HIV-1 latent infection activator, medicine and application

Abstract

The invention relates to an HIV-1 latent infection activator, a medicine and application. The invention relates to an HIV-1 (Human Immunodeficiency Virus-1) latent infection activator, which 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 birabresil or AZD5153 (Azodicarbonamide 5153). The invention also discloses a medicine which comprises any one of the HIV-1 latent infection activator and the HIV-1 inhibitor. The HIV-1 inhibitor is polypeptide EKL1C, and the amino acid sequence of the HIV-1 inhibitor is shown as SEQ ID NO: 1. The HIV-1 inhibitor is prepared from one or more of zidovudine, nevirapine or indinavir. The HIV-1 latent infection activator disclosed by the invention can activate latent HIV (human immunodeficiency virus), and is a novel, safe and efficient HIV-1 latent infection activator.

Description

Technical Field

[0001] The present application relates to the technical field of small molecule drugs, and particularly relates to an HIV-1 latent infection activator, a drug and uses thereof. Background Art

[0002] Acquired immune deficiency syndrome (AIDS), also known as AIDS, is a serious infectious disease caused by human immunodeficiency viruses (HIV), which poses a great threat to human health. Due to the extensive mutation and replication ability of the HIV virus, its ability to establish a lifelong infection, and the scale of the pandemic, the virus strains have diversified significantly over time, presenting great challenges for prevention and treatment. Currently, the effective treatment method for AIDS is combination antiretroviral therapy (cART). cART can effectively reduce viremia below the clinical detection limit. However, due to the existence of factors such as the latent viral reservoir (LVR), cART cannot completely eradicate HIV-1 in the body.

[0003] Latent infection is the main obstacle to the cure of HIV-1. To eliminate the latent HIV-1 virus reservoir and achieve a functional cure for AIDS, multiple strategies have been proposed, including the "shock and kill" strategy. The "Shock and kill" strategy mainly uses various latent infection activators or latency-reversing agents (LRAs) to reactivate latent HIV-1, and then clears it through antiretroviral therapy and host immune function. This strategy is effectively applied to the combined use of LRAs and antiviral drugs. The main purpose of LRAs is to specifically reactivate proviral transcription without having an adverse impact on cell homeostasis. However, various currently developed LRAs face 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 applying LRAs in combination with antiviral drugs to the "shock and kill" strategy is an effective treatment means to achieve a functional cure for AIDS.

[0004] ISX-9 (Isoxazole 9, CAS: 832115-62-5, molecular formula: C 11 H 10 N 2 O 2S) is a specific neural stem cell differentiation inducer that promotes NeuroD expression by activating voltage-gated Ca 2+ channels and NMDA receptors, and simultaneously regulates the Notch signaling pathway, showing significant activity in epicardial-derived cell differentiation 137. No one has reported its related use as an HIV-1 latent infection activator yet.

[0005] ML-327 (ML327, CAS: 1883510-31-3, molecular formula: C 19 H 18 N 4 O 4 ) belongs to isoxazoline small molecule inhibitors. Literature reports that it can inhibit tumor cell proliferation by antagonizing MYC transcriptional activity and show anti-metastatic effects in preclinical models such as breast cancer and colon cancer. No one has reported its related use as an HIV-1 latent infection activator yet. SUMMARY OF THE INVENTION

[0006] This application discloses an HIV-1 latent infection activator, a drug and its uses, exploring new safe and efficient HIV-1 latent infection activators, which can be combined 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 a first aspect, this application relates to an HIV-1 latent infection activator, which comprises ISX-9 and / or ML327.

[0008] Furthermore, the HIV-1 latent infection activator further comprises an HDAC inhibitor and / or a BET inhibitor.

[0009] Furthermore, the HDAC inhibitor is quinosinostat.

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

[0011] In a second aspect, this application relates to a drug, which comprises any of the above-mentioned HIV-1 latent infection activators and an HIV-1 inhibitor.

[0012] Furthermore, the HIV-1 inhibitor is polypeptide EKL1C, and its amino acid sequence is as shown in SEQ ID NO: 1.

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

[0014] Further, its dosage form is tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or freeze-dried powder injection.

[0015] In a third aspect, the present application relates to the use of any one of the above-mentioned HIV-1 latent infection activators, or any one of the above-mentioned drugs, in the preparation of drugs for preventing and / or treating diseases related to HIV virus infection.

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

[0017] The HIV-1 latent infection activator of the present application can activate latent HIV, and is a new, safe and highly effective HIV-1 latent infection activator. Further, when ISX-9 and / or ML327 in the HIV-1 latent infection activator of the present application are used in combination with HDAC inhibitors and / or BET inhibitors, the activation effect can be improved and a synergistic activation effect can be produced. Moreover, the HIV-1 latent infection activator of the present application does not cause the activation of T cells and the release of pro-inflammatory cytokines.

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

[0019] The present application can be used in the preparation of drugs for preventing and / or treating diseases related to HIV virus infection. When combined with the "shock and kill" strategy, it can increase the diversity and potential efficacy of treatment strategies. Description of the Drawings

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0021] Figure 1 It is the first-round screening result of Example 1;

[0022] Figure 2 It is the second-round screening result of Example 1;

[0023] Figure 3 It is the compound situation of the second-round screening of Example 1;

[0024] Figure 4 It is the detection result of the activation effect of ISX-9 and ML327 in Example 1;

[0025] Figure 5Detection results of in vitro activation effects of ISX-9 and ML327 in Example 2;

[0026] Figure 6 Cytotoxicity detection results of ISX-9 and ML327 in Example 3;

[0027] Figure 7 Analysis results of combined application of different LRAs in Example 4;

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

[0029] Figure 9 For CD4 in Example 5 + Activation detection results of T cells;

[0030] Figure 10 For CD4 in Example 6 + Cytokine TNF-α detection results of T cells;

[0031] Figure 11 For CD4 in Example 6 + Cytokine IFN-γ detection results of T cells;

[0032] Figure 12 For CD4 in Example 6 + Cytokine IL-10 detection results of T cells;

[0033] Figure 13 Drug inhibition activity detection results in Example 7. Detailed implementation manners

[0034] The implementation schemes of the present application will be described in detail below in combination with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present application and are not intended to limit the present application. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer.

[0035] An HIV-1 latent infection activator, which comprises ISX-9 and / or ML327. The chemical formula of ISX-9 is C 11 H 10 N 2 O 2 S, and the structural formula is as follows:

[0036]

[0037] The chemical formula of ML327 is C 19 H 18 N 4 O 4 , and the structural formula is as follows:

[0038]

[0039] The HIV-1 latent infection activator also includes an HDAC inhibitor and / or a BET inhibitor. The HDAC inhibitor is quinosat. The chemical formula of quinosat is C 21 H 26 N 6 O 2 , and the structural formula is as follows:

[0040]

[0041] The BET inhibitor is blisibimod or AZD5153. The chemical formula of blisibimod is C 25 H 22 C l N 5 O 2 S, and the structural formula is as follows:

[0042]

[0043] The chemical formula of AZD5153 is C 36 H 41 N 7 O 6 , and the structural formula is as follows:

[0044]

[0045] A drug, which comprises any one of the above-mentioned HIV-1 latent infection activators and an HIV-1 inhibitor. The HIV-1 inhibitor can be polypeptide EKL1C, and its amino acid sequence is shown as SEQ ID NO:1: NVTFLDLEYEMKKLEEAIKKLEESYIDLKELGTYEY. The HIV-1 inhibitor can also be one or more of zidovudine, nevirapine or indinavir. The drug dosage form is tablet, capsule, dropping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or freeze-dried powder injection.

[0046] The J-Lat A2 cell line and ACH-2 cell line in the examples were provided by the National Institutes of Health (NIH) AIDS Reagent Program. The J-Lat C11 cells were a gift from Professor Zhu Huanzhang of the School of Life Sciences, Fudan University. The above three cell lines are all latent HIV virus reservoir cell lines. PBMCs were derived from healthy human donors and provided by the Department of Gastroenterology, Jing'an District Central Hospital, Shanghai, with the ethical approval number: 2022-029. The 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 latent infection cell model J-Lat A2

[0049] Experimental method:

[0050] (1) Dilute the compounds 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 supplement with an equal volume of RPMI1640 medium containing 1% penicillin / streptomycin (purchased from Meilun Biology, China);

[0052] (3) Add J-Lat A2 cells to the diluted compound at a density of 5×10 4 cells / 100 μL / well, so that the final concentration of the compound is 10 μM;

[0053] (4) Set panobinostat and 10 ng / ml TNF-α as positive controls; 0.1% DMSO as negative control;

[0054] (5) Place the cells in an incubator at 37°C and 5% CO 2 for 48 h, and then observe the GFP expression of J-Lat A2 cells with an inverted fluorescence microscope to determine whether the compound can activate the HIV-1 virus reservoir.

[0055] Results and analysis:

[0056] As Figure 1 shown, through the first-round screening of the classical known active library using the J-Lat A2 cell line, 177 candidate molecules that can reactivate the GFP expression in J-Lat A2 cells were obtained in the first-round screening.

[0057] II. Screening of novel HIV-1 LRAs using the latent infection cell model ACH2

[0058] Experimental method:

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

[0060] (2) Add 50 μL of the compound at the specified concentration to a 96-well cell culture plate, and supplement with an equal volume of RPMI1640 medium containing 1% penicillin / streptomycin;

[0061] (3) Set panobinostat as the positive control; 0.1% DMSO as the negative control;

[0062] (4) Add 100 μL of 5×10 5 cells / mL of ACH2 cells to the cell culture plate, and place the cells in an incubator at 37°C and 5% CO 2 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 overnight at 4°C;

[0064] (6) Dilute human anti-HIV-1 IgG antibody (HIVIG) to 5 μg / mL with NaHCO 3 (pH 9.6) buffer and coat the ELISA plate at 50 μL / well overnight at 4°C;

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

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

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

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

[0069] (11) Wash the ELISA plate 3 times with PBST using an ELISA plate washer, add 50 μL of TMB chromogenic substrate to each well, and react at room temperature for 5 - 10 min;

[0070] (12) 50 μL / well of 1N H2 SO 4 Add the stop solution to the ELISA plate;

[0071] (13) Read the absorbance at 450 nm using a multi-detection microplate reader.

[0072] Results and analysis:

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

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

[0075] Experimental method:

[0076] (1) Dilute the compounds screened in the second round two-fold in a 96-well cell culture plate with a final concentration of 40 μM in the first well, set 8 dilution gradients, and set three replicate wells for each concentration;

[0077] (2) Add an equal volume of RPMI1640 medium containing 1% penicillin / streptomycin to each well;

[0078] (3) Set panobinostat as the positive control; 0.1% DMSO as the negative control;

[0079] (4) Add 100 μL of 5×10 5 / mL ACH2 cells and 50 μL of the compound at the specified concentration to the 96-well plate and incubate for 48 h.

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

[0081] Results and analysis:

[0082] Detect the activation effect of the screened compounds on the latent infection cell model ACH2. The results are as Figure 4As shown, the present application has identified two novel HIV-1 latent infection activators, ISX-9 and ML327, which 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 method:

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

[0087] (2) 50 μL of RPMI1640 medium containing 1% penicillin / streptomycin was added to each well in equal volume.

[0088] (3) 100 μL of 5×10 4 cells / mL of J-Lat A2 cells and J-Lat C11 cells were incubated with the compound at the designated concentration in a 96-well plate for 48 h.

[0089] (4) The expression of GFP-positive cells was detected using a BD LSRFortessa TM flow cytometer, 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. As Figure 5 shown, ISX-9 can induce up to 80% and 40% of GFP-positive cells in J-Lat A2 and J-Lat C11 cells, respectively, and 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 detection of ISX-9 and ML327 compounds.

[0094] Experimental method:

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

[0096] (2) 100 μL of 5×10 5ACH2, J-Lat A2, and J-Lat C11 cells at a density of 6 per mL, and resting CD4 + T cells at a density of 2 per mL were added to the diluted compounds and cultured at 37 °C under 5% CO

[0097] (3) The CCK8 reagent was diluted 5-fold with RPMI1640 medium containing 1% penicillin / streptomycin, 50 μL was added to each well, and the cells were further cultured at 37 °C for 4 - 6 h;

[0098] (4) The absorbance was measured at 450 nm using a microplate reader, and the cytotoxicity of different compounds to the cells was calculated. The formula for calculating cell viability = [(OD value of the experimental group - OD value of the negative group) / (OD value of the positive group - OD value of the negative group)] × 100%. The half-cytotoxic concentration (50% cytotoxicity concentration, CC50) of the compounds was calculated using Calcusyn software (Biosoft), and a curve graph was plotted using GraphPad Prism 8.0.

[0099] Results and Analysis:

[0100] As Figure 6 shown in 50 A, the CC Figure 6 values of ISX-9 on ACH2, J-Lat A2, and J-Lat C11 cells were 12.2 μM, 130.0 μM, and 33.8 μM, respectively. As 50 shown in

[0101] B, the CC

[0102] values of ML327 on ACH2, J-Lat A2, and J-Lat C11 cells were 8.4 μM, 74.0 μM, and 35.2 μM, respectively. The cytotoxicity of ISX-9 and ML327 on J-Lat A2 and J-Lat C11 cells was relatively low, and the reactivation efficiency would not be affected by drug toxicity.

[0103] Experimental Method:

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

[0105] (2) Combine the compounds into LRAs combinations according to different types: ISX-9-Quinostatin, ISX-9-Birabresib, ISX-9-AZD5153, ML327-Quinostatin, ML327-Birabresib, ML327-AZD5153, and add them to a 96-well cell culture plate at the final concentration in (1) for two-fold serial dilution, set 8 dilution gradients, and set three replicate wells for each concentration;

[0106] (3) Add 100 μL of J-Lat A2 cells at 5×10 4 cells / mL to a 96-well cell plate and culture them in an incubator at 37 °C and 5% CO 2 for 48 h;

[0107] (4) Detect the expression of GFP-positive cells using a BD LSRFortessa TM flow cytometer, and analyze the results using FlowJo software (v10.0.7);

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

[0109] Results and analysis:

[0110] In this application, the novel LRAs ISX-9 and ML327 screened were combined with other types of LRAs including HDACi Quinostatin and BETi Birabresib, AZD5153. The results are as Figure 7 shown in Table 1. LRAs combinations 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 in triplicate, and the experiment was repeated 3 times. Data from representative experiments are expressed as mean ± SD.

[0114] Example 5

[0115] I. Isolation and culture of PBMCs

[0116] Experimental method:

[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 human lymphocyte separation solution into a 50 mL centrifuge tube, and then carefully add an equal volume of peripheral blood along the side wall with a Pasteur pipette. Due to different densities, the human lymphocyte separation solution and peripheral blood will form an obvious stratification;

[0119] (3) Centrifuge at 2000 rpm for 20 min. Note that the acceleration is selected at gear 9 and the deceleration is selected as no brake;

[0120] (4) After centrifugation, different components will have obvious stratification. Carefully aspirate the PBMC cells into a clean 15 mL centrifuge tube with a pipette;

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

[0122] (6) Resuspend the cells with RPMI1640 medium containing 10% FBS and 1% penicillin / streptomycin for subsequent experiments.

[0123] II. Sorting of resting CD4 + T cells

[0124] Experimental method:

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

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

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

[0128] (4) After washing the cells twice with PBS containing 0.5% FBS, count and adjust the cell density to 10 7 cells / mL;

[0129] (5) Filter the cells with a flow tube with a filter to remove cell clumps, and place them on ice waiting for loading onto the machine;

[0130] (6) Prepare a collection tube and add 2 mL of RPMI1640 medium containing 3% penicillin / streptomycin;

[0131] (7) Flow cytometry sorting gating strategy: Gate the non-adherent PBMC cells on the diagonal of the sorting interface for subsequent gating, and continue to gate the CD4 + T cells with better viability of CD4 + T cells, and finally collect the resting CD4

[0132] Results and analysis:

[0133] The results are as Figure 8 shown. First, gate the general position of T cells in the first gate, then gate the non-adherent PBMC cells on the diagonal of the second gate, gate the CD4 + T cells with good viability in the third interface, gate out the non-HLA-DR monoclonal antibody in the fourth interface, and collect CD25-CD69-CD4+ T cells at the final sorting interface.

[0134] III. Activation detection of resting CD4 + T cells

[0135] Experimental method:

[0136] (1) Dilute the compound to be detected to a specific concentration in a 96-well cell culture plate, and set human T-activator CD3 / CD28 dynabeads, 100 ng / ml PMA, and 1 mM ionomycin as positive controls, and 0.1% DMSO as a negative control;

[0137] (2) Add 100 μL of resting CD4 + T cells at a density of 1×10 6 cells / mL to the diluted compound, and place the cells in an incubator at 37°C and 5% CO 2 for 48 h;

[0138] (3) Collect the cells into a flow tube and centrifuge at 1600 rpm at 4°C for 5 min;

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

[0140] (5) Resuspend the cells with 1 ml of PBS per tube, centrifuge at 1600 rpm at 4°C for 5 min, and discard the supernatant;

[0141] (6) Dilute CD4-FITC / CD25-APC / CD69-PE antibody 1:100 and Fixable Viability Dye eFluor 506 antibody 1:1000 with PBS, 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 according to the above step (5);

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

[0144] Results and analysis:

[0145] The results are as follows Figure 9 As shown, ISX-9, ML327, bilabrecib, AZD5153, quinostat, panobinostat and their combination did not show an increase in CD25 and CD69 positive cells, indicating that these compounds did not induce in vitro activation of T cells and had no effect on overall cell viability.

[0146] Example 6

[0147] ELISA to detect compound-induced resting CD4 + Cytokine secretion by T cells.

[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) Take out the microplate to be used from the sealed bag that has been equilibrated to room temperature, and put the remaining unused strips back into the aluminum foil bag for storage;

[0152] (4) Add different concentrations of standard substances and samples to be tested into a microplate, set up three replicate wells, 100 μL per well, seal the reaction wells 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, and repeat the washing process three times. After the last wash, pat all the liquid in the wells dry on absorbent paper.

[0154] (6) Add 100 μL of detection antibody to each well, seal the well 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 in the dark at room temperature for 20 min;

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

[0158] (10) Add 100 μL of chromogenic substrate to each well and incubate in the dark at room temperature 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 with a microplate reader within 30 min after adding the stop solution, and set 540 nm or 570 nm as the correction wavelength;

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

[0162] Results and analysis:

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

[0164] Example 7

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

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

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

[0168] (3) Add the premixed solutions of individual LRAs, LRAS, and HIV-1 antiviral drugs into a 96-well cell culture plate, and set up three replicate wells;

[0169] (4) Add 100 μL of ACH2 cells at a density of 5×10 5 cells / mL into the 96-well plate and culture them in an incubator at 37 °C with 5% CO 2 for 48 h;

[0170] (5) Add TZM-Bl cells into the 96-well cell culture plate at a density of 1×10 4 cells / 100 μL / well and culture them at 37 °C with 5% CO 2 for 12 - 16 h;

[0171] (6) Collect the supernatant of ACH2 cells and add it to the TZM-Bl cells, and incubate them at 37 °C with 5% CO 2 for 12 h;

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

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

[0174] (9) Take 30 μL of the cell lysate and add it to a 96-well white-bottom enzyme-linked immunosorbent assay (ELISA) plate from Costar, then add an equal volume of luciferase substrate, and detect the fluorescence of the samples with an enzyme-linked immunosorbent assay (ELISA) reader.

[0175] Results and analysis:

[0176] As Figure 13 shown, no virus activation or virus infection was detected in the PBS group. In the condition where there were only latent infection activators such as ISX-9, ML327, TNF-α, panobinostat, vorinostat, etc., and no anti-HIV-1 virus inhibitors, the reactivated HIV-1 could replicate and infect in TZM-Bl cells. When the activators were used in combination with three HIV-1 inhibitors, nevirapine, zidovudine, indinavir, and the fusion inhibitory polypeptide EKL1C, the replication of the activated HIV-1 on TZM-Bl cells was significantly inhibited.

[0177] Although the specific embodiments of the present 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 that have been disclosed, and these changes are all within the scope of protection of the present application. The full scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. An HIV-1 latent infection activator, characterized in that This includes ISX-9 and / or ML327.

2. The HIV-1 latent infection activator according to claim 1, characterized in that It also includes HDAC inhibitors and / or BET inhibitors.

3. The HIV-1 latent infection activator according to claim 2, characterized in that The HDAC inhibitor is quinostat.

4. The HIV-1 latent infection activator according to claim 2, characterized in that The BET inhibitor is bilabrecib or AZD5153.

5. A drug, characterized in that The invention comprises the HIV-1 latent infection activator and the HIV-1 inhibitor according to any one of claims 1 to 4.

6. The drug according to claim 5, characterized in that The HIV-1 inhibitor is a polypeptide EKL1C, and its amino acid sequence is shown in SEQ ID NO:

1.

7. The drug according to claim 5, characterized in that The HIV-1 inhibitor includes one or more of zidovudine, nevirapine or indinavir.

8. The drug according to claim 5, characterized in that The dosage form is tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent, suppository or lyophilized powder injection.

9. Use of the HIV-1 latent infection activator according to any one of claims 1 to 4, or the drug according to any one of claims 5 to 8, in the preparation of a drug for preventing and / or treating diseases associated with HIV virus infection.

10. The use according to claim 9, characterized in that The HIV virus is HIV-1 virus.

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