Molecular Modulation of Virus Capsid Assembly

描述（由申请人提供）：乙型肝炎病毒（HBV）是一种包膜DNA病毒，带有RNA中间体。病毒的核心在病毒mRNA和逆转录酶周围组装成一个蛋白质外壳。在细胞质中，被封装的RNA被转录成DNA，从而产生成熟的细胞核。全世界有3.6亿人患有慢性乙型肝炎病毒感染。乙肝病毒引起肝功能障碍、肝硬化和肝细胞癌。它每年造成近100万人死亡。虽然乙肝病毒可以通过接种疫苗来预防，但这种疫苗没有治疗作用。在可行的情况下，治疗的选择是使用逆转录酶抑制剂进行单药治疗。不幸的是，这种方法可能会选择耐药突变体。理想情况下，可以开发一个替代靶点，从而产生适合联合治疗的药物。我们已经提出HBV核心组装就是这样一个分子靶标。基于理论，我们建议最有效的方法将是加强组装，以结合非生产性复合体中的许多核心蛋白质。我们已经研究了几个具有组装增强活性的小分子家族，特别是杂芳基二氢嘧啶（HAPs）。在我们的研究过程中，我们已经检测了大约150种不同的潜在组装效应器，开发了高通量筛选，确定了HAP结合位点的晶体学，并开发了溶液方法来评估病毒对组装效应器的热力学和动力学响应。我们观察到在培养细胞中组装动力学和抗病毒效果之间有很强的相关性。现在，我们建议利用这些结果：(i)寻找新的组装效应器，确定替代结合位点和先导化合物，并改进现有的设计，（ii）定义组装效应器活性效应的结构和物理化学基础，（iii）研究核心蛋白对组装效应器产生抗性的能力。（iv）通过检查组装效应物如何改变细胞中HBV核心的分布来检查它们如何影响病毒与宿主之间的相互作用。这些独立的目标将使组装效应器从纯粹的学术研究转向临床应用。

DESCRIPTION (provided by applicant): Hepatitis B Virus (HBV) is an enveloped DNA virus with an RNA intermediate. The core of the virus assembles as a protein shell around a viral mRNA and reverse transcriptase. In the cytoplasm, the encapsidated RNA is transcribed into DNA to yield the mature core. Worldwide, 360 million people suffer from chronic HBV infection. HBV causes liver dysfunction, cirrhosis, and hepatocellular carcinoma. It contributes to almost one million deaths each year. Though HBV can be prevented by vaccination, the vaccine is not therapeutic. Where available, the treatment of choice is monotherapy with a reverse transcriptase inhibitor. Unfortunately, this approach may select for resistant mutants. Ideally, an alternative target can be developed that will yield drugs suitable for combination therapy. We have proposed that HBV core assembly is such a molecular target. Based on theory, we suggest that the most effective approach will be to enhance assembly to tie up many core proteins in unproductive complexes. We have investigated several families of small molecules with assembly enhancing activity, in particular heteroaryldihydro- pyrimidines (HAPs). In the course of our studies we have examined about 150 different potential assembly effectors, developed high throughput screens, determined the HAP binding site crystallographically, and developed solution approaches to evaluate thermodynamic and kinetic responses of viruses to assembly effectors. We observed a strong correlation between assembly kinetics and antiviral effect in cultured cells. Now we propose to capitalize on these results by (i) searching for new assembly effectors, identifying alternative binding sites and lead compounds, and improving on the design of those in hand, (ii) defining the structural and physical chemical basis for assembly effector activity effect, (iii) investigating the ability of the core protein for developing resistance to assembly effectors, and (iv) examining how assembly effectors affect interactions between virus and host by examining how they alter the distribution of HBV cores in cells. These independent aims will move assembly effectors from the subject of purely academic investigation towards clinical application.