阅读说明：本技术 汉黄芩素在制备乙酰转移酶抑制剂、巴豆酰化抑制剂和潜伏促进剂中的应用 (Application of wogonin in preparation of acetyltransferase inhibitor, crotonylation inhibitor and latency promoter ) 是由 夏瑾瑜 洪仲思 张海涛 李春娜 邓丽斯 黄明星 刘曦 丁立 邹幼清 蔡金锋 邓凯 于 2021-07-30 设计创作，主要内容包括：本发明属于中药活性成分应用技术领域,公开了汉黄芩素在制备乙酰转移酶抑制剂、巴豆酰化抑制剂和潜伏促进剂中的应用。本发明指出,汉黄芩素可抑制组蛋白乙酰转移酶的表达,以及抑制组蛋白H3/H4的巴豆酰化,并能起到抑制潜伏HIV-1再激活的功能,因此可应用于制备乙酰转移酶抑制剂、巴豆酰化抑制剂和潜伏促进剂。(The invention belongs to the technical field of application of active ingredients of traditional Chinese medicines, and discloses application of wogonin in preparation of acetyltransferase inhibitors, crotonylation inhibitors and latency promoters. The wogonin can inhibit the expression of histone acetyl transferase and crotonyl of histone H3/H4 and inhibit the reactivation of latent HIV-1, so that it may be used in preparing acetyl transferase inhibitor, crotonyl inhibitor and latent promoter.)

1. Application of wogonin in preparing acetyltransferase inhibitor is provided.

2. The use according to claim 1, wherein the acetyltransferase is P300.

3. Application of wogonin in preparing crotonylation inhibitor is provided.

4. Application of wogonin in preparing latency promoter is provided.

5. The use of claim 4, wherein the latency-enhancing agent comprises wogonin and triptolide.

Technical Field

The invention belongs to the technical field of application of active ingredients of traditional Chinese medicines, and particularly relates to application of wogonin in preparation of acetyltransferase inhibitors, crotonylation inhibitors and latency promoters.

Background

The scutellaria baicalensis is a common traditional Chinese medicine for clinically treating AIDS and complications thereof, and has the effects of clearing heat and drying dampness, purging fire and detoxifying, resisting oxidation, resisting tumors, resisting allergy, resisting bacteria and the like. A large number of research reports show that the flavonoid components (including baicalein, wogonin, baicalin and the like) are the most main chemical active components in the scutellaria baicalensis and are the basis for exerting pharmacological activity.

However, the common research suggests that baicalein is the key of the anti-HIV activity in scutellaria baicalensis, the action mechanism of the baicalein is probably to inhibit the replication of Human Immunodeficiency Virus (HIV) by inhibiting the activity of human immunodeficiency virus-1 reverse transcriptase, and moreover, the baicalein can be combined with a hydrophobic region of an catalytic core region of HIV-1 integrase to change the structure of the HIV-1 integrase which catalyzes the HIV to enter mitochondria of host cells, so that the baicalein has the anti-HIV effect. Wogonin is considered to have no good anti-AIDS function, and is only considered to have certain anti-inflammatory, anti-oxidation and anti-tumor activities.

Although the variety of AIDS treatments is wide at present, the AIDS cannot be completely cured, and the main obstacle is the existence of human immunodeficiency virus type 1 (HIV-1) latent storage. Therefore, the continuous inhibition of the activation of latent viruses to achieve a "blocking and blocking" scheme that does not rebound after the inactivation of Antiretroviral therapy (ART) is one of the strategies for the functional cure of aids proposed in recent years. The reported "blocking and blocking" agents, also known as Latency Promoters (LPAs), can slow the rebound of HIV-1 after ART is discontinued in cell and animal experiments, but still do not achieve the goal of clinical application, and there is a great need for more and more ideal HIV-1 inhibitory drugs.

Since the pharmacological properties of wogonin have not been sufficiently studied, there may be some unique pharmacological effects that have not been found to be utilized. Therefore, the application develops further research on wogonin to develop new pharmacological action and application of wogonin.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of wogonin in preparing acetyl transferase inhibitors, crotonylation inhibitors and latency promoters. The wogonin can inhibit the expression of histone acetyl transferase and crotonyl of histone H3/H4 and inhibit the reactivation of latent HIV-1, so that it may be used in preparing acetyl transferase inhibitor, crotonyl acylation inhibitor and latent promoter.

The invention provides an application of wogonin in preparing an acetyltransferase inhibitor. The acetyl transferase includes P300, CBP, Mof or Tip 60.

Preferably, the acetyltransferase is P300.

The invention also provides the application of wogonin in preparing crotonylation inhibitor.

The invention also provides the application of wogonin in preparing a latency promoter.

Preferably, the latency promoter comprises wogonin and triptolide. Research shows that wogonin and triptolide have the function of synergistically inhibiting reactivation of latent HIV-1.

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

the invention points out that a flavonoid compound, Wogonin (Wogonin), in the traditional Chinese medicine scutellaria baicalensis has the effect of inhibiting the expression of histone acetyltransferase, can reduce crotonylation of histone H3/H4, can also play a role in reactivating latent HIV-1, and has low toxicity and lasting effect. Therefore, wogonin can be applied to the preparation of acetyltransferase inhibitors, crotonylation inhibitors and latency promoters, and has important values and application prospects in scientific research and clinical practice. The present invention further indicates that the use of triptolide, a known latency promoter, in combination with wogonin, synergistically inhibits the reactivation of latent HIV-1.

Drawings

FIG. 1 shows the inhibition of crotonylation of histones by wogonin;

FIG. 2 shows the results of inhibition of histone acetyltransferase by wogonin;

FIG. 3 shows the reversal effect of CTPB on the effect of wogonin;

FIG. 4 shows the inhibition of latent HIV-1 reactivation by wogonin at various concentrations;

FIG. 5 shows the result of the "blocking" effect of wogonin on the reactivation of the latent pool;

FIG. 6 shows the inhibition of the reactivation of the HIV-1 infected peripheral leukemia virus reservoir by wogonin;

FIG. 7 shows the inhibition of reactivation of latent HIV-1 by wogonin in combination with triptolide;

FIG. 8 shows the specificity of wogonin to inhibit the reactivation function of latent HIV-1.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are only preferred embodiments of the present invention, and the claimed protection scope is not limited thereto, and any modification, substitution, combination made without departing from the spirit and principle of the present invention are included in the protection scope of the present invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Example 1

Inhibition of histone crotonylation by wogonin

The chromatin nucleosome is composed of 8 core histones (two monomers of H2A, H2B, H3 and H4) which can be epigenetically modified, and the epigenetics modification of the nucleosome can realize the regulation of HIV-1 transcription. Wherein acetylation of the core histone modifies the chromatin to loosen it for easier utilization by interacting proteins, thereby promoting HIV-1 transcriptional activation. Whereas histone acylation modification of the binding site of the HIV-1 Long Terminal Repeat (LTR) is crucial for the establishment of latency pools.

Recent studies have found that crotonylation and acetylation of histones loosen the binding of histones to the LTR region of HIV-1, and promote the transcriptional reactivation of latent HIV-1, and that crotonylation promotes the transcription of HIV-1 more efficiently. Vorinostat (SAHA) is a known Histone Deacetylase (HDAC) inhibitor that promotes histone crotylation by inhibiting HDAC, thereby activating the expression of latent HIV-1.

To investigate whether wogonin affects crotonylation of the LTR block protein of latent HIV-1, this example used 12-well cell culture plates, using J-Lat 10.6 cells with a culture system of 3mL (J-Lat 10.6 is a full length HIV-1 genome with non-functional Env gene frameshift mutation and GFP (green fluorescent protein) instead of the Nef gene, constructed by packaging retrovirus infection with Jurkat cells for HIV-R7/E-/GFP), cells were treated with different drugs according to the following groups: the DMSO group was used as a negative control group, the SAHA group (1. mu.M) was used as a positive control group, and wogonin (50. mu.M) + SAHA group (1. mu.M) were used alone as experimental groups. After 24 hours of treatment, the proteins were extracted and finally the histone H3/H4 was detected by immunoblotting for crotonylation and the experiment was repeated three times (see schematic experimental scheme A in FIG. 1). In FIG. 1, B is the Western blot image of histone H3 and histone H3/H4 crotonyl acylation. In FIG. 1, C-D are core histone H3 as reference, and the ratio of crotonylation to histone H3 band gray value was calculated separately for H3/H4. Results are expressed as mean ± standard deviation. "+" indicates p < 0.05, "+" indicates p < 0.01, and "ns" indicates no statistical difference (p > 0.05). Wogonin: wogonin, Cr: crotonylation, DMSO: dimethylsulfoxide, SAHA: vorinostat, WB: western blotting.

The results in FIG. 1 show that crotonylation H3/H4 was significantly down-regulated in the wogonin group relative to the negative control DMSO group; while H3/H4 crotonylation was also significantly inhibited in the wogonin + SAHA group relative to the positive control SAHA group. The results show that wogonin can inhibit crotonylation of H3/H4 histone of J-Lat 10.6 cell.

Example 2

Inhibition of histone acetyltransferase by wogonin

Crotonylation modification of histones is doubly regulated by histone deacetylase 1(HDAC1) and histone acetyltransferase P300, where P300 promotes acetylation and crotonylation modification of histones, whereas HDAC1 acts in contrast.

To investigate the upstream pathway of wogonin down-regulation of histone crotonylation, the present invention further explored the effect of wogonin on HDAC1 and P300 expression. Using 12-well cell culture plates, selecting J-Lat 10.6 cells with a culture system of 3mL, and applying different drugs to treat the cells according to the following groups: the DMSO group was used as a negative control group, while wogonin (50. mu.M, 100. mu.M, 150. mu.M) was used as an experimental group. After 24 hours of treatment, the expression of HDAC1 and P300 was detected by immunoblotting after protein extraction, and the experiment was repeated three times (see schematic experimental flow in a in fig. 2); in fig. 2B is an immunoblot image of HDAC1 and P300 expression. In fig. 2, C is shown by calculating the ratio of the gray scale values of HDAC1 and P300 bands to GAPDH band using GAPDH as a reference. Results are expressed as mean ± standard deviation. "+" indicates p < 0.05, "+" indicates p < 0.01, and "ns" indicates no statistical difference (p > 0.05). Wogonin: wogonin, P300: a histone acetyltransferase, HDAC 1: histone deacetylase 1, DMSO: dimethylsulfoxide, WB: western blotting.

The results in fig. 2 show that wogonin significantly inhibited histone acetyltransferase P300 expression compared to the negative control DMSO group, with increased inhibition with increasing wogonin concentration; while wogonin had no significant effect on HDAC1 expression. The above results indicate that wogonin specifically inhibits the expression of P300 with less effect on HDAC 1.

Example 3

The effect of wogonin on crotonylation and latent HIV-1 can be reversed by CTPB

CTPB is a specific small molecule activator of P300. To investigate whether P300 activation by CTPB could reverse the inhibition of crotonylation by wogonin, this example used immunoblotting to detect changes in H3/H4 crotonylation in J-Lat 10.6 cells. Using 12-well cell culture plates, selecting J-Lat 10.6 cells with a culture system of 3mL, treating the cells according to the following grouping: taking DMSO group as negative control group, taking wogonin (50 μ M, 100 μ M, 150 μ M) and wogonin (50 μ M, 100 μ M, 150 μ M) + CTPB (100M) as experimental group alone, treating for 24 hr, extracting protein, and detecting crotylation of H3/H4 group protein by immunoblotting (see schematic experimental flow in A in FIG. 3); in FIG. 3, B is an immunoblot image of histone H3 and crotonylation expression of H3/H4; taking DMSO as a negative control group, CTPB (100M) as a positive control group, and wogonin (50 μ M) + CTPB (100M) as experimental groups, treating for 24 hours, and detecting GFP expression by flow cytometry; in FIG. 3, C shows the statistics of percentage of GFP positive cells in J-Lat 10.6 cells compared to the negative control DMSO group, and the results are expressed as mean. + -. standard deviation, "+" indicates p < 0.05, "+" indicates p < 0.01, and "ns" indicates no statistical difference (p > 0.05). Wogonin: wogonin, Cr: crotonylation, DMSO: dimethylsulfoxide, CTPB: a selective small molecule activator of P300, WB: western blotting.

The results in FIG. 3 show that the simultaneous treatment of cells with CTPB potentiates the H3/H4 crotonylation, which is inhibited by wogonin, again compared to wogonin alone. By detecting the GFP (green fluorescent protein) expression in J-Lat 10.6 cells by selecting flow cytometry, the expression of the GFP in the wogonin group is reduced, the expression of the GFP in the positive control CTPB group is increased, and the expression of the GFP in the wogonin + CTPB group and the DMSO group is not statistically different. The above results indicate that the P300 agonist CTPB simultaneously reverses the inhibition of wogonin on crotonylation of histones and reactivation of latent HIV-1.

Example 4

Concentration-dependent inhibition of latent HIV-1 reactivation in J-Lat A2 cells by wogonin

The J-Lat A2 cell model is a cell strain constructed by Verdin laboratories in the United states and used for simulating HIV-1 latent state. It integrates HIV-1 Long Terminal Repeat (LTR) and Tat gene and green fluorescent protein GFP gene on the chromosome of Jurkat T cell. When the drug acts on J-Lat A2 cells, if activation occurs, HIV-1-LTR will initiate gene transcription, thereby stimulating GFP expression. The activation activity of the drug was judged by measuring the proportion of positive cells expressing GFP in all the cells examined by flow cytometry.

Using 48 well cell culture plates, 1mL for the J-Lat a2 cell culture system, the cells were treated with different drugs according to the following groupings: the DMSO group was used as a negative control group, the PMA group (10ng/mL) was used as a positive control group, and the experimental group was wogonin (1. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M) in combination with PMA (10 ng/mL). After the above groups were cultured for 24 hours, detection was performed (see schematic experimental flow in A in FIG. 4). FIG. 4, B is the inhibition of HIV-1 reactivation in J-Lat A2 cells by wogonin at various treatment concentrations, with bar graphs representing percent GFP positive cells, dot graphs representing percent viable cells, and counts expressed as mean. + -. standard deviation. As compared to the percentage of GFP positive cells in the positive control PMA group, "×" indicates p < 0.001 and "×" indicates p < 0.0001. Wogonin: wogonin, DMSO: dimethyl sulfoxide, PMA: phorbol-12-myristic-13-acetate, GFP: green fluorescent protein.

The results in FIG. 4 show that the GFP expression was significantly down-regulated for each treatment concentration of wogonin + PMA group compared to the positive control PMA group, a PKC (protein kinase C) activator, and that the higher the concentration of wogonin, the lower the proportion of GFP-positive cells activated by PMA, i.e., the function of wogonin to inhibit the reactivation of latent HIV-1 in J-Lat A2 cells showed significant concentration dependence. The concentration interval selected was 40. mu.M for the most potent inhibition of wogonin, and this concentration ensured good cell viability.

Example 5

Wogonin can inhibit the subsequent effect of reactivation of latent HIV-1 in J-Lat A2 cells "

The aim of the strategy of "blocking and blocking" aimed at eliminating the danger of the latent HIV-1 reservoir is to achieve a "blocking" of the latent HIV-1 reservoir by deepening the latency of the provirus, continuing the expression of the provirus until the natural death of the host cell, thus allowing the safe withdrawal of CART therapy (antigen receptor T cell immunotherapy), where "safe withdrawal" does not simply mean the withdrawal of CART therapy, and "blocking" would be of no significance if another drug is required to maintain the virus not rebound after withdrawal. Wogonin treated cells made latent HIV-1 provirus less susceptible to activation, demonstrating its "blocking" function in reactivation of the latent pool. If the provirus in the pretreated cells is still not readily activated after elution of the drug, a potential "blocking" effect is possible.

To verify the "blocking" function of wogonin, we treated cells with wogonin for different periods of time and eluted the drug, and then examined the expression of GFP. Because the cell states are different at different culture times, the percentage baseline of GFP positive cells in the control group is not uniform, and for comparison, the activation rate of the DMSO group is set as 100%, so that the inhibition effect of the experimental group is normalized. This example uses 48 well cell culture plates, 1mL J-Lat a2 cell culture system, using different drug-treated cells grouped as follows: setting DMSO group as control group and wogonin (40 μ M) as experimental group; after culturing for 48h, 96h and 144h respectively, eluting the drug (see the schematic diagram of the experimental flow A in figure 5); the percentage of GFP-positive cells was measured after further incubation for 24h with cells treated with PMA (10ng/mL) (as shown in B in FIG. 5) or without any drug (as shown in C in FIG. 5), wherein the GFP-positive rate of the wogonin group was shown after normalization to the DMSO group as 100%. Wogonin: wogonin, DMSO: dimethyl sulfoxide, PMA: phorbol-12-myristic-13-acetate, GFP: green fluorescent protein. Counts are expressed as mean ± standard deviation.

The results in FIG. 5 show that the longer the wogonin pretreatment time, the less readily the latent GFP in J-Lat A2 cells is activated by PMA, despite the subsequent elution of the drug; i.e., the longer the pretreatment time, the lower the background expression of latent GFP. Although elution removed only the drug from the medium and cell surface, the intracellular drug was further metabolized by a 24 hour incubation period after elution, and the longer the pretreatment time of wogonin, the less likely the latent HIV-1 was activated after elution, all of which indicate that wogonin may have the potential to "lock" the latent reservoir.

Example 6

Wogonin can inhibit reactivation of HIV-1 infected person's peripheral leukemia virus repository

Although the expression level of the virus can be evaluated by detecting the J-Lat cell GFP, the J-Lat cell is not completely in a resting state due to the excessively high proportion of 'latent cells', so that the real state of a latent reservoir in a patient cannot be completely simulated. To obtain the actual effect of wogonin on the expression of latent provirus in HIV-1 infected subjects, this example further demonstrates the ability of wogonin to inhibit reactivation of the latent reservoir by long-term treatment of peripheral blood of HIV-1 infected subjects without viremia with CART.

CD4+ T cells were purified from peripheral blood of HIV-1 infected patients using 12-well cell culture plates with a culture system of 3ml of CD4+ T cells, and the cells were treated with different drugs according to the following groups: the DMSO group was used as a negative control, the Anti-CD3/CD28 (2. mu.g/mL) as a positive control, and the wogonin (40. mu.M) + Anti-CD3/CD28 (2. mu.g/mL) and the single wogonin (40. mu.M) groups were used as experimental groups 1-2, respectively. After 3 days of drug treatment, a portion was stained intracellularly with P24, followed by flow cytometry to detect P24 positive cells. Another part of the cells were extracted for RNA and reverse transcribed into cDNA, and expression of HIV-1 transcripts was detected by VQA-PCR (see schematic A experimental flow chart in FIG. 6). In FIG. 6, B is a flow cytometry chart of an example of P24, and the numerical value in the chart indicates the percentage of P24 positive cells. In FIG. 6, C is a statistical chart of the percentage of P24 positive cells detected by flow cytometry (5 samples). In FIG. 6, D and E are statistical results of the VQA method for detecting the expression level of HIV-1 transcript in test group 1(9 samples) and test group 2(6 samples), respectively, the positive group and test group are expressed as multiples of the DMSO group, and the counts are expressed as the mean. + -. standard deviation. "+" indicates p < 0.05 and "+" indicates p < 0.01 "+" indicates p < 0.0001. Visual Quality Asset (VQA): viral quality assurance, Wogonin (i.e., WO): wogonin, DMSO: dimethylsulfoxide, P24: HIV-1 nucleocapsid protein, Anti-CD3/CD 28: anti-human CD3/CD28 monoclonal antibody.

The results in FIG. 6 show that the proportion of P24 positive cells in the experimental group (Anti-CD3/CD28+ wogonin) was significantly reduced compared to the Anti-CD3/CD28 group, compared to the DMSO group, which is a negative control, and the survival rate of cells was not statistically different among the three groups. Absolute fluorescent quantitative PCR (VQA-PCR) assay showed that the expression of HIV-1 transcript was significantly down-regulated in experiment 1(Anti-CD3/CD28+ wogonin) compared to the positive control Anti-CD3/CD28 group (see D in FIG. 6), and in experiment 2 (wogonin alone) compared to the negative control DMSO group (see E in FIG. 6). Therefore, the wogonin can obviously inhibit the reactivation of latent HIV-1 in peripheral blood cells of a patient.

Example 7

Efficacy evaluation of wogonin combined with triptolide

Triptolide can inhibit HIV-1 replication in vitro at the level of viral transcription, and is reported to be a traditional Chinese medicine extract for inhibiting reactivation of HIV-1 repository.

In this example, J-Lat 10.6 cells were selected to evaluate the efficacy of a combination of wogonin and triptolide. Using 48-well cell culture plates, selecting 1mL of J-Lat 10.6 cells as culture system, and applying different drugs to treat the cells according to the following groups: the DMSO group is used as a negative control group, the PMA (10ng/mL) group is used as a positive control group, the experimental group is a wogonin (40 μ M) + PMA (10ng/mL), triptolide (2nM) + PMA (10ng/mL) group, and wogonin (40 μ M) + triptolide (2nM) + PMA (10ng/mL) group. After 24 hours of culture, GFP positive cells were detected by flow cytometry (see schematic A experimental flow chart in FIG. 7). The bar graph in B in fig. 7 represents the percentage of GFP-positive cells, the bar graph represents the percentage of viable cells, and the counts are expressed as mean ± standard deviation. ". indicates p < 0.0001. Wogonin: wogonin, Triptolide: triptolide, DMSO: dimethyl sulfoxide, PMA: phorbol-12-myristic-13-acetate, GFP: green fluorescent protein.

The results in fig. 7 show that both wogonin and triptolide reduce the percentage of GFP-positive cells and the inhibition effect of both were significantly enhanced (single wogonin reduced the percentage of GFP-positive cells by about 63%, single triptolide reduced the percentage of GFP-positive cells by about 16.2%, and both reduced the percentage of GFP-positive cells by about 76.1%). The two medicines are prompted to generate a synergistic sensitization effect to inhibit the reactivation of latent HIV-1, triptolide inhibits the extension of HIV-1 proviral transcription by inhibiting Tat function, and wogonin may have different virus inhibition mechanisms. In clinical practice of traditional Chinese medicines, a compound consisting of a plurality of traditional Chinese medicines is commonly used for treating diseases, and the study on the inhibition effect of the combination of different compounds can provide some references for clinical prescription.

Example 8

Specificity of wogonin for inhibiting latent HIV-1 reactivation function

Baicalin, baicalein and wogonin are flavonoids derived from scutellaria baicalensis, and experiments prove whether only wogonin in the flavonoids has the function of inhibiting the reactivation of latent HIV-1.

Using 48-well cell culture plates, selecting 1mL of J-Lat a2 cells as culture system, and applying different drugs to treat the cells according to the following groups: the DMSO group was used as a negative control group, the PMA (10ng/mL) group was used as a positive control group, the experimental groups were wogonin (40. mu.M) + PMA (10ng/mL), baicalein (40. mu.M) + PMA (10ng/mL), baicalin (1. mu.M) + PMA (10ng/mL), and the three compounds were combined with PMA at the above concentrations. After 24 hours of culture, GFP positive cells were detected using flow cytometry (see schematic experimental flow diagram a in fig. 8). The bar graph shown in B in fig. 8 represents the percentage of GFP-positive cells, the dot graph represents the percentage of viable cells, and the counts are expressed as mean ± standard deviation. ns indicates no statistical difference (p > 0.05). Wogonin: wogonin, Baicalein: baicalein, Baicalin: baicalin, DMSO: dimethyl sulfoxide, PMA: phorbol-12-myristic-13-acetate, GFP: green fluorescent protein.

The results in FIG. 8 show that only wogonin can reduce GFP expression, and that the combination of the three does not enhance the function of suppressing reactivation (see B in FIG. 8). Both baicalin and baicalein have been reported to have anti-HIV-1 activity, but baicalin inhibits HIV-1 infection before virus entry, and baicalein is considered to be a class of HIV-1 integrase inhibitors. The above results indicate that only wogonin can inhibit the reactivation of latent HIV-1, and the above three flavonoids may have different anti-HIV-1 mechanisms.

The embodiments of the present application have been described in detail with reference to the drawings, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.