基于拆分荧光素酶报告模型的HBV cccDNA抑制药物高通量筛选及候选药物的作用机制研究

乙型肝炎病毒共价闭合环状DNA (HBV cccDNA)的持续稳定存在是慢乙肝难以治愈及停药后复发的关键原因，而现有治疗药物均不能有效清除cccDNA，缺乏高效的筛选模型限制了靶向药物的研发。在本项目前期工作中，申请人从病毒序列和报告基因等方面优化了病毒基因组改造策略，建立了拆分荧光素酶作为报告基因的cccDNA抑制药物筛选模型，并初步验证了该模型的可行性，获得具有抗病毒效果的先导化合物。在此基础上，本研究将进一步验证该模型用于高效药物筛选的稳定性，扩大药物筛选规模，对先导化合物进行结构优化，进一步在多种HBV体内外模型中评估候选化合物的安全性和有效性，并探索其作用机制；另一方面，尝试利用新型拆分荧光蛋白实现cccDNA的定量可视化与单细胞水平分析。预期成果将为慢性乙肝新药研究领域提供高通量药物筛选模型和候选化合物。

Chronic hepatitis B virus (HBV) infection represents a major global health problem, affecting an estimated 292 million persons worldwide and is associated with substantial morbidity and mortality because of clinical complications, such as liver cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) is the viral persistence reservoir and key obstacle for cure of chronic hepatitis B, and the licensed drugs fail to eliminate HBV cccDNA. It is hard to design drugs targeting HBV cccDNA based on theoretical guidance because the formation and maintenance mechanism of HBV cccDNA is complex. The lack of efficient drug screening models also limits the development of drugs. In previous study, we have optimized the virus modification strategy in terms of virus sequence, reporter gene and position of insertion, which supported the establishment of two cccDNA reporter models with split-NanoLuc as reporter gene. The feasibility of these models for high throughput screening was evaluated preliminarily and lead compounds with antiviral effect was obtained. We plan to confirm the stability of these model and screen for “hits” targeting cccDNA. In addition, we will verify the safety and effect of candidate drugs in various HBV research models and explore the mechanism; On the other hand, we attend to apply the novel split fluorescent protein to achieve the visualization and single-cell level analysis of cccDNA. The expected results will provide cell models for high-throughput screening and candidate drugs for chronic hepatitis B.