有效的非 HAP I 类 HBV 衣壳组装调节剂 ALG-005398 在体内降低 HBsA

StephenW

有效的非 HAP I 类 HBV 衣壳组装调节剂 ALG-005398 在体内降低 HBsAg 水平
乙型肝炎病毒
2021 年 11 月 22 日

Aligos Belgium 介绍了对 ALG-005398 的研究，这是一系列非杂芳基二氢嘧啶 (HAP) I 类衣壳组装调节剂的第一个代表，该调节剂是使用基于结构的药物设计和支架跳跃鉴定的。

为了治疗慢性乙型肝炎，I 类 CAM 除了抑制 HBV RNA 衣壳化和感染性 HBV 颗粒的形成外，还诱导 AAV-HBV 小鼠的乙型肝炎病毒 (HBV) 核心蛋白 (HBc) 聚集和持续降低 HBsAg。使用定量 PCR 在 HepG2.117 细胞中测定对 HBV DNA 的抗病毒活性。在 HBV 感染的原代人肝细胞 (PHH) 中研究了对 HBV DNA 的主要影响和对共价闭合环状 DNA (cccDNA) 建立的次要影响。在优化的对数下降试验中评估了几个数量级的抑制。

使用该测定，ALG-005398 (n = 3) 的 EC50、EC90、EC99 和 EC99.9 值分别确定为 1.70、4.80、8.12 和 13.7 nM，而 ALG-005398 的值为 59.6、204、458 和 989 nM。 RG-7907 (n = 2)，另一个 I 级 CAM。此外，ALG-005398 可有效抑制 HBV 感染的 PHH 中的 RNA 衣壳化和 cccDNA 的建立。对于 ALG-005398 (n = 3) 和 5.41/55，HBV DNA、HBsAg、HBeAg 和 HBV RNA 的 EC50/EC90 值分别为 9.29/83.6、167/650、97.3/353 和 128/446 nM。 848/> 10000、372/2730 和 294/> 分别为 RG-7907 (n = 2) 10000 nM。此外，ALG-005398 在体外诱导小异常衣壳的快速形成。与 RG-7907 相比，ALG-005398 与 HBc 的结合产生了快速寡聚化，最大猝灭略低，但斜率更陡（3.4 对 1.9）。

在对数下降试验中，与 RG-7907 相比，ALG-005398 的斜率更陡（2.3 对 1.5）。两种化合物都显着降低了蛋白质印迹中可检测到的 HBc 量，但 ALG-005398 在诱导 HBc 斑点方面的效力比 RG-7907 强大约 10 倍。 ALG-005398 (n = 4) 的 EC50 和 EC90 值分别为 34.7 和 192 nM，而 RG-7907 (n = 4) 的 EC50 和 EC90 值为 529 和 1970 nM。关于存在核 HBc 的细胞区室，证实了 HAP 诱导的 HBc 斑点与 PML 体的共定位。

结论是ALG-005398诱导的HBc斑点的核定位可能不同。未来可能进行评估的一个假设是，HBc 点定位于 PML 体等结构可能与 HBsAg 丢失有关，可能是通过翻译后修饰（例如，小的泛素样修饰符）或干扰素反应的激活。延迟抗原减少和缺乏明确的 ALT 升高可能表明 ALG-005398 与 HAP 的作用机制略有不同，Aligos 目前正在研究这种机制。在体内，ALG-005398 (30 mg/kg b.i.d.) 导致 HBV DNA 的多阶段减少，95 天后减少 6.4 log10 IU/mL。观察到血清 HBsAg 水平显着降低，随着起搏动力学降低 2.24 log10 IU/mL：在 ALG-005398 治疗的第 49 天和第 95 天之间观察到主要下降，ALT 变化很小。

在其他发现中，注意到 HBsAg 水平显着下降，但与 HBsAg 下降相比略有延迟； pgRNA 水平显着降低；肝内 HBsAg 在第 95 天免疫组化染色的肝切片中显示“显着消失”；并且 I 类 CAM 治疗的动物肝脏中 AAV-HBV 附加体的数量减少了 10 倍

StephenW

Potent non-HAP class I HBV capsid assembly modulator ALG-005398 reduces HBsAg levels in vivo
Hepatitis B virus
Nov. 22, 2021

Aligos Belgium presented research on ALG-005398, a first representative of a series of non-heteroaryldihydropyrimidines (HAPs) class I capsid-assembly modulators, that was identified using structure-based drug design and scaffold hopping.

To treat chronic hepatitis B, class I CAMs induce hepatitis B virus (HBV) core protein (HBc) aggregation and sustained HBsAg reduction in AAV-HBV mice, in addition to inhibiting HBV RNA encapsidation and formation of infectious HBV particles. Antiviral activity on HBV DNA was determined in HepG2.117 cells using quantitative PCR. Both the primary effect on HBV DNA and the secondary effect on covalently closed circular DNA (cccDNA) establishment were studied in HBV-infected primary human hepatocytes (PHHs). Inhibition over several orders of magnitude was assessed in an optimized log drop assay.

Using this assay, the EC50, EC90, EC99 and EC99.9 values were determined to be 1.70, 4.80, 8.12 and 13.7 nM, respectively, for ALG-005398 (n = 3) vs. 59.6, 204, 458 and 989 nM for RG-7907 (n = 2), another class I CAM. In addition, ALG-005398 potently inhibited RNA encapsidation and cccDNA establishment in HBV-infected PHHs. The EC50/EC90 values for HBV DNA, HBsAg, HBeAg and HBV RNA were 9.29/83.6, 167/650, 97.3/353 and 128/446 nM, respectively, for ALG-005398 (n = 3) vs. 5.41/55.3, 848/> 10000, 372/2730 and 294/> 10000 nM for RG-7907 (n = 2), respectively. Furthermore, ALG-005398 induced rapid formation of small aberrant capsids in vitro. Compared with RG-7907, binding of ALG-005398 to HBc produced rapid oligomerization, with slightly lower maximum quenching but a steeper slope (3.4 vs. 1.9).

In the log drop assay, a steeper slope was also observed for ALG-005398 compared with RG-7907 (2.3 vs. 1.5). Both compounds sharply reduced the amount of detectable HBc in Western blot, but ALG-005398 was approximately 10-fold more potent than RG-7907 in inducing HBc spots. The respective EC50 and EC90 values were 34.7 and 192 nM for ALG-005398 (n = 4) vs. 529 and 1970 nM for RG-7907 (n = 4). Regarding the cellular compartments in which the nuclear HBc exist, colocalization of HAP-induced HBc spots with PML bodies was confirmed.

It was concluded that the nuclear localization of HBc spots induced by ALG-005398 may be different. A hypothesis for possible future evaluation is that localization of the HBc spots to structures such as PML bodies might be related to HBsAg loss, perhaps through post-translational modification (for example, small ubiquitin-like modifier) or the activation of interferon responses. The delayed antigen reductions and lack of clear ALT elevation may suggest a slightly different mechanism of action for ALG-005398 vs. HAPs, which is being currently investigated by Aligos. In vivo, ALG-005398 (30 mg/kg b.i.d.) resulted in a multiphasic reduction in HBV DNA, with a 6.4 log10 IU/mL reduction after 95 days. A significant decrease in serum HBsAg levels was observed with a 2.24 log10 IU/mL reduction with paced kinetics: the main decline was observed between day 49 and 95 of ALG-005398 treatment, with only a minimal change in ALT.

Among other findings, significant decreases in HBsAg levels were noted, but with a slight delay compared with HBsAg decline; pgRNA levels were significantly reduced; intrahepatic HBsAg showed a 'dramatic disappearance' in immunohistochemically stained liver sections at day 95; and a 10-fold reduction occurred in the amount of AAV-HBV episome in the livers of class I CAM-treated animals