WO2019041127A1 - Cbx4作为hiv-1潜伏感染激活靶点的应用 - Google Patents

Cbx4作为hiv-1潜伏感染激活靶点的应用 Download PDF

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WO2019041127A1
WO2019041127A1 PCT/CN2017/099466 CN2017099466W WO2019041127A1 WO 2019041127 A1 WO2019041127 A1 WO 2019041127A1 CN 2017099466 W CN2017099466 W CN 2017099466W WO 2019041127 A1 WO2019041127 A1 WO 2019041127A1
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cbx4
hiv
cells
latent infection
protein
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张辉
陈灿灿
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Sun Yat Sen University
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  • the present invention relates to the field of disease-related functional targets, and in particular to the field of HIV-1 latent infection activating target technology, and more particularly to the use of CBX4 as a target for HIV-1 latent infection activation.
  • the HIV-1 virus is a lentivirus that infects cells of the human immune system. It attacks human T lymphocytes and destroys the body's immune system, eventually leading to the acquisition of the acquired immunodeficiency syndrome "AIDS.”
  • the virus was first discovered in 1981, and researchers have been working on the virus for more than 30 years, and hope to cure the disease.
  • the current conventional treatment method is a combination of cART and antiretroviral therapy, which can effectively inhibit the replication of the virus, thereby controlling the viral load in the peripheral blood of the patient to a lower level.
  • current general treatment methods can only control the virus to a certain extent and cannot be effectively eliminated.
  • HIV-1 can lurk in cells to form an HIV-1 reservoir, and conventional drugs cannot affect these reservoirs, and once the patient stops treatment, they are activated for various reasons, leading to repeated illnesses.
  • the existence of HIV-1 reservoir has become a huge obstacle to the cure of HIV-1.
  • How to effectively eliminate the HIV-1 reservoir is one of the research hotspots in the field of HIV-1.
  • Today's mainstream method "shock and kill” is the idea of killing first and then killing. Therefore, the development of safe and efficient HIV-1 latent infection activators is one of the urgent problems to be solved.
  • HDACi histone deacetylase inhibitor
  • HMTi histone methyltransferase inhibition Agent
  • DNMTi DNA methyltransferase inhibitor
  • PKC protein kinase C activator
  • CBX4 belongs to the CBX protein family and is an important component of the multi-comb protein complex PRC1.
  • the CBX protein family includes five members: CBX2, CBX4, CBX6, CBX7, and CBX8.
  • Their N-terminus has a CHROMO domain, which has the binding ability of H3K27 trimethylation; the C-terminus has a conserved region called C-Box, which is responsible for the assembly of the PRC1 complex.
  • CBX4 not only has a certain affinity for H3K27 trimethylation, but also exhibits significant affinity with H3K9 trimethylation.
  • the function of CBX4 reported in the prior art is mainly involved in the regulation of individual development, stem cell differentiation, and tumorigenesis. So far, no research has been published.
  • CBX4 is a brand new HIV-1 latent infection. Live target.
  • the present inventors have found through research that inhibition of CBX4 has good HIV-1 latent activation ability.
  • the present invention detects the transcription level of HIV-1 LTR by using a double luciferase reporter gene by knocking down the expression level of the CBX4 gene in TZM-bl cells, and found that knockdown of CBX4 can effectively promote the transcription of HIV-1 LTR.
  • the present invention then effectively knocks down the expression of CBX4 protein in the J-lat 10.6 HIV-1 latent infection cell model, and found that J-lat10.6 cells can be efficiently activated and GFP gene expression is up-regulated.
  • the invention also constructs a CBX4 expression plasmid, and overexpresses CBX4 protein in TZM-bl cells, and the double luciferase reporter gene detection shows that the transcriptional activity of HIV-1 LTR decreases correspondingly with the increase of CBX4 protein expression, and the relationship between the two There is a concentration gradient effect. Further detection revealed that decreased expression of CBX4 protein reduced the degree of enrichment of HIV-1 LTR H3K9 trimethylation and H3K27 trimethylation, thereby activating HIV-1 latent infection. Therefore, the present invention claims the use of CBX4 as a target for HIV-1 latent infection activation.
  • CBX4 can be used as a target for HIV-1 latent infection activation
  • compounds that inhibit the CBX4 gene or CBX family genes or derivatives thereof, or compounds that degrade CBX4 protein or CBX family proteins or derivatives thereof can be prepared as HIV-1 latent Infective activator.
  • the derivative is a pharmaceutically acceptable salt or ester of the compound.
  • the pharmaceutically acceptable salt is a sodium, potassium, calcium, iron, ferrous or zinc salt.
  • the pharmaceutically acceptable ester is ethyl or butyl ester.
  • the present invention has the following beneficial effects:
  • the present invention finds that CBX4 can be used as a target for HIV-1 latent infection activation, and provides a strong theoretical basis and practical basis for further development of HIV-1 related drugs.
  • Figure 1 shows that the expression of CBX4 gene in TZM-bl cells can be efficiently knocked down, which can effectively promote the transcriptional activity of HIV-1 LTR.
  • Figure A shows the knockdown efficiency of si-CBX4 in this cell line by q-PCR;
  • Panel B is a method for detecting LTR transcriptional activity using a dual luciferase reporter gene and performing analysis.
  • Figure 2 shows that the CBX4 gene in shRNA-CBX4 knockdown of J-lat 10.6 HIV-1 latently infected cells can effectively activate the HIV-1 latent infection repository.
  • Figure A shows the use of q-PCR to detect shRNA-CBX4 in J- The efficiency of knockdown of CBX4 in the lat 10.6 cell line;
  • Figure B is the use of flow cytometry The activation effect of HIV-1 latent infection in this cell model was examined after CBX4 knockdown.
  • Figure 3 shows the construction of CBX4 expression plasmid. Overexpression of CBX4 protein in TZM-bl cells can inhibit the transcription of LTR of HIV-1.
  • Figure A shows the pcDNA3.1-CBX4-FLAG overexpression plasmid constructed by Western Blot. Expression effect; Panel B is a gradient effect between LTR transcriptional activity and CBX4 overexpression using dual luciferase reporter assays.
  • Figure 4 shows that decreased expression of CBX4 protein reduces the degree of enrichment of H3K9 trimethylation of HIV-1 LTR (panel A) and H3K27 trimethylation (panel B), thereby activating HIV-1 latent infection.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used are, if not specified, commercially available reagents and materials.
  • TZM-bl cells were seeded in 24-well plates.
  • the medium used was complete medium: high glucose DMEM, 10% fetal bovine serum and 1% double antibody, culture conditions were 5% carbon dioxide, 37 ° C;
  • siRNA-CBX4 can effectively knock down the endogenous CBX4 gene, and its knockdown efficiency is about 80%. In contrast, knockdown of CBX4 can effectively increase the transcription level of HIV-1 LTR.
  • the well-grown human kidney cell line 239T cells were inoculated into a 10 cm flat bottom plate.
  • the medium used was complete medium: high glucose DMEM, 10% fetal bovine serum and 1% double antibody, culture conditions were 5% carbon dioxide, 37 ° C;
  • the amount of virus is about 2-3 ⁇ 10 6 /ml cells infected by virus in each 10 cm plate;
  • the pro-infective agent polybrene polybrene
  • the well-grown TZM-bl cells were plated in 6-well transparent plates at 1 ⁇ 10 6 cells per well.
  • the medium used was complete medium: high glucose DMEM, 10% fetal bovine serum and 1% double antibody.
  • the culture condition is 5% carbon dioxide, 37 ° C;
  • the well-grown TZM-bl cells were plated in 24-well transparent plates at 1 ⁇ 105 cells per well.
  • the medium used was complete medium: high glucose DMEM, 10% fetal bovine serum and 1% double antibody.
  • the culture condition is 5% carbon dioxide, 37 ° C;
  • CBX4 protein expression will reduce the H3K9 trimethylation of HIV-1 LTR and the enrichment of H3K27 trimethylation.
  • the specific experimental methods are as follows:
  • the well-grown TZM-bl was taken and inoculated into a 10 cm flat bottom plate.
  • the medium used was complete medium: high glucose DMEM, 10% fetal bovine serum and 1% double antibody, culture conditions were 5% carbon dioxide, 37 ° C;
  • CBX4 is an effective target for the activation of HIV-1 latent infection
  • a compound that inhibits the CBX4 (and CBX family) gene or a protein that degrades the CBX4 (and CBX family) protein and particularly a pharmaceutically acceptable salt or ester thereof, such as Sodium, potassium, calcium, iron, ferrous, zinc, ethyl, butyl and the like can also activate HIV-1 latent infection, which is convenient for the follow-up treatment of HIV.

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Abstract

本发明公开了CBX4作为HIV-1潜伏感染激活靶点的应用。

Description

CBX4作为HIV-1潜伏感染激活靶点的应用 技术领域
本发明涉及疾病相关功能靶点技术领域，具体地，涉及HIV-1潜伏感染激活靶点技术领域，更具体地，涉及CBX4作为HIV-1潜伏感染激活靶点的应用。
背景技术
HIV-1病毒是一种感染人类免疫系统细胞的慢病毒，它通过攻击人体T淋巴细胞，破坏人体免疫系统，最终导致获得性免疫缺陷综合征“艾滋病”的产生。该病毒最初发现于1981年，在此之后的三十余年研究人员一直致力于对该病毒控制及清除，期望能够治愈该疾病。目前的常规治疗方法为简称cART的联合抗逆转录病毒疗法，该治疗方法能够有效的抑制病毒的复制，从而将病人外周血中的病毒载量控制到较低的水平。但是目前的通用治疗方法只能够在一定程度上控制病毒，并不能有效的清除。究其原因是因为HIV-1可潜伏在细胞内形成HIV-1储藏库，常规药物不能影响这些储藏库，并且病人一旦停止治疗，它们会因为各种原因被激活，从而导致病情反复。综上所述，HIV-1储藏库的存在已成为HIV-1治愈的巨大障碍，如何有效的清除HIV-1储藏库是目前HIV-1领域的研究热点之一。当今的主流方法“shock and kill”是采用先激活后杀灭的思路。因此，研发安全、高效的HIV-1潜伏感染激活剂是当前亟待解决的问题之一。目前已有多种HIV-1潜伏感染激活剂被开发，根据它们的作用靶点可以分为以下几种类型，包括：组蛋白去乙酰化酶抑制剂(HDACi)、组蛋白甲基转移酶抑制剂(HMTi)、DNA甲基转移酶抑制剂(DNMTi)、蛋白激酶C激活剂(PKC)、Bromodomain抑制剂。
CBX4属于CBX蛋白家族，是多梳蛋白复合体PRC1的重要组分。CBX蛋白家族包括：CBX2，CBX4，CBX6，CBX7，CBX8五个成员。它们的N端拥有一个CHROMO结构域，这个结构域具有H3K27三甲基化的结合能力；C端有一个保守区域叫做C-Box，它负责PRC1复合体的组装。其中CBX4不仅对H3K27三甲基化有一定的亲和性，其与H3K9三甲基化也表现出显著的亲和力。现有技术中报道的CBX4的功能主要为参与调控个体发育、干细胞分化以及肿瘤的发生。截至目前为止尚未有研究未公开CBX4是一个全新的HIV-1潜伏感染激 活作用靶点。
发明内容
本发明的目的在于提供CBX4作为HIV-1潜伏感染激活全新靶点的应用。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
本发明通过研究发现抑制CBX4具有良好的HIV-1潜伏激活能力。本发明通过敲低TZM-bl细胞中的CBX4基因的表达量，使用双荧光素酶报告基因检测HIV-1的LTR的转录水平，结果发现敲低CBX4能够有效促进HIV-1 LTR的转录。本发明继而在J-lat 10.6 HIV-1潜伏感染细胞模型中有效敲低CBX4蛋白的表达情况，发现J-lat10.6细胞能够有效被激活，GFP基因表达上调。本发明还构建CBX4表达质粒，在TZM-bl细胞中过表达CBX4蛋白，双荧光素酶报告基因检测发现，随着CBX4蛋白表达增加，HIV-1的LTR的转录活性相应降低，两者的关系存在浓度梯度效应。进一步检测发现CBX4蛋白表达降低会降低HIV-1 LTR H3K9三甲基化，H3K27三甲基化的富集程度，从而激活HIV-1潜伏感染。所以，本发明要求保护CBX4作为HIV-1潜伏感染激活靶点的应用。
因为CBX4可以作为HIV-1潜伏感染激活靶点，所以，抑制CBX4基因或CBX家族基因的化合物或其衍生物，或降解CBX4蛋白或CBX家族蛋白的化合物或其衍生物可以制备成HIV-1潜伏感染激活剂。
所述衍生物为化合物在药学上可接受的盐或酯。
所述药学上可接受的盐为钠、钾、钙、铁、亚铁或锌盐。
所述药学上可接受的酯为乙酯或丁酯。
与现有技术相比，本发明具有如下有益效果：
本发明首次发现CBX4可以作为HIV-1潜伏感染激活靶点，为进一步研发HIV-1相关药物提供了强有力的理论基础和实践基础。
附图说明
图1为高效敲低TZM-bl细胞中的CBX4基因的表达，能够有效促进HIV-1LTR的转录活性，其中，图A为通过q-PCR检测si-CBX4在该细胞系中的敲低效率；图B为利用双荧光素酶报告基因检测LTR转录活性并进行分析。
图2为使用shRNA-CBX4敲低J-lat 10.6 HIV-1潜伏感染细胞中的CBX4基因能够有效激活HIV-1潜伏感染储存库，其中，图A为利用q-PCR检测shRNA-CBX4在J-lat 10.6细胞系中敲低CBX4的效率；图B为运用流式细胞术 检测CBX4敲低后，该细胞模型中HIV-1潜伏感染的激活效果。
图3为构建CBX4表达质粒，在TZM-bl细胞中过表达CBX4蛋白能够抑制HIV-1的LTR的转录，其中，图A为通过Western Blot技术检测构建的pcDNA3.1-CBX4-FLAG过表达质粒的表达效果；图B为利用双荧光素酶报告基因检测随着LTR转录活性与CBX4过表达之间的梯度效应。
图4为CBX4蛋白表达降低会降低HIV-1 LTR的H3K9三甲基化(图A)，H3K27三甲基化(图B)的富集程度，从而激活HIV-1潜伏感染。
具体实施方式
下面结合说明书附图和具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
实施例1
敲低CBX4基因表达能够有效促进HIV-1 LTR的转录活性，具体实验方法如下：
(1)取生长良好的TZM-bl细胞，接种于24孔板中。使用的培养基是完全培养基：高糖DMEM，10％胎牛血清以及1％双抗，培养条件是5％二氧化碳、37℃；
(2)24h贴壁后，分别共同转染pcDNA3.1-Tat质粒和siRNA-CBX4或siRNA-NC，如1)的条件继续培养；
(3)48h后，收取细胞沉淀，取一种一部分使用Trizol法提取RNA，反转获得cDNA，使用CBX4特异性Q-PCR引物检测CBX4的mRNA表达水平，确定siRNA-CBX4的敲低效率；
(4)取剩余细胞，裂解后利用双荧光素梅报告基因检测方法，检测不同转染处理组中的HIV-1 LTR的转录活性；
(5)对比CBX4敲低后，HIV-1 LTR的转录活性。
实验结果如图1所示。从图中可以看出，在TZM-bl细胞中，siRNA-CBX4能够有效敲低细胞内源的CBX4基因，其敲低效率大约为80％左右。对比发现，敲低CBX4能够有效的提升HIV-1 LTR的转录水平。
实施例2
抑制J-lat 10.6 HIV-1潜伏感染细胞中的CBX4有效激活HIV-1潜伏感染储存库，具体实验方法如下：
(1)合成特定碱基序列，退火构建针对CBX4的shRNA，PLKO.1-CBX4并测序确认序列正确。
(2)取生长良好的人肾上细胞株239T细胞，接种于10cm平底板中。使用的培养基是完全培养基：高糖DMEM，10％胎牛血清以及1％双抗，培养条件是5％二氧化碳、37℃；
(3)24h贴壁后，转染PLKO.1-CBX4或PLKO.1-NC和psPAX2、VSV-G至细胞中，如2)的条件继续培养；
(4)48h后，收取上清，使用PEG-6000离心浓缩病毒颗粒，使用ELISA P24试剂盒检测病毒P24；
(5)取生长良好的J-lat10.6细胞，计数后，用4)中得到的病毒进行感染，病毒用量约为每一个10cm板中的病毒感染2-3×106/ml细胞；(感染时使用促感染试剂polybrene)；
(6)感染6h后换液，使用PBS清洗三次，弃上清，使用1640培养基(10％胎牛血清，1％双抗)培养，培养条件是5％二氧化碳、37℃；
(7)第二天，在培养基中加入适量嘌呤霉素，浓度约为1-2μg/ml，5％二氧化碳、37℃培养2周，定期更换含有嘌呤霉素的1640培养基，以获得稳定感染shRNA-CBX4的J-lat 10.6细胞；
(8)取部分细胞使用Trizol裂解提RNA,反转获得cDNA，使用CBX4特异性Q-PCR引物检测CBX4的mRNA表达水平；
(9)使用流式细胞仪检测相应细胞的GFP表达水平，并作统计图分析结果。
实验结果如图2所示。从图中可以看出，在J-lat 10.6 HIV-1潜伏感染细胞有效敲低CBX4后能够有效激活HIV-1潜伏感染储存库。
实施例3
在TZM-bl细胞中过表达CBX4蛋白能够抑制HIV-1的LTR的转录活性，具体实验方法如下：
(1)订购CBX4cDNA质粒模板，设计合适的引物，构建CBX4过表达质粒，pcDNA3.1-CBX4-FLAG；
(2)取生长良好的TZM-bl细胞铺于6孔透明板中，每孔1×106细胞,使用的培养基是完全培养基：高糖DMEM，10％胎牛血清以及1％双抗，培养条件是5％二氧化碳、37℃；
(3)24h贴壁后，转染pcDNA3.1-CBX4-FLAG质粒，如2)的条件继续培养；
(4)48h后，收取细胞，使用RIPA裂解液裂解细胞，通过western blot检测CBX4过表达水平，确定pcDNA3.1-CBX4-FLAG过表达质粒的效果；
(5)取生长良好的TZM-bl细胞铺于24孔透明板中，每孔1×105细胞，使用的培养基是完全培养基：高糖DMEM，10％胎牛血清以及1％双抗，培养条件是5％二氧化碳、37℃；
(6)24h贴壁后，分别转染不同浓度的pcDNA3.1-CBX4-FLAG质粒和pcDNA3.1-Tat质粒，pcDNA3.1-CBX4-FLAG梯度设定从0-500ng，用pcDNA3.1调节质粒的一致性，如2)的条件继续培养；
(7)48h后，收取细胞，使用裂解液裂解利用双荧光素梅报告基因检测方法，检测不同转染处理组中的HIV-1 LTR的转录活性。
实验结果如图3所示。从图中可以看出，随着CBX4蛋白表达量的上升，HIV-1 LTR的转录活性逐渐降低。
实施例4
CBX4蛋白表达降低会降低HIV-1 LTR的H3K9三甲基化，H3K27三甲基化的富集程度，具体实验方法如下：
(1)取生长良好的TZM-bl，接种于10cm平底板中。使用的培养基是完全培养基：高糖DMEM，10％胎牛血清以及1％双抗，培养条件是5％二氧化碳、37℃；
(2)24h贴壁后，分别共同转染pcDNA3.1-Tat质粒和siRNA-CBX4或siRNA-NC，如1)的条件继续培养；
(3)48h后，收取细胞沉淀，利用H3K9三甲基化和H3K27三甲基化ChIP抗体，通过ChIP检测HIV-1 LTR的H3K9三甲基化和H3K27三甲基化的富集程度；
(4)取生长良好的J-lat10.6细胞，计数后用shRNA-SCR和shRNA-CBX4分别感染细胞，病毒用量约为每一个10cm板中的病毒感染2～3×106/ml细胞；(感染时使用促感染试剂polybrene)；
(5)感染6h后换液，使用PBS清洗三次，弃上清，使用1640培养基(10％胎牛血清，1％双抗)培养，培养条件是5％二氧化碳、37℃；
(6)第二天，在培养基中加入适量嘌呤霉素，浓度约为1～2μg/ml，5％二氧化碳、37℃培养2周，定期更换含有嘌呤霉素的1640培养基，以获得稳定感染shRNA-CBX4的J-lat 10.6细胞；
(7)收取细胞沉淀，利用H3K9三甲基化和H3K27三甲基化ChIP抗体，通过ChIP检测HIV-1 LTR的H3K9三甲基化和H3K27三甲基化的富集程度；
(8)对比分析，CBX4敲低前后对HIV-1 LTR的H3K9三甲基化和H3K27三甲基化的富集程度。
CBX4是HIV-1潜伏感染激活的有效靶点，抑制CBX4(及CBX家族)基因或降解CBX4(及CBX家族)蛋白的化合物及其衍生物，特别是其为药学可接受的盐、酯，如其钠、钾、钙、铁、亚铁、锌盐、乙酯、丁酯等同样可以激活HIV-1潜伏感染，便于HIV的后续治疗。

Claims (5)

  1. CBX4作为HIV-1潜伏感染激活靶点的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述应用具体为将抑制CBX4基因或CBX家族基因的化合物或其衍生物，或降解CBX4蛋白或CBX家族蛋白的化合物或其衍生物制备成HIV-1潜伏感染激活剂。
  3. 根据权利要求2所述的应用，其特征在于，所述衍生物为化合物在药学上可接受的盐或酯。
  4. 根据权利要求3所述的应用，其特征在于，所述药学上可接受的盐为钠、钾、钙、铁、亚铁或锌盐。
  5. 根据权利要求3所述的应用，其特征在于，所述药学上可接受的酯为乙酯或丁酯。
PCT/CN2017/099466 2017-08-29 2017-08-29 Cbx4作为hiv-1潜伏感染激活靶点的应用 Ceased WO2019041127A1 (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7842123B2 (en) * 2006-10-31 2010-11-30 Milo Ehud Extraction of gas from infused liquid

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7842123B2 (en) * 2006-10-31 2010-11-30 Milo Ehud Extraction of gas from infused liquid

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
WANG, BOQING: "Biological Function of CBX4 and Its Prognostic Significance and Corresponding Molecular Mechanisms in Hepatocellular Carcinoma", CDFD, 15 February 2015 (2015-02-15), pages E072 - 79 *
WANG, XIN ET AL.: "Role of CBX4 in the Colorectal Carcinoma Metastasis-Response", CANCER RESEARCH, vol. 76, no. 24, 18 November 2016 (2016-11-18), pages 7277 - 7289 *
ZENG, KAI ET AL.: "Research Progress of CBX Protein and Tumor", PRACTICAL ONCOLOGY JOURNAL, vol. 31, no. 2, 28 April 2017 (2017-04-28), pages 165 - 169 *
ZHENG, CHANGLI ET AL.: "MicroRNA-195 Functions as a Tumor Suppressor by Inhibiting CBX4 in Hepatocellular Carcinoma", ONCOLOGY REPORTS, vol. 33, no. 3, 19 January 2015 (2015-01-19), pages 1115 - 1122, XP055579900 *

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