有效的第二代CpAMs的临床前概，能够抑制HBV感染细胞的hb凹

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ABSTRACT FINAL ID: 922
TITLE: Preclinical Profile of Potent Second Generation CpAMs Capable of Inhibiting the Generation of HBsAg, HBeAg, pgRNA and cccDNA in HBV Infected Cells
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ABSTRACT BODY:
Background: Clinical cure remains elusive in chronic HBV (CHB) patients, despite prolonged treatment periods with current available therapies. Core Protein Allosteric Modulators (CpAMs) are a novel class of direct acting antivirals with the potential to help improve cure rates by targeting multiple aspects of the viral life cycle. Here we characterize potent 2nd generation CpAMs that exhibit favorable drug properties and inhibit cccDNA establishment and replenishment via multiple mechanisms.

Methods: CpAM antiviral activities were determined using the HepAD38 cell line (Gt D), and infected HepG2-NTCP cells and Primary Human Hepatocytes (PHH). Pan-genotypic activity was established using transient transfection assays or HBV stable cell lines. Combination studies were performed using a range of inhibitory concentrations in HepAD38 cells. HBV DNA levels were quantified by Taqman PCR (qPCR) using primers and a probe specific to the HBV core gene. HBeAg and HBsAg were quantified by ELISA. HBV total RNA/encapsidated pgRNA, Cp, capsids, and capsid-associated core DNA were detected by Northern Blot or b-DNA, Western Blot, Enzyme Immunoassay (EIA), and Southern Blot, respectively, as previously described.

Results: Representative 2nd generation CpAMs exhibited potent inhibition of viral DNA replication in HepAD38 cells (EC50s <30 nM), and reductions in HBeAg, HBsAg and pgRNA in infected HepG2-NTCP cells and PHH (EC50s <300 nM). Antiviral activity extended to other major HBV genotypes in stably or transiently transfected HepG2 cells. No significant activity was exhibited against a panel of other viruses (EC50s >10 µM) or cell lines in cytotoxicity assays (CC50s >10 µM), indicating that the compounds are selective inhibitors of HBV. Lead molecules possess promising physical properties, low drug-drug interaction potential and favorable PK profiles in multiple species. Mechanism of action studies suggest that the enhanced potency of these new CpAMs is likely due to inhibition of multiple steps of the HBV life cycle, including acceleration of aberrant capsid assembly, disruption of pre-formed capsids and premature disassembly during trafficking of incoming mature capsids and rcDNA containing capsids to the nucleus during infection.

Conclusions: Novel 2nd generation pan-genotypic CpAMs demonstrate potent HBV antiviral activity, particularly against cccDNA generation and its surrogate markers. They appear to work via newly identified mechanisms and possess promising drugable properties. The potency and favorable preclinical profile support advancement of this next generation of CpAMs into clinical development.

antiHBVren

摘要最终ID：922
标题：有效的第二代CpAMs能够抑制HBV感染细胞中HBsAg，HBeAg，pgRNA和cccDNA产生的临床前特征
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摘要身体：
背景：尽管目前可用的治疗方案延长了治疗时间，但慢性HBV（CHB）患者的临床治愈依然难以实现。核心蛋白质变构调节剂（CpAMs）是一类新型的直接作用抗病毒药物，可通过靶向病毒生命周期的多个方面来帮助提高治愈率。在这里，我们描述了有效的第二代CpAMs，其表现出良好的药物性质并且通过多种机制抑制cccDNA建立和补充。

方法：使用HepAD38细胞系（Gt D）和感染的HepG2-NTCP细胞和原代人肝细胞（PHH）确定CpAM抗病毒活性。使用瞬时转染试验或HBV稳定的细胞系建立泛基因型活性。使用HepAD38细胞中的一系列抑制浓度进行组合研究。使用引物和对HBV核心基因特异性的探针，通过Taqman PCR（qPCR）定量HBV DNA水平。通过ELISA定量HBeAg和HBsAg。如前所述，通过Northern印迹或b-DNA，Western印迹，酶免疫测定（EIA）和Southern印迹分别检测HBV总RNA /衣壳化pgRNA，Cp，衣壳和衣壳相关核心DNA。

结果：代表性的第二代CpAMs在HepAD38细胞（EC50s <30nM）中表现出对病毒DNA复制的有效抑制，并且在感染的HepG2-NTCP细胞和PHH（EC50s <300nM）中HBeAg，HBsAg和pgRNA的减少。在稳定或瞬时转染的HepG2细胞中，抗​​病毒活性延伸至其他主要的HBV基因型。在细胞毒性测定（CC50s>10μM）中对一组其他病毒（EC50>10μM）或细胞系没有显示显着的活性，表明这些化合物是HBV的选择性抑制剂。铅分子具有有希望的物理特性，低药物相互作用潜力和多种物种中有利的PK谱。作用机制研究表明，这些新的CpAMs的增强效力可能是由于抑制了HBV生命周期的多个步骤，包括异常衣壳装配的加速，预成形衣壳的破坏以及在输入成熟衣壳期间的过早拆卸和在感染过程中rcDNA含有核衣壳。

结论：新的第二代泛基因型CpAMs表现出强有力的HBV抗病毒活性，特别是针对cccDNA生成及其替代标记。它们似乎通过新鉴定的机制起作用并且具有有希望的可药用性质。效力和有利的临床前概况支持将这种新一代CpAMs推进到临床开发中。