CN110960669B

CN110960669B - HIV-1 latent infection activator thiostrepton

Info

Publication number: CN110960669B
Application number: CN201911243091.0A
Authority: CN
Inventors: 邓凯; 彭雯
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-12-06
Filing date: 2019-12-06
Publication date: 2022-01-21
Anticipated expiration: 2039-12-06
Also published as: CN110960669A

Abstract

The invention discloses a latent HIV-1 infection activator, i.e. thiostrepton, and utilizes the thiostrepton and/or pharmaceutically acceptable salt thereof as the latent HIV-1 infection activator. The invention provides a novel small molecular compound Thiostrepton (Thiostrepton), which can effectively activate latent HIV-1 infected cells, has small toxic and side effects, does not cause the activation of global T cells, is expected to be developed into a novel latent activator in the future and enter clinical tests, and has good popularization and application values.

Description

HIV-1 latent infection activator thiostrepton

Technical Field

The invention relates to the technical field of prevention and treatment of HIV (human immunodeficiency virus), in particular to a latent HIV-1 infection activator thiostrepton.

Background

Human Immunodeficiency Virus (HIV) was discovered in 1981 and subsequently demonstrated to cause Acquired Immunodeficiency Syndrome (AIDs), a retrovirus that is extremely harmful to humans. After HIV enters the body, CD4 lymphocytes of the human immune system are mainly attacked, so that the number of CD4 cells in the body is sharply reduced, the immune cells are greatly damaged, the immune system of the body is collapsed, and therefore high-incidence of various opportunistic infections and cancers is caused, and finally death of infected patients is caused.

The emergence of combined antiretroviral therapy (cART) effectively controls HIV-1 virus and represents a milestone in modern medicine. ART can persistently suppress HIV-1 replication, restore the immune system, prevent disease progression, and reduce the risk of viral transmission to uninfected individuals.

Since HIV-1 is able to integrate into the host cell genome, forming a stable viral latency pool and persisting for a long time, the viral load in patients rapidly rebounds once dosing is discontinued. The existence of viral latency pools is the main reason for the difficulty of curing HIV-1. Currently, a number of therapeutic strategies are proposed to target the viral latency pool, one of which is called "activation-killing", which is proposed to specifically activate HIV-1 virus in the reservoir by latent infection-reversing drugs (activators), and then ultimately achieve a functional cure for aids by viral cytopathic effects or specific killing of cells by the body's immune system. Many latent infection activators are effective in inducing virus production in different HIV-1 latent models, some of which have been approved for clinical trials.

There is no evidence in various clinical trials that latent infection activators are capable of safely and efficiently inducing reactivation of latent HIV-1. Because multiple molecular mechanisms maintain the latent reservoir of HIV-1, suboptimal efficacy or action of latent infection activators on a single mechanism results in incomplete reactivation. In addition, PKC agonists have been shown to be effective in inducing HIV-1 activation in vivo and in vitro, but can cause global activation of T cells, cytokine storms, and life-threatening inflammatory responses due to nonspecific induction. Another latent infection activator, SAHA, impairs the function of HIV-1 specific cytotoxic lymphocytes and has been shown to fail to reduce the HIV-1 latent reservoir. Therefore, there is an urgent need to find new compounds with higher efficacy and low toxicity.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a latent HIV-1 infection activator, namely thiostrepton.

The first object of the present invention is to provide the use of thiostrepton and/or its pharmaceutically acceptable salt as HIV-1 latent infection activator.

The second purpose of the invention is to provide the application of thiostrepton and/or pharmaceutically acceptable salt thereof in preparing HIV-1 latent infection activator.

The third purpose of the invention is to provide an HIV-1 latent infection activator.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the structural formula of thiostrepton is shown in a formula (I), and the thiostrepton is a polypeptide antibiotic separated from streptomycin coelicolor. It has great activity not only against gram-positive bacteria, malaria and fungi, but also has been reported in recent years to have anticancer activity. The invention researches the optimal action concentration, the drug safety, the action mechanism and the like of the HIV-1 virus.

The present invention therefore claims the use of thiostrepton and/or its pharmaceutically acceptable salts as activators of HIV-1 latent infection.

The invention also claims the application of thiostrepton and/or the pharmaceutically acceptable salt thereof in preparing HIV-1 latent infection activators.

The invention further claims an HIV-1 latent infection activator which contains thiostrepton and/or pharmaceutically acceptable salt thereof.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a novel small molecular compound Thiostrepton (Thiostrepton), which can effectively activate latent HIV-1 infected cells, has small toxic and side effects, does not cause the activation of global T cells, is expected to be developed into a novel latent activator in the future and enter clinical tests, and has good popularization and application values.

Drawings

FIG. 1 is a flow chart of double staining of CD4 and Bcl-2 on Bcl-2 transduced cells.

FIG. 2 shows the GFP negative cell flow sorting strategy and purity

FIG. 3 is a compound capable of activating latent HIV-1.

FIG. 4 is a graph showing the test of the concentration of the optimum action of X4.

FIG. 5 is a graph showing a comparison of the reactivation efficiency of thiostrepton at different concentrations in the J-Lat10.6 cell line.

FIG. 6 shows the apoptosis of cells after the action of thiostrepton detected by Annexin V/PI double staining method.

FIG. 7 shows the CCK8 method for detecting the effect of thiostrepton on cell activity.

FIG. 8 is a graph showing the effect of thiostrepton on the expression of cell surface HIV-1 co-receptors CXCR4 and CCR 5.

FIG. 9 is a graph showing the effect of thiostrepton on the expression of CD25, CD69 and HLA-DR, markers for surface activation of CD4+ T lymphocytes.

FIG. 10 shows the results of thiostrepton acting on ex vivo samples of HIV-1 infected patients.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Sample source

Healthy human peripheral blood leucocyte (concentrated cell) from Guangzhou city blood center, and is negative to conventional tests of hepatitis B (HBsAg), Hepatitis C (HCV), AIDS (HIV), treponema pallidum and treponema pallidum (Syphilis).

Patient samples peripheral blood was from the fifth hospital of Zhongshan university, Zhuhai, with patient selection based on a viral load of less than 50 copies per milliliter, and CD4+ T cells of more than 400 cells per microliter, maintaining this criterion for more than two years.

Example 1 construction of HIV-1 Primary cell latent infection model

First, Bcl-2 transduction and amplification

Firstly, screening CD4+ T cells in normal human PBMCs by using magnetic beads, infecting lentivirus EB-FLV carrying Anti-apoptotic gene Bcl-2 after Anti-CD3/28 and IL-2 are activated, culturing for 3-4 weeks under the condition of no TCR stimulation and exogenous cytokine existence, and removing dead cells by a Ficoll density gradient centrifugation method to obtain a primary CD4+ T cell line capable of stably expressing Bcl-2.

(I) Experimental method

1. PBMC from healthy donors were isolated, CD4+ T cells were obtained from BD negative selection beads, resuspended in STCM at a cell density of 1X 10⁶Perml, activation was performed in 6-well plates.

2. Collecting activated CD4+ T cells, centrifuging to obtain a proper amount of STCM (cell concentration is 2-4 multiplied by 10) for resuspension⁶Per ml), adding Bcl-2-carrying lentivirus EB-FLV concentrated solution (20-30 multiplied by 10 per milliliter)⁶The amount of the virus obtained by packaging each cell in 3 10cm culture dishes) was added, and the mixture was thoroughly shaken and mixed.

3. The cell-virus mixture was transferred to a V-type loading well, and the gun pipetted the mixture into a round bottom 96 well plate at 100 μ l per well.

4.1200g, and centrifuged at 30 ℃ for 2 hours.

5. The 96-well plate was removed and supplemented with 100. mu.l of STCM per well and incubated overnight at 37 ℃.

6. The cells were blown up and mixed well, transferred from the 96-well plate to a culture flask, supplemented with sufficient STCM to give a cell density of 0.5X 10⁶Cells/ml.

After 2 days of incubation at 7.37 ℃, the cell suspension was transferred to a 50ml centrifuge tube and trypan blue counted and centrifuged simultaneously at 350g for 10 minutes at 4 ℃.

8. Resuspending the cells with CFM at a cell density of 1-2X 10⁶Mixing the cells/ml, culturing at 37 deg.C for 3 weeks

9. The cultured cells were collected, centrifuged at 350g for 10 minutes at room temperature, the supernatant was discarded, and then resuspended in CFM incubated at 37 ℃ at a maximum of 10 per 9ml⁸And (4) cells.

10. After passing through Mini Ficoll, 4.5ml of human lymphocyte separating medium is transferred into a 15ml centrifuge tube, then 9ml of cell suspension is carefully added along the tube wall, and then the cell suspension is centrifuged at 335g for 10 minutes at room temperature, and the speed is reduced to 0.

11. And (3) reducing dead cells to the bottom of the tube, taking the cell layer between the lymphocyte separation liquid and the CFM as live cells, sucking the live cells in the middle layer into a 50ml centrifuge tube, adding WM (WM) to 40ml, reversing, uniformly mixing, and centrifuging at 350g at room temperature for 10 minutes.

12. Add appropriate WM heavy suspension cell, trypan blue count, at the same time centrifugation, so that stably expressing Bcl-2 primary CD4+ T cells, thereafter Bcl-2 cells.

13. Bcl-2 cells were resuspended in STCM to a cell density of 1X 10⁶Cells/ml, activated in 6-well plates.

After 14.3 days, the cells were harvested, centrifuged, and CFM resuspended at 1X 10⁶The final concentration of each cell per ml was 100U/ml by adding human cytokine IL-2.

Cultured at 15.37 ℃ and counted every 2 days, and cell density was maintained at10 by supplementing CFM⁶Cells/ml, supplemented with IL-2 to maintain a final concentration of 100U/mlAnd amplifying for 7-10 days.

16. And repeating the steps 9-12 to obtain the amplified Bcl-2 cells, and activating or freezing the cells according to the experimental requirements.

The primary CD4+ T cell line obtained was then stained with flow-through antibodies CD4 and Bcl-2 to confirm the successful introduction of Bcl-2.

(II) results of the experiment

The experimental results showed that the expression levels of both Bcl-2 and CD4 were high, almost close to 100%, in the obtained primary CD4+ T cells (fig. 1). The high expression of the primary CD4+ T cell Bcl-2 provides important guarantee for the subsequent long-term latent establishment process.

Second, infection and latent cell sorting of HIV-1 pseudoviruses

To further obtain HIV-1 latently infected cells, we reactivate the primary CD4+ T cell line with Bcl-2 over-expression using Anti-CD3/28 and IL-2, then infect the HIV-1 pseudovirus with GFP reporter gene, culture for more than 4 weeks without cytokines and other stimulators, allow the activated cells to return to resting state, and finally, select GFP negative cells by flow sorting, thus completing the establishment of HIV-1 primary cell latently infected model.

(I) Experimental method

STCM resuspension of Bcl-2 cells activated in 6-well plates.

2. Centrifugally collecting cells, after the STCM is resuspended, adding the resuspended green fluorescent protein gene, and then adding the HIV-1 pseudovirus CM6 (the virus dosage is 500-800 ng/10) carrying the green fluorescent protein gene⁶) The cell-virus mixture was transferred to a 96-well plate, and centrifuged at 1200g at 30 ℃ for 2 hours to infect the cells.

3. Supplementing STCM 100 μ l per well, culturing at 37 deg.C overnight, transferring cells from 96-well plate to culture flask, and supplementing STCM to 0.5 × 10 according to cell number⁶Cells/ml.

Culturing at 4.37 deg.C for 2 days, counting cells, and collecting 1 × 10 cells⁶After washing with WM, the cells were washed with a flow cytometer and then the virus infection rate was measured with the flow cytometer, and the remaining cells were centrifuged at 350g for 10 minutes at 4 ℃.

5, resuspending the cells in the CFM,the cell density is controlled to be 1-2 × 10⁶Culturing the cells/ml at 37 ℃ for 4-6 weeks until the cells are rested, taking half of the cell suspension at 2-3 weeks, centrifuging the cell suspension at 350g and 4 ℃, re-suspending the cells by using fresh CFM with the same volume, and transferring the cells to an original culture flask.

6. Removing dead cells after passing through Mini Ficoll, counting the obtained living cells, centrifuging, and performing centrifugation according to the weight ratio of 15-20 multiplied by 10⁶Cells/ml were resuspended in WM and transferred to a capped flow tube.

7. The flow cytometric cell sorter sorts GFP-cells, which contained HIV-1 latently infected cells and uninfected cells, and resuspended in CFM after centrifugation, cultured at 37 ℃ for subsequent experiments.

Sorting of GFP negative cells was performed as shown in FIG. 2.

8. GFP-cells selected by flow-sorting were stimulated with PMA/Ionomycin, Anti-CD3/28, SAHA and Brysotatin-1, respectively, and the percentage of GFP-positive cells was measured 2 days later. The dosage of PMA and Ionomycin is 50ng/ml and 1 mu M respectively, the dosage of Anti-CD3 and Anti-CD28 is 1 mu g/ml, the dosage of SAHA and Brysotatin-1 is 1 mu M respectively, and an unworked control group is arranged. The control group and the treatment group were each provided with 3 duplicate wells.

(II) results of the experiment

And (3) activating the CD4+ T cells stably expressing Bcl-2 again, infecting the HIV-1 pseudovirus with GFP, culturing for 4-6 weeks, and sorting GFP negative cells containing uninfected and latent infected cells when the cells return to a resting state. The purity of the obtained cells is close to 100 percent after the sorting of GFP negative cells.

As shown in the figure, when cells are co-stimulated by strong activators PMA and Ionomycin, 2.47% of the cells can be induced to express GFP, while Anti-CD3/28 group GFP positive cells can also reach 1.87%, but the non-stimulated group GFP positive rate is only 0.105%, which indicates that the GFP negative cells are rich in HIV-1 latent infected cells. Meanwhile, the cells are stimulated by the classical activators SAHA and Brostatin-1 respectively, and the cells can be induced to express GFP by 1.48 percent and 1.23 percent, which further proves the reliability of the constructed model for screening the latent infection activator.

EXAMPLE 2 Thioselins activation of latent HIV-1

First, experiment method

Flow cytometry-based stimulation of selected GFP-negative cells was used for activation and cellular GFP expression was detected by flow cytometry. The negative control was DMSO and the positive control was PMA/Ionomycin, with controls using reported latent activators such as panobinostat, bryostatin, disulfiram, JQ 1.

Second, experimental results

Typically, reactivation of GFP-negative cells in this model induces expression of GFP fluorescence by 1% to 6% of the cells. After the PMA/Ionomycin group reactivated cell GFP positive rate is ensured to reach at least more than 1%, the activation effect of the small molecular compound thiostrepton on latent HIV-1 is detected.

The results show that thiostrepton acts stronger than other known activator controls, even higher than the PMA/Ionomycin group (FIG. 3).

EXAMPLE 3 screening of the optimal working concentration of thiostrepton

First, experiment method

To determine the optimal working concentration of thiostrepton, different drug concentrations were set for activation experiments, including 7 groups of 100nM, 250nM, 500nM, 1. mu.M, 2.5. mu.M, 5. mu.M and 10. mu.M, with the group treated with dilution DMSO as a negative control and PHA as a positive control. The action time is 3 days.

Second, experimental results

The results show that the activation efficiency gradually increases with increasing thiostrepton dosage, the reactivation ratio reaches the maximum at 1. mu.M, and the activation effect of the donor1 is even higher than that of the positive control group at the concentration of 1. mu.M. And then the activation rate began to gradually decrease as the concentration of the drug increased (fig. 4). This result indicates that the optimal working concentration of thiostrepton is 1. mu.M, at which the maximum ratio of activation of HIV-1 latently infected cells is achieved.

Example 4 detection of the reactivation efficiency of thiostrepton in the J-LatA10.6 cell line

The ability of thiostrepton to reactivate latent virus was further demonstrated by the use of another HIV-1 latent infected cell line, J-Lat10.6 cells.

First, experiment method

The latent activation ability of X4 was verified by using another latently infected cell line, J-Lat 10.6. Cells were first stimulated with thiostrepton at various concentrations for 48h, and then cellular GFP expression, the level of GFP expression representing the level of reactivation of HIV-1, was detected by flow.

Second, experimental results

As with HIV-1 primary cell latent infection model results, thiostrepton can also obviously activate the latent infected cells in J-Lat10.6 cells, and besides positive controls PMA and TNF-alpha can effectively activate the latent HIV-1 in J-Lat10.6, thiostrepton also has obvious activation effect on the latent infected viruses in the cell line. And similar to the result of the Bcl-2 cell latent infection model, in J-La 10.6 cells, along with the increase of the thiostrepton drug concentration, the reactivation ratio of the latent infected cells is gradually increased, the optimal activation effect is achieved at1 mu M, the GFP positive cell ratio is about 55%, and the difference has statistical significance compared with other drug concentration groups (figure 5). It is strongly demonstrated that thiostrepton can induce potent reactivation of latent HIV-1, and that 1. mu.M is the optimal drug administration concentration.

Example 5 drug safety evaluation

Drug safety evaluation is an important component in the screening work of activators. Although thiostrepton is effective in activating latent HIV-1 provirus, its effect on cell viability will also affect its potential for future development as a latent activator.

One-and two-staining method for detecting apoptosis of cell after thiostrepton action

1. Experimental methods

The apoptosis of the cells after the thiostrepton action was detected by Annexin V/PI double staining (shown in FIG. 6A). Thiostrepton and other activators are respectively added into freshly separated human PBMCs, flow antibody staining is carried out after 3 days, and the positive rates of cells PI and Annexin V of different drug groups are detected.

2. Results of the experiment

The results show that PMA/Ionomycin caused a large increase in the rate of apoptosis compared to the untreated group. SAHA, one of the commonly used HIV-1 latent infection activators, also causes apoptosis of partial cells at the optimal drug concentration, wherein the expression of Annexin V is obviously higher than that of a negative control group, and the results of the two groups are statistically different. However, neither 1 μ M thiostrepton nor 0.5 μ M Disulfiam caused significant apoptotic responses, and the positive rates of PI and Annexin V in both groups were comparable to the untreated group, and the positive rates were comparable to the untreated group, and the proportion of PI-positive cells in the thiostrepton group was slightly lower than that in the Disulfiam group (FIG. 6B). From the above results, we can know that thiostrepton has a small cytotoxic effect and that its use does not induce a large amount of apoptosis.

Second, CCK8 method for detecting cell viability

1. Experimental methods

1. Preparing cell suspension from healthy human PBMCs, and controlling the cell density to be 1-2 multiplied by 10⁶Cells/ml.

2. Thiostrepton and other activators were added and incubated at 37 ℃ for 3 days.

3. Plating into 96-well plates: the same samples were replicated in 3 replicates, with an appropriate number of plated cells, about 100ul of cell suspension per well.

4. 10ul of CCK8 was added and cultured for 4 hours.

6. Absorbance at 450nm was measured.

2. Results of the experiment

The results show (FIG. 7) that the optimal drug administration concentration of thiostrepton, 1 μ M, had less effect on cell activity.

Example 6 Effect of thiostrepton treatment on the susceptibility of cells to HIV-1

HIV-1 requires the co-action of the co-receptors CXCR4 or CCR5 in addition to the receptor CD4 when infecting cells. It has been shown that the expression of peripheral blood CD4+ T lymphocyte cell surface co-receptor is closely related to HIV susceptibility and disease progression.

First, experiment method

To determine whether thiostrepton, while activating latently infected cells, would cause an increase in the susceptibility of the cells to the virus, the expression of cell surface CCR5 and CXCR4 was examined using flow cytometry on primary CD4+ T cells after thiostrepton treatment.

Second, experimental results

Except that the PMA/Ionomycin group is over-cytotoxic and the expression of CXCR4 is obviously reduced, the expression of CXCR4 of three groups of thiostrepton, Disulfiram and SAHA cells is not obviously changed, and no statistical difference exists compared with a negative control group (DMSO) (FIG. 8A). Likewise, the use of thiostrepton did not affect the expression of cellular CCR5 (fig. 8B).

Example 7 the use of thiostrepton did not trigger global T cell activation

In addition to the specific activation of latent HIV-1 provirus, the widespread activation of latent infected cells can also induce the transcription and replication of latent HIV-1. However, if the latent infection activator causes global activation of CD4+ T cells while activating the latent virus, it induces the cells to secrete a large amount of cytokines, causing cytokine storm syndrome and causing severe damage to the body. PKC activators such as PMA, while having a strong ability to activate latent HIV-1, have been limited in their use because they cause extensive T cell activation. Thus, whether it would cause global T cell activation was assessed in this study by examining the expression of cell surface activation markers CD25, CD69, and HLA-DR after thiostrepton action.

First, experiment method

And (3) adding thiostrepton, SAHA, Disulfiam and PMA/Ionomycin to treat freshly separated primary CD4+ T cells, centrifuging after 3 days, collecting the cells, and detecting the expression levels of activation markers CD25, CD69 and HLA-DR on the cell surface by using a flow cytometer through flow antibody staining.

Second, experimental results

The results are shown in FIG. 9, where the positive control PMA/Ionomycin treated, both CD25 and CD69 were significantly increased, while the thiostrepton treated, CD69, CD25 and HLA-DR expression levels were not significantly changed, and the CD25 and HLA-DR expression levels were even lower than those of SAHA and Disulfiam control groups. While the expression of CD69 was slightly higher than that of the two groups, there was no statistical difference compared to the negative control group. This result indicates that X4 does not trigger non-specific T cell activation when activating HIV-1 latent in primary CD4+ T cells.

EXAMPLE 8 Effect of thiostrepton on patients on Long-term dosing

First, experiment method

CD4+ T cells were extracted for drug activation testing in HIV infected patients with cART controlled viremia for more than two years and viral loads below the clinical limit of detection for long term use. Thiostrepton (1 mu M) is selected to stimulate CD4+ T cells for 48h, cell RNA is extracted and subjected to reverse transcription, and then a qPCR experiment is carried out to detect the expression condition of HIV RNA. PMA/Ionomycin is adopted as a positive control drug.

Second, experimental results

The results show (fig. 10) that a significant increase in HIV transcript levels was also observed in isolated CD4+ T cells from chronically dosed patients after stimulation with thiostrepton.

Claims

1. Use of thiostrepton and/or its pharmaceutically acceptable salt for preparing HIV-1 latent infection activator.