Small molecule inhibitors of HBx that decrease hepatitis B virus replication

慢性乙肝病毒感染是全球第七大和第五大致死原因 癌症的起因。乙型肝炎病毒慢性感染的关键是核定位的乙肝病毒表型共价闭合 驱动乙肝病毒转录和复制的环状DNA(CcDNA)。肝细胞癌的风险与 从cccDNA复制的水平。治疗乙肝需要消除ccDNA，这在目前是不可行的。 然而，实现功能性治愈，即乙肝表面抗原(HBs)的持续丢失，可能会 通过沉默cccDNA是可能的。目前的治疗方法，用干扰素α或终身治疗 对核糖核酸类似物的维护，功能治愈率低，突显了对新乙肝病毒的迫切需要 治疗学。HBx蛋白是开发抗病毒药物的一个很好的候选药物，因为它对 病毒生命周期中的作用--病毒转录的调节，沉默的病毒限制因子的降解 CccDNA，并干扰宿主细胞的许多过程。尽管HBX扮演着核心角色，但它还没有成为 抗病毒治疗的靶点。我们的中心假设是，靶向HBx将抑制病毒复制，沉默 CccDNA，并促进乙肝病毒的功能性治疗。我们提出的研究建立在几个最新进展的基础上。第一, 我们已经证明HBx与细胞DDB1相互作用，以介导已知为沉默的细胞蛋白质的降解 CccDNA。其次，我们利用人类肝干细胞建立了一个真实的乙肝病毒感染模型。 有机化合物(HLOS)。最后，我们与药物发现中心(CDD)建立了合作关系，网址为 贝勒医学院已经开发了50个图书馆，其中包含超过50亿个新的DNA条形码小 类药物分子(DEC-Tec)。我们现在提出一个高度创新的项目，最终确定新的乙肝病毒 针对HBX的抗病毒药物。在目标1中，将通过针对DEC-1的亲和选择来筛选纯化的HBx蛋白 TEC文库。结合蛋白将通过DNA测序进行鉴定，并与新的目标蛋白进行验证。考虑 由于HBX与100多种细胞蛋白相互作用，我们预计会鉴定出多个HBX结合子。结构-活性 将使用计算软件确定相互关系(SAR)，并测量粘结剂对HBX的亲和力。 将寻求具有SAR和高亲和力的化合物。在目标2中，将对高亲和力HBX结合剂进行筛选 在裂解荧光素酶实验中测量的抑制关键HBX-DDB1相互作用的能力。一种化合物 然后，将在HLO复制模型中测试抑制HBx-DDB1抑制乙肝复制的能力 与已知的核(T)类似物恩替卡韦比较。成功的HBX活页夹将作为线索 未来的体内评价，包括毒性、药物代谢和药代动力学。其他高亲和力粘合剂 可以被结合到蛋白水解靶向嵌合分子(PROTACs)中，以实现蛋白酶体介导 退化。在世界范围内，超过2.57亿人慢性感染乙肝病毒，约100万人 每年死于肝硬变或癌症。拟议中的研究具有发现 治疗慢性乙肝的新方法，预防乙肝相关癌症，并导致功能性治愈。

Chronic infection with hepatitis B virus (HBV) is the 7th leading cause of death worldwide and the 5th leading cause of cancer. The key to HBV chronic infection is the nuclear localized HBV episomal covalently closed circular DNA (cccDNA) that drives HBV transcription and replication. The risk of HCC is directly related to the level of replication from the cccDNA. An HBV cure requires eliminating cccDNA, which is not feasible at present. However, achieving a functional cure, defined as a sustained loss of hepatitis B surface antigen (HBsAg), may be possible by silencing the cccDNA. The current therapies, treatment with interferon alpha or life-long maintenance on nucleos(t)ide analogs, have low functional cure rates highlighting the critical need for new HBV therapeutics. The HBV HBx protein is an excellent candidate for the development of antivirals due to its critical roles in the virus life cycle – regulation of viral transcription, degradation of viral restriction factors that silence cccDNA, and interference with many host cellular processes. Despite its central role, HBx has yet to be the target of antiviral therapy. Our central hypothesis is that targeting HBx will inhibit viral replication, silence cccDNA, and facilitate an HBV functional cure. Our proposed studies build on several recent advances. First, we have shown HBx interacts with cellular DDB1 to mediate the degradation of cellular proteins known to silence cccDNA. Second, we have developed an authentic HBV infection model using human liver stem cell-derived organoids (HLOs). Finally, we have established a collaboration with the Center for Drug Discovery (CDD) at Baylor College of Medicine that has developed 50 libraries containing over 5 billion novel DNA-bar coded small drug-like molecules (DEC-Tec). We now propose a highly innovative project to ultimately identify new HBV antivirals that target HBx. In Aim 1, purified HBx protein will be screened by affinity selection against the DEC- Tec libraries. Binders will be identified by DNA sequencing and validated with fresh target protein. Considering that HBx interacts with over 100 cellular proteins, we expect to identify multiple HBx binders. Structure-activity relationship (SAR) will be determined using computational software, and the binder's affinity for HBx measured. Compounds with SAR and high affinity will be pursued. In Aim 2, high affinity HBx binders will be screened for the ability to inhibit the critical HBx-DDB1 interaction as measured in a split luciferase assay. Compounds that inhibit HBx-DDB1 will then be tested for the ability to inhibit HBV replication in the HLO replication model and compared against the known nucleos(t)ide analogue entecavir. Successful HBx binders will serve as leads for future in vivo evaluation that includes toxicity, drug metabolism, and pharmacokinetics. Other high affinity binders can be incorporated into proteolysis targeting chimeric molecules (PROTACs) for proteasome-mediated degradation. Worldwide, over 257 million people are chronically infected with HBV and approximately 1 million die each year of liver cirrhosis or cancer. The proposed studies hold tremendous potential for the discovery of novel approaches to treat chronic HBV, prevent HBV-associated cancer, and lead to a functional cure.