EASL2013 HAPS 衣壳蛋白抑制剂影cccDNA的功能

StephenW

Abstract 371
     
HAPS HEPATITIS B VIRUS (HBV) CAPSID INHIBITORS AFFECT CORE PROTEIN INTERACTION WITH THE MINICHROMOSOME AND TARGET CCCDNA FUNCTION   
HAPS乙型肝炎病毒（HBV）衣壳蛋白抑制剂影响核心蛋白的相互作用的微小染色体目标cccDNA的功能
L. Belloni1,2,3*, L. Li4, G.A. Palumbo1,2, S.R. Chirapu5, L. Calvo1, M. Finn5, A. Zlotnick4, M. Levrero1,2,3
1Dip. di Medicina Interna (DMISM), 2EAL Inserm U 785, 3Life Nanosciences Laboratory, Sapienza University, Rome, Italy, 4Dept of Molecular & Cellular Biochemistry, Indiana University, Bloomington, IN, 5Dept Chemistry, Scripps Research Inst, La Jolla, CA, USA. *[email protected]

Background: HBV capsid assembly is critical for RNA packaging, reverse transcription, and intracellular trafficking and represents an attractive new therapeutic target. Core proteins (Cp) have been shown to bind the nuclear cccDNA, possibly contributing to the regulation of its function and stability. Hetero-aryl-dihydropyrimidines (HAPs), a new class of antivirals inhibiting HBV replication in vitro and in vivo, enhance the rate and the extent of core protein (Cp) assembly and, at high concentration, stabilize preferentially non-capsid polymers of Cp. Here we investigated the impact of HAP12 on cccDNA formation, levels and transcription as part of its antiviral activity against HBV.
Methods: Capsid-associated HBV-DNA (TaqMan real-time PCR), cccDNA (TaqMan real-time PCR) and pgRNA levels (quantitative real-time PCR with specific primers), were assessed in: a) HepG2 cells transfected with full length HBV genomes; b) the HepG2 H1.3 HBV stable clone; c) the inducible AD38 stable HBV cell line, left untreated or treated with the hetero-aryl-dihydropyrimidine HAP12 at 1-5 microM.
Results: HAP12 treatment of cells transfected with wild type linear HBV genomes showed a complete suppression of HBV replication at 72 and 96 hrs with a peak >50% reduction of pgRNA transcription at 96 hours, without significant changes in cccDNA levels. The strong HAP12 inhibitory effect on pgRNA transcription and HBV replication as well as the lack of significant effect on steady state cccDNA levels was confirmed in both the HepG2 H1.3 stable cell line and in the AD38 HBV inducible cell line. We confirmed, using the cccDNA ChIP assay that HBc is recruited onto the cccDNA in HBV replicating cells. HAP12 treament strongly inhibits cccDNA HBc occupancy in HepG2 H1.3 cells and, in agreement with the inhibition of cccDNA transcription and pgRNA production, a sharp decrease in cccDNA-bound H3 histone acetylation.
Conclusions: HAPs binding to HBV capsid, in addition to conformational change resulting in a core protein that does not support HBV replication, also target the nuclear cccDNA. We show that inappropriate assembly and effective cytoplasmic trapping of Cp induced by HAPs affects Cp interaction with the HBV minichromosome and cccDNA function.


Assigned speakers:
Dr. Laura Belloni, Sapienza University , Rome , Italy

Assigned in sessions:
25.04.2013, 09:00-18:00, Poster Session, P01-07a, Category 07a: Viral Hepatitis B & D: Experimental, Poster Area

StephenW

背景：乙肝病毒衣壳的装配是至关重要的，反转录，RNA包装和胞内运输，是一个有吸引力的新的治疗靶点。已经示出的核心蛋白（Cp）的绑定核的共价闭合环状DNA，可能有助于它的功能和稳定性的调节。杂芳基 - 二氢嘧啶（HAPs）等，在体外和体内的抗病毒药物抑制HBV复制的一类新的，增强的速度和核心蛋白​​（Cp）的组件的程度，并在高浓度下，优先非衣壳聚合物的Cp的稳定。在这里，我们调查的影响，HAP12 cccDNA的形成，其对乙肝病毒的抗病毒活性的水平和转录。
方法：衣壳相关HBV-DNA（荧光定量实时PCR），，cccDNA的（TaqMan实时PCR）和pgRNA水平（实时定量PCR特异性引物），进行了评估：A）转染细胞全长HBV基因组，B）H1.3 HBV的HepG2细胞稳定克隆，C）的诱导AD38稳定的HBV细胞系，不及时治疗或治疗的杂芳基 - 二氢HAP12在1-5 MICROM。
结果：HAP12治疗与野生型线性HBV基因组转染的细胞表现出一个完整的抑制HBV复制具有峰值在72和96小时> 50％在96小时减少pgRNA转录，而不cccDNA水平的显着变化。上的pgRNA转录和HBV复制的抑制作用强HAP12以及对HepG2 H1.3稳定的细胞系，并在AD38 HBV诱导的细胞被证实在缺乏稳定状态cccDNA水平显着影响。我们确认，使用的cccDNA的芯片检测HBc阳性招募到cccDNA的乙肝病毒复制的细胞。 HAP12 treament强烈抑制cccDNA的HBc阳性占用的在肝癌H1.3细胞，并与抑制cccDNA的转录和pgRNA cccDNA的绑定H3组蛋白乙酰化生产，大幅减少。
结论：结合到HBV衣壳，在除了不支持HBV复制的核心蛋白，也针对核的共价闭合环状DNA的构象变化导致的有害气体污染物。我们发现，不当引起的有害空气污​​染物会影响组装和有效的细胞质捕获CP CP交互与HBV微小染色体与cccDNA功能。

9病成医

呼唤宁静温泉!

疯一点好

宁静温泉是男孩还是女孩啊，我们需要你，斯提芬的文章也需要你。

StephenW

News新闻
   一个公司正在开发衣壳蛋白抑制剂


Biotechnology company adds new investor in its fight against Hepatitis B virus

March 25, 2013 6:17 pm by Stephanie Baum | 0 Comments

deal, agreementA biotechnology company seeking to eradicate the Hepatitis B virus has added a new investor to its backers to close a $25 million Series A round.

Novira Therapeutics in Radnor, Pennsylvania added Versant Ventures — a venture firm that backs early stage life science companies, according to a company statement. Versant will invest $7.5 million in Novira as part of the deal. The investment follows an initial close of the Series A round in August last year.

Novira is developing capsid inhibitors that attack the shell protecting the virus’ genetic material. It is also developing other Hepatitis B treatments. It is expecting to start clinical trials in 2014.

In an e-mail to clarify the financing, Chief Scientific Officer and President Osvaldo Flores said the financing round followed a two-step closing model in which investors 5AM and Canaan led the first closing in August with a partial syndicate. That was followed by a second closing announced after Versant agreed to be part of the syndicate. Flores added that the financing round was increased to $25 million from $23 million because the slot for the third venture group was increased to match that of the lead investors. The $25 million amount also takes into account the participation of seed investors in the round.

Newborns, young children and immunocompromised patients frequently become chronically infected with Hepatitis B. About 400 million people worldwide suffer from chronic HBV infection. Up to 25 percent with chronic cases of Hepatitis B die from carcinoma or liver cancer, according to the Centers for Disease Control. Vaccinations for infants and non-vaccinated children under 18 is the most common way to prevent transmission. The most common ways the virus is transmitted are through unprotected sex and blood transfusions. About 600,000 people die each year from the virus, according to the World Health Organization.

In an interview last August Flores told MedCity News: “’We believe our drug either alone or in combination with others has the potential to lead to better cure rates” than the treatments that are currently available.

Read more: http://medcitynews.com/2013/03/b ... irus/#ixzz2QFV0s78V

生物技术公司增添了新的投资者，在其对抗B型肝炎病毒

2013年3月25日6:17 P​​M由斯蒂芬妮·鲍姆| 0评论

交易，的agreementA生物科技公司寻求根除乙肝病毒又增加了一个新的投资者，其支持者关闭了2500万美元的第一轮融资。

在拉德诺Novira治疗，宾夕法尼亚州增加了Versant的风险投资公司 - 备份早期生命科​​学公司的风险投资公司，根据公司的声明。 Versant的，作为交易的一部分，将投资750万美元，Novira。的投资在去年8月最初的A系列轮结束之后。

Novira发展衣壳蛋白抑制剂，攻击病毒的遗传物质的外壳保护。它也正在开发其他的B型肝炎的治疗。预计在2014年开始临床试验。

在一封邮件澄清融资，首席科学官兼总裁奥斯瓦尔多·弗洛雷斯说了此轮融资后两个步骤的关闭模式，即投资者5AM和迦南导致部分集团的首个截止8月。其次是第二次收市价宣布Versant的同意后，该集团的一部分。弗洛雷斯说，一轮融资是从$ 23亿美元增加至2500万美元，因为插槽的第三次创业，增加匹配的铅投资者的。 2500万美元的金额也考虑到圆形的种子投资者的参与。

新生儿，幼儿和免疫功能低下的患者经常成为慢性感染B型肝炎，关于全世界有400万人患慢性HBV感染者。高达25％死于癌或肝癌与B型肝炎慢性病例，根据美国疾病控制中心的。婴幼儿疫苗接种和未接种疫苗的儿童，18岁以下是最常见的方式，以防止传染。最常见的病毒的传播方式是通过不安全的性行为和输血。约60万人，每年死于该病毒，根据世界卫生组织。

去年八月弗洛雷斯在接受记者采访时告诉MedCity新闻：“我们相信，我们单独或与其他药物有可能导致更佳的治愈率比目前可用的治疗方法，是的。

咬牙硬挺

都是好消息。

宁静温泉

我是男的，哈哈

这一篇太难了，无数多的医学专业名词，看了几遍都没看明白。
这个研究是关于将HAP作用于衣壳蛋白，探讨对HBVDNA、pgRNA和cccDNA的影响。让我疑惑不解的是，文章里说cccDNA的水平没有受到影响，但是标题和结论又说，这个方法确实对抑制cccDNA的功能有作用。莫非是说这个方法不会降低cccDNA的水平，但是会印象cccDNA的活性（或者别的功能）？

期待高手解答， 我也会继续研究这个。。。

StephenW

回复 宁静温泉 的帖子

I don't understand the abstract myself. Initially, scientists think HAP reduces viral load by interfering with the capsid made of HBc protin that is essential for forming a new virion. The scientists now discover that HAP also indirectly interferes with the transcription of cccDNA, thereby further reduces production of new virions.
Just my own understanding.

宁静温泉

谢谢StephenW！查了些资料，确实，到目前为止，大多数的研究都是关于用HAP来误导衣壳蛋白的组装，从而阻止HBVDNA的复制。相对于核苷类似物的治疗方案，这个方法具有更好的稳定性（低抗药性）。
这个研究似乎提到了其对cccDNA也存在的某些作用，但是从中我没有看到更详细的说明。
期待后续报道。

咬牙硬挺

感谢二位的辛苦付出