WO2010067980A2 - Histone deacetylase inhibitor that reactivates hiv-1 proviruses from latently hiv-infected cells - Google Patents

Abstract

The present invention relates to a histone deacetylase (HDAC) inhibitor represented by chemical formula 1 that enables reactivation of HIV-1 proviruses from latently HIV-infected cells, and a method for reactivating HIV-1 provirus from latently HIV-infected cells using the inhibitor. The HDAC inhibitor exhibits low cytotoxicity and high stability, and thus can effectively reactivate latent HIV-1 proviruses from a CD4+ T cell reservoir. Accordingly, the HDAC inhibitor is processed together with an HAART cocktail therapeutic drug such as a reverse transcriptase inhibitor AZT, thereby efficiently reducing or removing the latent HIV reservoir.

Description

Histone deacetylase inhibitors that reactivate the HIV-1 provirus in latent HIV infected cells

The present invention relates to HDAC inhibitors capable of efficiently reactivating HIV-1 provirus in latent HIV infected cells and methods of reactivating HIV-1 provirus from latent HIV infected cells using the inhibitor.

Histones are the central proteins that make up chromatin, acting as a winding axis of DNA, helping to condense DNA and play an important role in regulating gene expression (Strahl et al , 2000, Nature 403: 41-45; Shahbazian et al , 2007 , Annu Rev Biochem 76: 75-100). In addition, histones are susceptible to chromatin modifications that regulate gene expression by acetylation, methylation, and the like, and changes in gene expression are also associated with the latent nature of HIV (Khorasanizadeh, 2004, Cell 116). : 259-272).

Several methods have been attempted to treat HIV (Human Immunodeficiency Virus) and AIDS patients, but this is inactive, ie HIV-1 provirus is fully inserted but viral gene transcription is suppressed. The presence of latent infected CD4 + T memory cells did not result in significant therapeutic effects. Multidrug therapy, such as Highly Active Anti-Retroviral Therapy (HAART), was able to reduce blood HIV-1 viral replication activity to levels below detection levels, but did not eliminate HIV-1 latent infectious agents. It has been reported that it would take more than 60 years to effectively remove them (Finzi et al , 1997; Science 278: 1295-1300; Siliciano et al, 2003; Journal of Virology 77: 4938-4949). Therefore, it is possible to treat the HIV-1 virus effectively latent only after inducing active transcriptional activity that induces HIV-1 provirus production by conferring activity on inactive CD4 + T cells that are HIV-1 provirus latent. .

Histone deacetylase (HDAC) inhibits the expression of the HIV-1 gene from memory CD4 + T cells, an HIV-1 latent infectious pathogen (Ylisastigui et al , 2005, Journal of Infectious Diseases, 190: 1429-1437). That is, HDAC binds to the HIV-1 LTR promoter and inhibits HIV-1 virus replication, thereby enabling the production of latent HIV-1 infected cells as HIV-1 reservoirs. Therefore, to effectively remove inactive HIV-1 latent memory T cells from HIV and AIDS patients, HDAC activity must be inhibited. Several HDAC inhibitors (HDACi) have already been considered for the removal of HIV-1 pathogens present in the host, but treatment with them has several limitations. For example, valproic acid (Valporic acid) or suberoylanilide hydroxamic acid (SAHA) is relatively stable with low cytotoxicity, while it is not effective for reactivation of HIV-1 provirus. Trichostatin A or PXD-101 It was judged unsuitable to use them as viral therapeutics by causing too high cytotoxicity compared to good HIV-1 provirus reactivation capacity. Accordingly, there is a need for the development of new HDAC inhibitors with higher efficiency and stability to treat chronic HIV and AIDS patients.

An object of the present invention is to provide an HDAC inhibitor capable of efficiently reactivating HIV-1 provirus in a latent HIV infected cell and a method for reactivating HIV-1 provirus using the same. Accordingly, the HDAC inhibitor can be treated with a cocktail therapy drug of HARRT, including other transcriptase inhibitors, such as AZT, or other AIDS agents, thereby making it possible to efficiently reduce or eliminate latent HIV pathogens. .

The present invention relates to a histone deacetylase inhibitor of formula 1 for reactivating HIV-1 provirus from latent HIV infected cells.

Formula 1

In Chemical Formula 1,

Each R ₁ is independently C _1-3 alkyl, hydroxyC _1-2 alkyl, haloC _1-2 alkyl, piperidinyl, morpholinyl, cyanomethyl, piperazinyl, diC _1-2 alkylamino C _1-2 alkyl, diC _1-2 alkylaminoC _1-2 alkyl, piperidinylC _1-2 alkyl, morpholinoC _1-2 alkyl, piperazinoC _1-2 alkyl, pyrrolidinyl, C and C _1-2 alkyl optionally substituted with one or more substituents selected from the group consisting of _{1-2-alkyl-pyrrolidinyl,}

R ₂ is hydrogen or methyl.

Preferably, said latent HIV infected cells are ACH2 cells or J1.1 cells.

More preferably, the latent HIV infected cells are ACH2 cells.

Also, preferably, the compound of Formula 1

(E) -N8-hydroxy-N1, N1-dimethyl-2-((naphthalen-1-yl oxy) methyl) octendiamide,

(E) -N1- (2- (dimethylamino) ethyl) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) octendiamide,

(E) -N1- (2- (dimethylamino) ethyl) -N8-hydroxy-N1-methyl-2-((naphthalen-1-yl oxy) methyl) octendiamide,

(E) -N1- (2- (diethylamino) ethyl) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) octendiamide,

(E) -N1- (2- (diethylamino) ethyl) -N8-hydroxy-N1-methyl-2-((naphthalen-1-yl oxy) methyl) octendiamide,

(E) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) -N1- (2- (pyrrolidin-1-yl) ethyl) octendiamide,

(E) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) -N1- (2- (piperidin-1-yl) ethyl) octendiamide,

(E) -N8-hydroxy-N1- (2-morpholinoethyl) -2-((naphthalen-1-yl oxy) methyl) octenamide,

(E) -N-hydroxy-8- (4-methylpiperazin-1-yl) -7-((naphthalen-1-yl oxy) methyl) -8-oxooctenamide,

(E) -N8-hydroxy-N1- (2- (4-methylpiperazin-1-yl) ethyl) -2-((naphthalen-1-yl oxy) methyl) octendiamide,

(E) -N1- (cyanomethyl) -N8-hydroxy-N1-methyl-2-((naphthalen-1-yl oxy) methyl) octenamide,

(E) -N8-hydroxy-N1- (2-hydroxyethyl) -N1-methyl-2-((naphthalen-1-yl oxy) methyl) octendiamide,

(E) -N8-hydroxy-N1-methyl-N1- (1-methylpyrrolidin-3-yl) -2-((naphthalen-1-yl oxy) methyl) octendiamide,

(E) -N1- (3- (dimethylamino) propyl) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) octendiamide, and

(E) -N-hydroxy-8-morpholino-7-((naphthalen-1-yl oxy) methyl) -8-oxooctendiiamide derivative.

The present invention also relates to a method of reactivating HIV-1 provirus from latent HIV infected cells using the histone deacetylase inhibitor.

The HDAC inhibitors of the present invention have the ability to reactivate HIV-1 proviruses with low efficacy compared to the high stability of conventional HDAC inhibitors, or have high reactivation ability but at the same time stability and efficacy in treating HIV due to high cytotoxicity Compared with failure to obtain, relatively low cytotoxicity and high HIV-1 proviral reactivation result in more effective reactivation of latent HIV-1 virus from CD4 + T cells, an HIV-1 chronic infectious pathogen. Accordingly, by treating the HDAC inhibitor with the currently used HIV therapeutic agent, reverse transcriptase inhibitor and protease inhibitor, the virus pathogen of the latent HIV-infected cells can be efficiently removed by suppressing the virus released into the extracellular space or cytoplasm. I could do it.

1 shows cytotoxicity (cell viability) after 48 hours of HDAC inhibitor treatment (n = 6).

2 shows the ability of HIV-1 p24 antigen production by reactivation of HIV-1 provirus from chronically infected cells (n = 6).

3 depicts cell viability and HIV-1 p24 antigen production capacity in ACH2 cells against SAHA.

4 depicts cell viability and HIV-1 p24 antigen production capacity in ACH2 cells for PXD-101.

5 depicts cell viability and HIV-1 p24 antigen production capacity in ACH2 cells for CG0005.

6 shows cell viability and HIV-1 p24 antigen production capacity in ACH2 cells for CG0006.

7 shows cell viability measured after treatment of HDAC inhibitors at a concentration of 0.14 μM single treatment.

FIG. 8 shows HIV-1 p24 antigen production capacity measured after treatment of HDAC inhibitors at a concentration of 0.14 μM single treatment.

9 shows cell viability and HIV-1 p24 antigen production capacity in J 1.1 cells against SAHA.

FIG. 10 shows cell viability and HIV-1 p24 antigen production capacity in J 1.1 cells for PXD-101.

FIG. 11 depicts cell viability and HIV-1 p24 antigen production capacity in J 1.1 cells for CG0005.

Figure 12 depicts cell viability and HIV-1 p24 antigen production capacity in J 1.1 cells for CG0006.

FIG. 13 shows changes in HIV-1 p24 antigen inside ACH2 cells after HDAC inhibitor treatment.

Figure 14 shows the change in expression of CD28, an immune cell activation marker after HDAC inhibitor treatment.

FIG. 15 shows the change in expression of signal material (RANTES) related to physiological signal transduction associated with response inside ACH2 cells after HDAC inhibitor treatment.

Figure 16 shows changes in the expression of signaling materials (PD-1 receptor and PD-L1 ligand) involved in the transmission of physiological signals related to responses inside ACH2 cells after HDAC inhibitor treatment.

17 depicts acetylation of histone H3 protein measured by ELISA method after treatment with HDAC inhibitor.

FIG. 18 depicts acetylation and methylation in each portion of histone H3 protein, represented by Western Blot after treatment with HDAC inhibitors.

19 shows changes in phosphorylation of NFkB transcription factors associated with the expression of HIV-1 provirus after treatment with HDAC inhibitors.

Below, The invention is illustrated in detail by the following examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Culture of Cells and Measurement of Cytotoxicity of HDAC Inhibitors

Latent HIV-1 infected cell lines J1.1 and ACH2 cells were treated with HDAC inhibitors to reactivate viral replication of HIV-1 pathogens.

ACH-2 and J1.1 cells, chronically infected with HIV-1 virus, CD4 + T cell lines derived from A3.01 cells and Jurkat cells, were used for the National Institute of Health AIDS Research Reagent. Program). 2- (aryloxymethyl) -oct-2-edioic acid 8-hydroxyamide 1-[(1-alkyl-piperidin-4-yl) -amide] (2) -(aryloxymethyl) -oct-2-enedioic acid 8-hydroxyamide 1-[(1-alkyl-piperidin-4-yl) -amide]) (hereinafter 'CG0005' and 'CG0006', Crystal Genomics, Seoul, Korea ).

The cells were treated with an HDAC inhibitor, and 48 hours later, the toxicity of the cells was measured using an MTT assay (MTT assay, Roche). As a result, CG0006 showed low cytotoxicity (CD ₅₀ : 0.1-0.3 μM) compared to SAHA (CD ₅₀ : 0.3 μM) or PXD-101 (CD ₅₀ : 0.1-0.3 μM). The results of cytotoxicity experiments showing the stability of CG0005 and CG0006 in comparison with SAHA, PXD-101 are shown in FIG. 1.

Example 2. HIV-1 Provirus Reactivation by Treatment of HDAC Inhibitors

<Measurement of HIV-1 Proviral Activation Through HDAC Inhibitor Treatment>

In order to measure HIV-1 proviral reactivation by HDAC inhibitors, the cells were treated with HDAC inhibitors, and then the amount of HIV-1 p24 antigen generated in the cell culture was measured using the Vironostika HIV-1 Antigen MicroELISA Kit (BioMerieux). (HIV-1 Gag p24 antigen ELISA). At this time, the J1.1 and ACH2 cell lines treated with CG0005 and CG0006 produced more HIV-1 p24 antigen at lower concentration than the positive control treated with SAHA and PXD-101 as a positive control. ED ₅₀ was about 0.1 μM for CG0005 and about 0.05 μM for CG0006.

Figure 2 is the result of measuring the amount of HIV-1 p24 antigen discharged to the cell culture in accordance with HIV-1 provirus reactivation by ELISA method. In Figures 3 to 6, the cell viability and HIV-1 p24 antigen generation capacity in ACH2 cells for each HDAC inhibitor based on the results of Figures 1 and 2, CD ₅₀ (Cytotoxic dose 50) and ED ₅₀ (Effective) dose 50). For CG0005 and CG0006, more than 50% of cells survived at higher concentrations (2 μM) compared to SAHA or PXD-101, and at much lower concentrations (0.05-0.1 μM), higher HIV- compared to SAHA or PXD-101. 1 provirus reactivation capacity is shown (FIGS. 5, 6). 7 and 8 are the results of comparing the cytotoxicity and HIV p24 antigen production at a single treatment concentration of 0.14 μM determined in consideration of the stability and reactivation effect of CG0005 and CG0006.

In the cytotoxicity and HIV-1 provirus reactivation assay using another type of T immune cell line, J1.1 cells, CG0005 and CG0006 were similar to those of the ACH2 cell line. ED ₅₀ was better than SAHA and PXD-101 (0.1 μM), and the amount of HIV-1 p24 antigen upon reactivation was also superior to PXD-101 or SAHA (FIGS. 9 to 12).

Next, to measure the intracellular HIV-1 proviral reactivation ability by HDAC inhibitor treatment, after treatment with HDAC inhibitor for 24-48 hours, the percentage of cells expressing intracellular HIV-1 p24 antigen was measured by flow cytometry. Measured. After treatment with HDAC inhibitor, the cultured cells were pretreated with Perm / Fix reagent (BD Sciences) for 15 minutes, stained with KC57 (p24) -RD1 antibody (Beckman Coulter) for 20 minutes, and then FC500 flow cytometer (FC500 Flow cytometer). , Beckman Coulter) was used to measure the cells. After 24 hours of treatment, the percentage of cells expressing HIV-1 p24 antigen in the cells treated with CG0005 and CG0006 increased rapidly. After 48 hours, the percentage of cells expressing HIV-1 p24 antigen was somewhat higher than that of 24 hours. It was reduced, but remained at a high level compared to the control or cells treated with SAHA and PXD-101 inhibitors.

In Fig. 13, the circled parts indicate HIV-1 chronically infected cells (cells infected with HIV-1 provirus but the HIV-1 virus has not replicated), and arrows indicate intracellular HIV-1 reactivated by HDAC inhibitors. The process proceeds to a group of cells expressing the p24 antigen. Compared with SAHA or PXD-101, CG0005 and CG0006 showed reactivation of intracellular HIV-1 provirus significantly after 24 hours of treatment.

In order to determine whether immune cell activation was induced by HDAC inhibitor treatment, CD28 expression patterns of activation markers on the surface of immune cells were measured by flow cytometry. Cells cultured after drug treatment were stained with CD28-ECD antibody (Beckman Coulter) for 20 minutes without pretreatment, and analyzed using FC500 flow cytometer (FC500 Flow cytometer, Beckman Coulter).

FIG. 14 shows the extent to which CD28 expression, an immune cell surface activation marker, is increased by each HDAC inhibitor treatment. Gray sections show CD28 expression in control cells not treated with HDAC inhibitors, and white sections show CD28 expression in cells treated with HDAC inhibitors. CD28 expression was increased after HDAC inhibitor treatment, and CD28 expression was significantly higher in cells treated with CG0005 and CG0006. This trend is consistent up to 48 hours after treatment.

Example 3 RANTES Production Measurement and PD-1, PD-L1 Measurement

After 24 hours of drug treatment, the changes in the expression level of PD-1 and its ligand PD-L1, which are related to intracellular production and cell death, of chemokine RANTES, a ligand of HIV-1 co-receptor CCR5, were measured. After drug treatment, the cultured cells were divided into two plastic test tubes, pretreated with Perm / Fix reagent (BD Sciences) for 15 minutes, and then for 20 minutes, test tube 1 was RANTES-PE (BD Sciences), test tube 2 was CD279 ( After staining with a mixture of PD-1) -PE and CD274 (PD-L1) -FITC (BD Sciences), they were analyzed using an FC500 flow cytometer (Beckman Coulter).

As a result, the intracellular RANTES production tended to increase in PXD-101 and CG0006 treated cells compared to SAHA. In addition, the expression of apoptosis markers PD-1 receptor (Programmed Death receptor 1) and PD-L1 ligand (Programmed Death Receptor 1 ligand) in ACH2 cells, HIV-1 chronically infected cell lines, compared to non HIV-1 infected cells The aspect of change and the recovery of this change by HDAC inhibitor treatment were measured.

In FIG. 15, the gray colored peaks indicate intracellular RANTES expression levels in ACH2 cells, which are chronically infected cell lines without drug treatment, and the white areas indicate increased intracellular RANTES expression levels by HDAC inhibitors. CG0005 and CG0006 showed locally increased RANTES expression compared to SAHA.

FIG. 16 shows the distribution of PD-1 (the yellow portion at the bottom right of each diagram) and PD-L1 (the red portion at the top left of the diagram), compared to A3.01 cells, which are not infected cells. In contrast to the relatively high proportion of PD-L1 ligand-expressing cells and half the low PD-1 receptor-expressing cells in chronically infected cell lines, ACH2 cells, these changes caused by chronic infection when treated with HDAC inhibitors were observed. The percentage of receptor expressing cells was higher and the ratio of PD-L1 expressing cells was somewhat lower, i.e. recovery in the opposite direction was shown.

Example 4 Measurement of Acetylation of Histone H3 Protein and Electrophoretic Mobility Shift Assay (EMSA)

The degree of acetylation of histone H3 protein, which is known to be involved in HIV-1 provirus reactivation from HIV-1 chronically infected cells, was measured using an ELISA kit from Cell Signaling. Treatment of CG0005 and CG0006 significantly increased acetylation of histone H3 (FIG. 17), and correspondingly to the change in acetylation, the amount of HIV-1 p24 antigen also increased as shown in FIG. HIV-1 p24 antigen in cell culture was measured using the Vironostika HIV-1 Antigen MicroELISA Kit (BioMerieux) described above.

To investigate the acetylation changes of histone H3 protein in relation to HIV-1 provirus activation, the primary antibody Acetyl-Histone H3 (Lys9) (# 9671, Cell signaling Technology), Acetyl-Histone H3 (Lys27) (# 07 Immunoblot using a Santa Cruz secondary antibody corresponding to -360, Upstate) and each primary antibody, and b-actin (# sc-1616) from Santa Cruz was used as a loading control. As a result, acetylation of histone H3 occurred at two lysine sites (K9 and K27). Treatment of HDAC inhibitors markedly increased acetylation of histone protein H3 (FIG. 18).

As a result of the measurement of nuclear fator kappa B (NFkB) using Panomics' electrophoretic mobility shift assay (EMSA) kit, the degree of phosphorylation (p65-S ²⁷⁶ ) at the serine 276 residue of NFkB p65 increased and the total amount of NFkB complex also increased. (FIG. 19). NFkB plays a role in the transcription process during the re-expression of the HIV virus hiding in the host genome. The expression of NFkB and 276 serine phosphorylation of NFkB transcription factors were increased by treatment with inhibitors. Treatment of CG0005 and CG0006 of the present invention significantly increased the total amount of NFkB complexes than that of other inhibitors.

As such, CG0005 and CG0006 of the present invention, compared with other conventional inhibitors, induce the activation of NFkB involved in promoting acetylation of histone H3 protein and increasing transcriptional activity upon HIV reactivation, thereby preventing from latent HIV infected cells. It was confirmed that it has the ability to reactivate HIV-1 provirus.

Claims (5)

Histone deacetylase inhibitors of Formula 1 for reactivating HIV-1 provirus from latent HIV infected cells: Formula 1

In Chemical Formula 1, Each R ₁ is independently C _1-3 alkyl, hydroxyC _1-2 alkyl, haloC _1-2 alkyl, piperidinyl, morpholinyl, cyanomethyl, piperazinyl, diC _1-2 alkylamino C _1-2 alkyl, diC _1-2 alkylaminoC _1-2 alkyl, piperidinylC _1-2 alkyl, morpholinoC _1-2 alkyl, piperazinoC _1-2 alkyl, pyrrolidinyl, C and C _1-2 alkyl optionally substituted with one or more substituents selected from the group consisting of _{1-2-alkyl-pyrrolidinyl,} R ₂ is hydrogen or methyl. The method of claim 1, A histone deacetylase inhibitor, characterized in that the latent HIV infected cells are ACH2 cells or J1.1 cells. The method of claim 2, A histone deacetylase inhibitor, characterized in that the latent HIV infected cells are ACH2 cells. The method according to any one of claims 1 to 3, Compound of Formula 1 (E) -N8-hydroxy-N1, N1-dimethyl-2-((naphthalen-1-yl oxy) methyl) octendiamide, (E) -N1- (2- (dimethylamino) ethyl) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) octendiamide, (E) -N1- (2- (dimethylamino) ethyl) -N8-hydroxy-N1-methyl-2-((naphthalen-1-yl oxy) methyl) octendiamide, (E) -N1- (2- (diethylamino) ethyl) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) octendiamide, (E) -N1- (2- (diethylamino) ethyl) -N8-hydroxy-N1-methyl-2-((naphthalen-1-yl oxy) methyl) octendiamide, (E) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) -N1- (2- (pyrrolidin-1-yl) ethyl) octendiamide, (E) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) -N1- (2- (piperidin-1-yl) ethyl) octendiamide, (E) -N8-hydroxy-N1- (2-morpholinoethyl) -2-((naphthalen-1-yl oxy) methyl) octenamide, (E) -N-hydroxy-8- (4-methylpiperazin-1-yl) -7-((naphthalen-1-yl oxy) methyl) -8-oxooctenamide, (E) -N8-hydroxy-N1- (2- (4-methylpiperazin-1-yl) ethyl) -2-((naphthalen-1-yl oxy) methyl) octendiamide, (E) -N1- (cyanomethyl) -N8-hydroxy-N1-methyl-2-((naphthalen-1-yl oxy) methyl) octenamide, (E) -N8-hydroxy-N1- (2-hydroxyethyl) -N1-methyl-2-((naphthalen-1-yl oxy) methyl) octenamide, (E) -N8-hydroxy-N1-methyl-N1- (1-methylpyrrolidin-3-yl) -2-((naphthalen-1-yl oxy) methyl) octendiamide, (E) -N1- (3- (dimethylamino) propyl) -N8-hydroxy-2-((naphthalen-1-yl oxy) methyl) octendiamide, and Histone deacetylase, characterized in that it is selected from the group consisting of (E) -N-hydroxy-8-morpholino-7-((naphthalen-1-yl oxy) methyl) -8-oxooctendiiamide derivatives Inhibitors. A method of reactivating HIV-1 provirus from latent HIV infected cells using the histone deacetylase inhibitor of claim 1.

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