HCV NS5B POLYMERASE MUTATIONS: BIOLOGY/PHARMACOLOGY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。据估计，美国有400万到1000万人患有慢性丙型肝炎。尽管丙型肝炎的治疗已经有所改善，但由于禁忌症，估计只有20%的患者能够接受目前的治疗。开发新的丙型肝炎治疗方法或疫苗仍然是一项未解决的重大公共卫生挑战。本研究将验证的假设是，由于HCV的高突变率而产生的HCV NS5B聚合酶变异可能导致对NS5B聚合酶抑制剂的耐药性，鉴定这些突变体对于开发有效的新抗病毒药物至关重要。鉴定对模型聚合酶抑制剂耐药的NS5B变异将有助于开发针对NS5B聚合酶中对HCV复制至关重要的基序的抑制剂。此外，NS5B的一些变体可能在确定NS5B、RNA模板和抑制剂的三元配合物的结构方面具有更有利的特性。美国疾病控制与预防中心（J. Med. Virol.）的同事获得的HCV序列数据的生物信息学分析取得了进展。此外，一种超过20毫克的NS5B变体已被纯化，目前正用于共晶生长研究。明年的具体目标是：确定慢性HCV感染期间发生的其他NS5B突变，并选择几个变体进行研究；进一步完善重组HCV NS5B聚合酶蛋白的表达和纯化方法，用于晶体生长研究；启动至少两种其他NS5B变体、寡核苷酸模板和抑制剂的晶体生长研究。这将允许基于结构的方法用于开发治疗性NS5B抑制剂，其目标是将HCV蛋白的优势（Hagedorn实验室）与NIH资助申请的药物化学专业知识联系起来。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. An estimated four to ten million people in the United States have chronic hepatitis C. Although treatment for HCV has improved only an estimated 20% of patients are able to take current therapies because of contraindications. Developing new therapeutics or vaccines for hepatitis C remains a major unsolved public health challenge. The hypothesis that this study will test is that variants of the HCV NS5B polymerase, arising due to the high mutation rate of HCV, can result in resistance to NS5B polymerase inhibitors and that identifying these mutants will be essential for developing effective new antivirals. Identifying variants of NS5B which are resistant to model polymerase inhibitors should aid in developing inhibitors that target motifs in the NS5B polymerase that are essential for HCV replication. In addition, some variants of NS5B may have more favorable properties for determining the structure of a ternary complex of NS5B, an RNA template, and an inhibitor. Progress has been made on the bioinformatics analysis of HCV sequence data obtained by colleagues at the CDC (J. Med. Virol.). In addition, over 20 mg of one NS5B variant was purified and is currently being used in co-crystal growth studies. The Specific Objectives for the next year are: To identify additional NS5B mutations occurring during chronic HCV infection and select several variants to study; To further improve methods of expressing and purifying sufficient quantities of recombinant HCV NS5B polymerase proteins for crystal growth studies; and To initiate crystal growth studies of at least two other NS5B variants, an oligonucleotide template, and an inhibitor. This will permit structure based approaches to be used in developing therapeutic NS5B inhibitors with the goal of linking strengths regarding HCV proteins (Hagedorn Lab) with expertise in Medicinal Chemistry for an NIH grant applications.