|
- 现金
- 275 元
- 精华
- 0
- 帖子
- 146
- 注册时间
- 2016-4-7
- 最后登录
- 2018-4-19
|
Assembly慢乙肝新药ABI-H0731能抑制病毒复制和阻断cccDNA形成
来源：肝脏巴士（HeparBus）微信 时间：2017/02/20
专注于新型乙肝药物及人体微生物菌群失调疾病口服生物疗法开发的生物科技公司 Assembly Biosciences 近日宣布，公司在上海举办的2017亚太肝病学会年会上公布了一项旗下用于慢乙肝治疗的在研新药 ABI-H0731 的Phase 1a 期研究临床数据。ABI-H0731 是 Assembly 公司开发用于治疗慢性HBV感染的主要核心蛋白变构调节剂（CpAM），现已完成该药的 Phase 1a/1b 期临床的用药剂量范围确定部分研究。
Phase 1a 期研究主要评估了ABI-H0731在健康志愿者中的安全性，耐受性和药代动力学。部分该研究的有利结果将允许公司将ABI-H0731推进到1b期研究，1b期研究将评估其在慢性HBV感染患者中的安全性，药代动力学和初步抗病毒功效。1b期试验预计将于今年晚些时候开始。公司打算在2017年的科学会议上报告1a期研究的结果。
肝脏巴士（HeparBus）微信公众号小编也从该会议中获得了公司的相关口头发言内容，以下为研究数据的相信介绍。
HBV核心蛋白（Cp）涉及HBV生命周期的多个步骤，包括cccDNA的形成，扩增和维持。已有研究发现一种新型的直接作用HBV抗病毒剂，核心蛋白变构修饰剂（CpAM），能靶向HBV核心蛋白并降低HBV复制和cccDNA水平。Assembly Biosciences 公司研究人员报告了使用各种生物化学和细胞测定方法来确定的两个原型CpAM（GLS4和AT-130）的作用机制，这两个药物为 Assembly Biosciences 公司专有的CpAMs。
方法：体外衣壳装配测定：BOPIPY FL标记Cp150用于荧光猝灭衣壳组装测定法测试潜在的CpAMs。 CpAM的分子图谱：用抑制剂处理HepAD38细胞并同时诱导刺激。分别使用Northern印迹，Western印迹（蛋白质印迹），免疫测定（EIA）和Southern印迹检测HBV总RNA /衣壳化的pgRNA，Cp，衣壳和衣壳相关的核心DNA。使用bDNA探针（Affimetrix）定量HBV DNA / pgRNA。使用ELISA测定法定量HBe和HBs抗原。使用标准免疫染色方法显现HBc。cccDNA Southern印迹：收集细胞并通过Hirt修饰方法提取染色体外DNA。
结果显示，与较早公布的数据表明不同类别的CpAM功能不同，抑制衣壳组装（HAP）或导致错误组装（AT-130），此次的研究数据表明所有CpAM会诱导空衣壳化，在体外（装配测定和EM）和体内（在HepAD38中使用EIA测定）表现出正常或异常体积。在HepAD38细胞中CpAM处理过的核心蛋白也重新分布，通过Cp免疫染色显示为点状或大聚集体。蛋白质印迹（Western blot）分析表明，CpAM可以改变Cp的磷酸化并导致细胞中不溶性Cp的积累。 HAP还诱导加速的Cp降解。虽然所有CpAM有效阻止pgRNA衣壳化和HBV DNA复制，只有某些类别的CpAM能阻止HBV感染和cccDNA形成在重复感染期间。cccDNA形成作用呈现时间依赖性但为可持续的，这种作用导致细胞内HBV RNA转录和HBsAg和HBeAg的分泌呈现剂量依赖性减少。
研究结论认为：CpAM代表着一类新型HBV 直接作用抗病毒药物（DAAs），其诱导HBV核心蛋白的不适当寡聚化，进而导致空衣壳形成。在HBV重新感染期间，一些CpAM还可以通过阻断rcDNA递送至细胞核并形成cccDNA，通过替代标志物HBeAg，HBsAg和pgRNA水平和直接cccDNA检测的方式。总之，这些研究证明某些类别的CpAM可以干扰病毒生命周期中的多个步骤，干扰cccDNA的正常长期维持【这是目前核苷（酸）类似物达不到的】。因此，当与目前的治疗药物联合使用时，CpAM具有显著增加HBV患者治愈率的潜力。
编号：LB003
Dual mechanism of actions of novel HBV core protein allosteric modifiers (CpAMs): inhibiting viral replication and blocking cccDNA formation
Qi Huang1, Dawei Cai1, Pao-Chen Li1, Alex Mercier1, Renuka Kumar1, Emily Connelly1, Yuhua Zong1, Ran Yan1, Xiulan Zhou1, Yi Zhou1, Lida Guo1, Ariel Tang1, Geoffrey Chen1,Esteban Carabajal1, Katherine Nabel1, Lichun Li1,Steve Dunkelbarger1, Sarah Katen1, Jason Deer1, Earl May1, Uri Lopatin1, Adam Zlotnick1,2, Richard Colonno1
1Assembly Biosciences, Carmel, USA; 2Indiana University,Bloomington, USA
Background: HBV core protein (Cp) is involved in multiple steps of the HBV life cycle, including the formation, amplification and maintenance of cccDNA. A novel class of direct acting HBV antivirals, Core Protein Allosteric Modifiers (CpAMs), have been discovered to target HBV core protein and reduce HBV replication and cccDNA levels. Here we report on the mechanism of actions of two prototype CpAMs (GLS4 and AT-130) and Assembly Biosciences proprietary CpAMs using a variety of biochemical and cellular assays.
Methods: In vitro capsid assembly assay: BOPIPY-FL-labeled Cp150 were used in the fluorescence quenching Cpasid assembly assay to test potential CpAMs. Molecular profiling of CpAMs: HepAD38 cells were treated with inhibitors and induced simultaneously. HBV total RNA/encapsidated pgRNA, Cp, capsid, and capsid associated core DNA were detected by Northern Blot, Western Blot, Enzyme Immunoassay (EIA), and Southern Blot, respectively. HBV DNA/pgRNA were quantified using bDNA probes (Affimetrix). HBe and HBs antigens were quantified using ELISA assays. HBc were visualized using Standard Immunostaining method. cccDNA Southern Blots: cells were harvested and extrachromosomal DNA extracted by a Hirt-modified method.
Result: In contrast to earlier published data suggesting different class of CpAMs function differently, either inhibits capsid assembly (HAP)or leads to mis-assembly (AT-130), our data demonstrated that all CpAMs induced empty capsids, either as normal or aberrant size in vitro (assembly assay and EM) and in vivo (EIA assays in HepAD38). Treatment of CpAMs in HepAD38 cells also re-distribute of Core Protein, shown as punctate or large aggregates by Cp immunostaining. Western blot analysis indicated that CpAMs may alter the phosphorylation of Cp and cause accumulation of insoluble Cp in cells. HAPs also induce accelerated Cp degradation. While all CpAMs effectively block pgRNA encapsidation and HBV DNA replication, only certain classes of CpAM prevent HBV infection and cccDNA formation during de novo infection. The effect of cccDNA formation is time-dependent but sustainable, resulting in dose-dependent reduction of intracellular HBV RNA transcription and secretion of HBsAg and HBeAg.
Conclusion: CpAMs represent novel HBV DAAs that induce inappropriat oligomerization of HBV core protein, resulting in empty capsid formation. Some CpAMs also prevent delivery of rcDNA to the nucleus and establishment of cccDNA during HBV de novo infection, based on surrogate markers HBeAg, HBsAg and pgRNA levels, and direct cccDNA detection. In conclusion, we demonstrate that certain class of CpAMs can interfere with multiple steps in viral life cycle, perturb the normal maintenance of cccDNA longevity (key deficiency of current nucleos(t)ide therapy). Therefore, CpAMs have the potential to significantly increase cure rates in HBV patients when used in combination with current therapies.
登入可以评论
0条评论 |
|
发表于 2017-2-20 11:37
