin RPMI 1640 medium;

[0131] (7) Flow cytometry sorting and gating strategy: Non-adhesive PBMCs on the diagonal of the sorting interface are gated for subsequent gating, and CD4+Fixable Viability Dye eFluor 506-HLA-DR Monoclonal Antibody (LN3) and APC-eFluor 780- CD4+ cells with good activity are further gated. + T cells were finally collected, including resting CD25-CD69- CD4+ cells. + T cells.

[0132] Results and Analysis:

[0133] The results are as follows Figure 8 As shown, first, the approximate location of T cells is circled in the first gating area. Then, non-adhesive PBMCs are selected on the diagonal of the second gating area. Finally, highly viable CD4 cells are selected in the third gating area. + T cells were selected at the fourth interface to identify non-HLA-DR monoclonal antibodies, and CD25-CD69-CD4+ T cells were collected at the final sorting interface.

[0134] III. Resting CD4 + T cell activation detection

[0135] Experimental methods:

[0136] (1) The compound to be tested was diluted to a specific concentration in a 96-well cell culture plate. Human T-activator CD3 / CD28 dynabeads, 100 ng / ml PMA and 1 mM ionomycin were set as positive controls and 0.1% DMSO was set as a negative control.

[0137] (2) Place 100 μL of resting CD4 + T cells were administered at a dose of 1×10 6 Add cells at a density of 1 cell / mL to the diluted compound and incubate the cells in a 37°C, 5% CO2 incubator for 48 hours.

[0138] (3) Collect the cells into flow cytometry tubes and centrifuge at 1600 rpm at 4℃ for 5 min;

[0139] (4) Collect the cell supernatant into a 1.5 mL EP tube, label it and store it at -80℃ for subsequent cytokine assays;

[0140] (5) Add 1 ml of PBS to each tube to resuspend the cells, centrifuge at 1600 rpm at 4℃ for 5 min, and discard the supernatant;

[0141] (6) Dilute CD4-FITC / CD25-APC / CD69-PE antibody with PBS at a ratio of 1:100, and dilute Fixed Viability Dye eFluor 506 antibody at a ratio of 1:1000. Add 50 μL to each tube and incubate at 4°C in the dark for 30 min.

[0142] (7) Wash the cells twice with PBS following step (5) above;

[0143] (8) Resuspend cells in 300 μL PBS and detect CD4 by flow cytometry. + The activation status of T cells was analyzed using FlowJo software (v10.0.7).

[0144] Results and Analysis:

[0145] The results are as follows Figure 9 As shown, ISX-9, ML327, bibraxib, AZD5153, quinolone, perbisitone, and their combined use did not show an increase in CD25 and CD69 positive cells, indicating that these compounds do not induce T cell activation in vitro and have no effect on overall cell viability.

[0146] Example 6

[0147] ELISA detection of compound-induced resting CD4 + T cell cytokine secretion.

[0148] Experimental methods:

[0149] (1) Place the reagents in the kit at room temperature and let stand for 15 minutes;

[0150] (2) Dilute the standard in a 1.5 mL EP tube according to the kit instructions, and dilute the cell supernatant to be tested 1:10;

[0151] (3) Remove the microplates needed from the sealed bag that has been brought to room temperature, and put the unused remaining strips back into the aluminum foil bag for storage.

[0152] (4) Add different concentrations of standard and test sample into microplate, set up three replicate wells, 100 μL per well, seal the reaction well with sealing tape, and incubate at room temperature for 2 h.

[0153] (5) Discard the liquid in the wells, add 300 μL of washing solution to each well, repeat the washing process 3 times, and after the last washing, pat all the liquid in the wells dry on absorbent paper.

[0154] (6) Add 100 μL of detection antibody to each well, seal the reaction wells with sealing tape, and incubate at room temperature for 2 h;

[0155] (7) Repeat step (5);

[0156] (8) Add 100 μL of SA-HRP to each well, seal the reaction wells with sealing tape, and incubate at room temperature in the dark for 20 min.

[0157] (9) Repeat step (5);

[0158] (10) Add 100 μL of chromogenic substrate to each well and incubate at room temperature in the dark for 20 min;

[0159] (11) Add 50 μL of stop solution to each well to terminate the reaction;

[0160] (12) Measure the absorbance at 450 nm using an ELISA reader within 30 min after adding the stop solution, and set 540 nm or 570 nm as the calibration wavelength.

[0161] (13) Calculate cytokine concentrations: Correct the absorbance values ​​of the standards and samples (450nm-540nm / 570nm), take the average of the three replicates, and subtract the average zero standard OD value. Create a standard curve in Excel using the standard concentrations and OD values, and generate a linear regression equation to obtain the sample concentration. If the sample has been diluted, the final concentration needs to be multiplied by the dilution factor.

[0162] Results and Analysis:

[0163] like Figure 10 , Figure 11 and Figure 12 As shown, the results indicate that ISX-9 and ML327, used alone or in combination with HDACi and BETi, do not induce resting CD4 at doses sufficient to trigger pathway activation. + Cytokine release from T cells.

[0164] Example 7

[0165] Assay for the inhibitory activity of ISX-9, ML327 and HIV-1 inhibitors in combination.

[0166] (1) Dilute HIV-1LRAs in 1.5ml EP tubes to make the final concentration of ISX-9 and ML327 10μM. Set 10ng / mL TNF-α, 1μM Pabistat and 1μM Vorinostat as positive controls and PBS as negative control.

[0167] (2) Add EKL1C to the corresponding LRAs to make the final concentration of EKL1C 5 μM. Set NVP, AZT, and IDV as positive controls and PBS as negative controls.

[0168] (3) Add the individual LRAs, LRAS and HIV-1 antiviral drug premix to a 96-well cell culture plate and set up three replicate wells;

[0169] (4) 100 μL of a density of 5 × 10 5 100 cells / mL of LACH2 cells were added to 96-well plates and cultured in an incubator at 37°C and 5% CO2 for 48 hours.

[0170] (5) TZM-Bl cells were administered at a rate of 1×10 4 Add cells at a density of 100 μL / well to a 96-well cell culture plate and incubate at 37°C and 5% CO2 for 12-16 h.

[0171] (6) Collect the supernatant of ACH2 cells and add it to TZM-Bl cells, and incubate at 37°C and 5% CO2 for 12 h.

[0172] (7) Discard the original culture medium of TZM-Bl cells and replace it with fresh DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin (purchased from Meilun Biotechnology, China).

[0173] (8) After culturing for another 48 hours, discard the cell supernatant, wash the cells twice with PBS, add 40 μL of diluted cell culture lysis buffer (purchased from Promega, USA) to each well, and lyse on a horizontal shaker at room temperature for 30 min.

[0174] (9) Take 30 μL of cell lysis buffer and add it to a Costa 96-well white-background microplate. Then add an equal volume of luciferase substrate and use a microplate reader to detect the fluorescence of the sample.

[0175] Results and Analysis:

[0176] like Figure 13 As shown, no viral activation or infection was detected in the PBS group. Under conditions where only latent infection activators such as ISX-9, ML327, TNF-α, papilostat, and vorinostat were used, without any anti-HIV-1 inhibitors, reactivated HIV-1 could replicate and infect TZM-Bl cells. When the activators were combined with three HIV-1 inhibitors—nevirapine, zidovudine, and indinavir—and the fusion inhibitory peptide EKL1C, the replication of activated HIV-1 in TZM-Bl cells was significantly inhibited.

[0177] Although specific embodiments of this application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and such changes are all within the scope of protection of this application. The full scope of this application is given by the appended claims and any equivalents.

Claims

1. Use of ISX-9 and / or ML327 in the preparation of an HIV-1 latent infection activator.

2. Use according to claim 1, characterized in that, The HIV-1 latent infection activator further includes quinostat, birabresib or AZD5153.

3. An HIV-1 latent infection activator, characterized by, It comprises component one selected from ISX-9 or ML327 and component two selected from quinostat, birabresib or AZD5153.

4. A medicament, characterized by comprising: It comprises the HIV-1 latent infection activator of claim 3 and an HIV-1 inhibitor.

5. The medicament according to claim 4, characterized in that, The HIV-1 inhibitor is polypeptide EKL1C, the amino acid sequence of which is shown as SEQ ID NO:

1.

6. The medicament according to claim 4, characterized in that, The HIV-1 inhibitor includes one or more of zidovudine, nevirapine or indinavir.

7. The medicament according to claim 4, characterized in that, The dosage form is tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal, suppository or freeze-dried powder injection.

8. Use of the medicament of any one of claims 4-7 in the preparation of a medicament for preventing and / or treating HIV-1 virus infection.

Images