Application of micromolecule PRT4165 in preparation of anti-HIV latency drugs

Abstract

The invention belongs to the field of medicines, and relates to an application of a small molecule drug in anti-HIV-1 latency. The invention provides an application of a micromolecule PTR4165 in preparation of an anti-HIV-1 latency drug. The anti-HIV-1 latency drug is a drug for promoting the transcription activation of a previrus in an HIV infected cell. The micromolecule PTR4165 can be combined with an HIV potential activator to be used as a pharmaceutical composition for promoting the transcription activation of a pre-virus in an HIV infected cell; the HIV potential activator comprises a histone deacetylase inhibitor SAHA, a BET inhibitor JQ1, a PKC agonist and a DNA methylation inhibitor 5-aza. The PTR4165 provided by the invention has a transcriptional activation effect on latent HIV in HIV infected cells no matter the PTR4165 is independently used or combined with a known activator, and can assist in deeper removal of HIV viruses.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to the application of a compound in promoting HIV transcriptional activation and for the treatment intervention of HIV latency. Background Art

[0002] AIDS, namely Acquired Immunodeficiency Syndrome (AIDS), is an infectious disease caused by the infection of the immune system with the Human Immunodeficiency Virus (HIV-1), which seriously affects the physical and mental health of humans. In 2018, approximately 36.9 million people worldwide were infected with HIV-1, among which the number of new infections was 1.7 million and the number of deaths was 660,000. AIDS has become a serious public health problem and social problem in the world today. Currently, the main clinical treatment method for AIDS is highly active antiretroviral therapy (ART), which can reduce the plasma HIV-1 level below the clinical detection line, greatly improving the quality of life of patients and enabling them to obtain a lifespan close to normal.

[0003] However, relying solely on ART cannot completely eliminate the virus in patients, mainly due to the existence of viral reservoirs. Viral reservoirs are established in the early stage after infection and mainly exist in long-lived resting CD4 + memory T cells, and the proviruses in the reservoirs are mainly transcriptionally silent. However, when the environment is suitable, the silent proviruses will start transcription and translation to produce infectious virus particles. Therefore, to completely cure AIDS, the viral latent reservoir must be completely eliminated. For this purpose, researchers have proposed a "shock and kill" strategy, that is, first use a method that can activate latent HIV proviruses to promote virus transcription, and under immune pressure or drug action, eliminate the latently activated infected cells, so as to achieve a functional cure of AIDS.

[0004] Currently, for latent HIV, a variety of different types of latency-reversing agents (LRAs) have been reported, including histone deacetylase inhibitors, histone methyltransferase inhibitors, Bromodomain (BET) inhibitors, PKC agonists, etc. Summary of the Invention

[0005] The purpose of the present invention is to provide a drug for promoting HIV-1 transcriptional activation.

[0006] Another purpose of the present invention is to provide the application of this compound in the preparation of anti-HIV latency drugs.

[0007] On the one hand, the present invention provides the use of the small molecule PTR4165 in the preparation of anti-HIV-1 latency drugs.

[0008] The chemical formula of PRT4165 is C 15 H 9 NO 2 , with a molecular weight of 235.24 and a CAS number of 31083-55-3. The structure is as follows:

[0009]

[0010] PRT4165 is a Bmi 1 / Ring1A inhibitor and can also inhibit PRC1-mediated H2A ubiquitination in vivo and in vitro. It is currently mainly used in related biological research, such as in the fields of cell biology, oncology, etc., to explore the mechanism of action of Bmi 1 / Ring1A protein in cell proliferation, differentiation, apoptosis, and tumorigenesis and development, providing a theoretical basis and potential drug targets for the treatment of related diseases.

[0011] The present invention has obtained a new use of PRT4165, that is, its use in the preparation of anti-HIV-1 latency drugs, and the anti-HIV-1 latency drugs refer to drugs that promote the transcriptional activation of proviruses in HIV-infected cells.

[0012] Preferably, the anti-HIV-1 latency drugs refer to drugs or reagents that inhibit the PRC1 complex, or drugs or reagents that have a mono-ubiquitination effect on H2AK119. The research of the present invention shows that PTR4165 can inhibit PRC1-mediated H2AK119ub1 near the latent HIV-1 LTR, thereby promoting the activation of latent HIV. Such drugs inhibit the H2AK119ub modification mediated by the PRC1 complex.

[0013] Preferably, the small molecule PRT4165 can be used to promote the transcriptional activation of proviruses in HIV-infected cells; including the following steps:

[0014] S1, adding the small molecule to the culture medium of HIV-infected cells; and / or

[0015] S2, incubating the small molecule with HIV-infected cells.

[0016] PRT4165 can be applied to various HIV-infected cells to promote the activation of latent HIV in HIV-infected cells, so that the detection system or related drugs can identify and eliminate these activated HIV. Preferably, the cells are selected from, but not limited to: human mononuclear cells, human macrophages, human CD4 +One or more of T lymphocytes, human mast cells, human dendritic cells, human follicular dendritic cells, human hematopoietic progenitor cells, human natural killer cells, human neurons, and oligodendrocytes.

[0017] Preferably, the final concentration for the use of the small molecule PTR4165 when activating latent HIV in HIV-infected cells is 5 - 20 μmol / L, more preferably 8 - 15 μmol / L. In a preferred example of the present invention, the use concentration of the small molecule PTR4165 is 10 μmol / L.

[0018] Furthermore, the small molecule PTR4165 can be used in combination with a potential HIV activator as a pharmaceutical composition for promoting proviral transcriptional activation in HIV-infected cells. The potential HIV activator includes, but is not limited to: the activator is histone deacetylase inhibitor SAHA, BET inhibitor JQ1, PKC agonist, and DNA methylation inhibitor 5-aza. According to the molar ratio, when used in combination, the dosage ratio of PTR4165 to histone deacetylase inhibitor SAHA is 1:(0.01 - 0.10); the dosage ratio of PTR4165 to BET inhibitor JQ1 when used in combination is 1:(0.001 - 0.005); the dosage ratio of PTR4165 to PKC agonist when used in combination is 1:(0.001 - 0.005); the dosage ratio of PTR4165 to DNA methylation inhibitor 5-aza when used in combination is 1:(0.05 - 0.20).

[0019] On the other hand, the present invention provides a pharmaceutical composition, and the active ingredients of the pharmaceutical composition include the small molecule PTR4165 and a potential HIV activator.

[0020] Preferably, the potential HIV activator includes, but is not limited to: the activator is histone deacetylase inhibitor SAHA, BET inhibitor JQ1, PKC agonist, and DNA methylation inhibitor 5-aza.

[0021] Preferably, in terms of molar ratio, when used in combination, the dosage ratio of PTR4165 to histone deacetylase inhibitor SAHA is 1:(0.01 - 0.10); the dosage ratio of PTR4165 to BET inhibitor JQ1 is 1:(0.001 - 0.005); the dosage ratio of PTR4165 to PKC agonist is 1:(0.001 - 0.005); the dosage ratio of PTR4165 to DNA methylation inhibitor 5-aza is 1:(0.05 - 0.20). For example, in a preferred embodiment of the present invention, when PTR4165 is respectively combined with SAHA (0.50 μM), PEP005 (0.01 μM), JQ1 (0.01 μM) or 5-aza (1 μM) to treat HIV latent cells, significant effects are achieved. Brief Description of the Drawings

[0022] To more clearly illustrate the technical solutions of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, each of the following drawings is for some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0023] Figure 1 It is the result of PRT4165 promoting the activation of HIV-1 in C11 and J-lat 10.6 cell lines.

[0024] Figure 2 It is the result of PRT4165 promoting the activation of HIV-1 in the virus latent cell line ACH2 and the primary CD4 + T lymphocyte latent model.

[0025] Figure 3 It is the result that PTR4165 has a good synergistic effect with reported potential activators.

[0026] Figure 4 It is the diagram of the effect of PRT4165 on the proliferation and apoptosis of PBMC. The CCK8 assay is used to detect the cell proliferation activity, and flow cytometry is used to detect the cell apoptosis level.

[0027] Figure 5 It is the image diagram of the effect of PRT4165 on the cell apoptosis level. Detailed Embodiments

[0028] The research results of the present invention show that the small molecule compound PRT4165 can activate latent HIV by inhibiting H2AK119ub modification and PRC1-mediated H2AK119ub, and can be used alone. Moreover, PTR4165 has a good synergistic activation effect with the reported potential activators. The experimental results show that SAHA (0.50 μM), BET inhibitor JQ1 (0.01 μM), PKC agonist (0.01 μM), DNA methylation inhibitor 5-aza (1 μM) and PTR4615 have a good synergistic activation effect, which further confirms that PTR4165 can be used as a good synergistic activator drug.

[0029] The main reagents and materials used in the present invention are as follows:

[0030] The primary CD4 + T cells used in the present invention were isolated from donated peripheral blood samples obtained from the Department of Hematology, Changhai Hospital, Shanghai. According to the method reported in previous literature, CD4 + T cells from healthy donors were isolated. The HIV-infected CD4 + T cells used in this experiment were isolated from the peripheral blood of cells donated by HIV-1 uninfected patients who visited the Shanghai Public Health Center. In the experiment of infecting healthy human CD4 + T cells with HIV, the HIV was isolated from the peripheral blood donated by AIDS patients. Whether the primary CD4 + T cells were infected or the HIV was activated was determined by detecting the reporter gene nanoluciferase in the cells.

[0031] The C11 and J-Lat 10.6 cell lines used in the present invention are HIV-1 latent infection models, which contain an integrated latent HIV-GFP reporter genome. The ACH2 cell line used is a clone of CEM cells latently infected with HIV-1, and each cell contains a single copy of proviral DNA.

[0032] The present invention includes the following contents:

[0033] (1) The effect of the small molecule inhibitor PTR4165 on HIV-1 latency in C11 and J-Lat 10.6 cell lines

[0034] The present invention first treated the C11 and J-lat 10.6 cell lines with different concentrations of PTR4165 for 24 hours and found that PTR4165 can not only activate the C11 and J-Lat 10.6 cells, but also activate them in a concentration-dependent manner.

[0035] (2) Effect of the small molecule inhibitor PTR4165 on HIV-1 latency in true virus latent cell lines

[0036] The present invention repeated the above experiment in the true virus latent cell line ACH2 and obtained similar results.

[0037] (3) Effect of the small molecule inhibitor PTR4165 on HIV-1 latency in primary latent cell models

[0038] To further confirm the effect of PTR4165 in primary cell models, a primary CD4 + T lymphocyte latent model was constructed and similar results were obtained in cell lines.

[0039] (4) Exploration of the synergistic effect of PTR4165 with reported potential activators.

[0040] The present invention selected different potential activators, including the histone deacetylase inhibitor SAHA (0.50 μM), the BET inhibitor JQ1 (0.01 μM), the PKC agonist (0.01 μM), and the DNA methylation inhibitor 5-aza (1 μM), to attempt to synergize with PTR4165 (10 μM). We found that JQ1, SAHA, and 5-aza had good synergistic activation effects with PTR4615, and the best synergistic activation effect was with PEP005. Then, we constructed a primary CD4+ T lymphocyte latent model again and confirmed that PTR4165 alone had a slight activation effect on the primary latent model, but combined with PEP005 had a good synergistic activation effect, further confirming that PTR4165 could be used as a good synergistic activator drug.

[0041] (5) Toxicity detection of PRT4165 in primary PBMCs cells

[0042] The present invention first detected the cell proliferation activity in PBMC cells by CCK8. The results showed that PRT5164 (10 uM) had little effect on the proliferation activity of PBMCs. In addition, PRT4165 did not significantly induce apoptosis. It was shown that the small molecule drug PRT4165 had the low toxicity characteristics that a latent activator suitable for clinical research should have.

[0043] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0044] Example 1: Effect of the small molecule inhibitor PTR4165 on HIV-1 latency in C11 and J-Lat 10.6 cell lines

[0045] C11 and J-Lat 10.6 cells were treated with different concentrations of the small molecule inhibitor PRT4165 for 48 h, and the cells were collected and washed with phosphate-buffered saline (PBS). The cells were stored in PBS before analysis on a BD LSRII flow cytometer. FlowJo software was used for flow cytometry analysis. The GFP expression level represents the level of latent HIV-1 activation.

[0046] The results showed that after adding PRT4165 to the culture medium of C11 cells at 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM, the percentages of GFP-positive cells were 2.60%, 3.03%, 3.60%, 5.34%, 11.14%, and 12.97%, respectively. The control was DMSO, with a value of 2.84%. After adding PRT4165 to the culture medium of J-Lat 10.6 cells at 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM, the percentages of GFP-positive cells were 1.51%, 1.75%, 3.73%, 5.22%, 8.51%, and 10.46%, respectively. The control was DMSO, with a value of 1.48%.

[0047] As the concentration of the small molecule inhibitor PRT4165 increased, the HIV-1 transcription level in C11 and J-Lat 10.6 cells also increased, and there were statistically significant differences when the concentration was equal to 1 μM and higher. The results of this example indicate that the small molecule inhibitor PRT4165 has a transcriptional activation effect on the HIV-1 provirus in C11 and J-Lat 10.6 cells.

[0048] Example 2: Effect of the small molecule inhibitor PTR4165 on HIV-1 latency in a true virus latent cell line

[0049] On the first day, 2 x 10 5 cells were seeded into a 24-well plate, and the next day, ACH2 cells were treated with different concentrations of PRT4165 for 48 h. After 48 h, the cell supernatant was collected, and the production of HIV-1 was measured by quantifying p24 in the culture supernatant using a p24 ELISA kit.

[0050] The results showed that after adding PRT4165 to the culture medium of ACH2 cells at 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM, the fold changes in the secretion of HIV-1 p24 were 0.98, 1.08, 1.74, 2.59, 3.60, and 5.37, respectively. The control was DMSO, with a value of 1.

[0051] With the increase in the concentration of the small molecule inhibitor PRT4165, the HIV-1 transcription level in ACH2 cells also increases, and there are statistical differences when the concentration is equal to 1 μM and higher. The results of the present invention suggest that the small molecule inhibitor PRT4165 has a transcriptional activation effect on the HIV-1 provirus in ACH2 cells.

[0052] Example 3 Effect of the small molecule inhibitor PTR4165 on latent HIV in primary cell models

[0053] PBMCs were obtained from donated healthy human peripheral blood by density gradient centrifugation, and Naive CD4 + T cells were isolated using a commercial kit. Naive CD4 + T cells were activated using αCD3 / CD28 magnetic beads. Three days later, primary CD4 + T lymphocytes were infected with HIV-1 pseudovirus with VSV-G envelope carrying luciferase. The cells were cultured for another 7 days, during which the concentration of IL-2 in the medium was gradually reduced to construct a primary HIV-1 latent cell model. After the successful construction of the model, cells were treated with different concentrations of PRT4165 for 48 h, and the cells were collected to detect the change in the expression level of luciferase in the cells using an enzyme-linked immunosorbent assay (ELISA) reader.

[0054] The results showed that after adding PRT4165 at 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM to the medium of CD4 + T cells, the fold change of luciferase was 1.01, 1.23, 1.61, 2.06, 2.74, and 3.38, respectively, with DMSO as the control and the value being 1. Different concentrations of PRT4165 have a transcriptional activation effect on the HIV-1 provirus in primary CD4 + T HIV-1 latent cells and show a dose-dependent manner. This suggests that PRT4165 is a potential drug for inhibiting the transcriptional activation of the HIV-1 provirus.

[0055] Example 4 Exploration of the synergistic effect of PTR4165 with reported potential activators

[0056] Using the C11 latent cell line or constructing a primary HIV-1 latent cell model as described in Example 3, C11 cells or primary CD4 +THIV-1 latent cells. The effect of latent HIV-1 activation was determined by detecting the GFP level in C11 cells by flow cytometry or the luciferase level in HIV-1 latent cells by microplate reader.

[0057] The results showed that after adding SAHA (0.50 μM), PEP005 (0.01 μM), JQ1 (0.01 μM), 5-aza (1 μM) to the culture medium of C11 cells and then adding PRT4165 (10 μM), the proportions of GFP-positive cells changed to 32.87%, 61.68%, 35.67%, 18.8%. Taking the case without adding PRT4165 as the control and with other experimental conditions being the same, the rates of GFP-positive cells in C11 were 21.50%, 36.77%, 21.13%, 9.93%. Repeating the above experiment in the primary HIV-1 latent cell model, after adding SAHA (0.50 μM), PEP005 (0.01 μM), JQ1 (0.01 μM), 5-aza (1 μM) and then adding PRT4165 (10 μM), the fold changes of luciferase expression levels were 5.78, 11.68, 6.48, 4.79. Taking the case without adding PRT4165 as the control and with other experimental conditions being the same, the rates of GFP-positive cells in C11 were 4.15, 7.11, 4.12, 2.47. The above results indicate that PRT4165 has good synergistic activation effects with JQ1, SAHA, 5-aza and PEP005.

[0058] Example 5 Toxicity detection of PRT4165 in primary CD4 + T cells

[0059] PBMC were obtained from the peripheral blood of healthy donors by density gradient centrifugation, and commercial human primary CD4 + T cells were used to sort CD4 + T cells. CD4 + T cells were activated with αCD3 / CD28 magnetic beads, and the cells were harvested after 3 days. The harvested cells were seeded in 96-well plates at a density of 0.5x10 4 cells / well, and primary CD4 + T cells were treated with PRT4165 (10 μM) for 0 h, 24 h, 48 h, 72 h, and 10% CCK-8 solution was added to the culture medium. The cells were incubated at 37 °C for 1 h. The absorbance of OD450 nm was measured by a microplate reader to determine the cell proliferation level.

[0060] The results showed that primary CD4 +After 24h, 48h, and 72h, there was almost no difference in OD values ​​between the three conditions: without PRT4165 (10μM), with only solvent DMSO, or without DMSO and PRT4165 (10μM) (MOCK). + There was no statistical difference in the proliferation level of T cells, indicating that the small molecule drug PRT4165 had no significant effect on primary CD4 + There was no effect on T cell proliferation.

[0061] Primary CD4 + T cells. The harvested cells were plated at 1x10 6 The density of cells / well was inoculated in 6-well plates, and primary CD4 + T cells for 72 hours. After 3 days, the cells were collected into 1.5 ml EP tubes and centrifuged at 300 g for 5 minutes. The cells were washed twice with 500 ul PBS and stained with Annexin V-Alexa Fluor488 / PI cell apoptosis detection kit. The proportion of Annexin V-Alexa Fluor 488 positive cells was then determined by flow cytometry and analyzed by FlowJo software. The results showed that the primary CD4 + There was no statistical difference in the level of T cell apoptosis, indicating that the small molecule drug PRT4165 would not cause primary CD4 + T cell apoptosis.

[0062] The above-described embodiments are only specific implementation methods of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be thought of by any technician familiar with the art within the technical scope disclosed in the present application without creative work should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims in the present application.

Claims

1. Use of a small molecule in the preparation of an anti-HIV-1 latency drug, characterized in that: The small molecule is PTR4165, and its structure is as follows:

2. Use of the small molecule according to claim 1 in the preparation of anti-HIV-1 latency drugs, characterized in that: The anti-HIV-1 latent drug refers to a drug that promotes the transcriptional activation of proviruses in HIV-infected cells.

3. Use of the small molecule according to claim 1 in the preparation of anti-HIV-1 latency drugs, characterized in that: The anti-HIV-1 latency drug refers to a drug or agent that inhibits the PRC1 complex, or a drug or agent that has a monoubiquitination effect on H2AK119.

4. Use of the small molecule according to claim 1 in the preparation of an anti-HIV-1 latency drug, characterized in that: The small molecule is used to promote proviral transcriptional activation in HIV-infected cells; The following steps are involved: S1, adding the small molecule to the culture matrix of HIV-infected cells; and / or S2, incubating the small molecule with HIV-infected cells.

5. Use of the small molecule according to claim 4 in the preparation of anti-HIV-1 latency drugs, characterized in that: The cells are selected from but not limited to: human mononuclear cells, human macrophages, human CD4 + One or more of T lymphocytes, human mast cells, human dendritic cells, human follicular dendritic cells, human hematopoietic progenitor cells, human natural killer cells, human neurons, and oligodendrocytes.

6. Use of the small molecule according to claim 4 in the preparation of anti-HIV-1 latency drugs, characterized in that: The final concentration of the small molecule PTR4165 was 5-20 μmol / L.

7. Use of the small molecule according to claim 1 in the preparation of an anti-HIV-1 latency drug, characterized in that: The small molecule is used in combination with a potential HIV activator as a pharmaceutical composition that promotes proviral transcriptional activation in HIV-infected cells; the potential HIV activator includes but is not limited to: the activator is a histone deacetylase inhibitor SAHA, a BET inhibitor JQ1 and a PKC agonist, and a DNA methylation inhibitor 5-aza.

8. Use of the small molecule according to claim 7 in the preparation of an anti-HIV-1 latency drug, characterized in that: According to the ratio of the amount of substances, the ratio of PTR4165 to the histone deacetylase inhibitor SAHA when used in combination is 1:(0.01-0.10); When used in combination, the dosage ratio of PTR4165 to BET inhibitor JQ1 is 1:(0.001-0.005); When used in combination, the dosage ratio of PTR4165 to PKC agonist is 1:(0.001-0.005); When used in combination, the dosage ratio of PTR4165 and DNA methylation inhibitor 5-aza is 1:(0.05-0.20).

9. A pharmaceutical composition, characterized in that The active ingredients of the pharmaceutical composition include small molecule PTR4165 and HIV potential activator.

10. The pharmaceutical composition according to claim 9, characterized in that The HIV potential activators include but are not limited to: the activators are histone deacetylase inhibitor SAHA, BET inhibitor JQ1 and PKC agonist, and DNA methylation inhibitor 5-aza.

Images