HCV NS4B: Translating molecular virology into novel antiviral therapies

HCV NS4B：将分子病毒学转化为新型抗病毒疗法

• 批准号: 8417695
• 负责人: JEFFREY S GLENN
• 金额: $ 37.26万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8417695
• 关键词: Amino Acids; Antihistamines; Antiviral Agents; Antiviral Therapy; Arginine; Atomic Force Microscopy; Binding; Biochemical; Cells; Characteristics; Chemicals; Clinical; Data; Development; Drug resistance; Electron Microscopy; Elements; Evaluation; Exhibits; Future; Generations; Genetic; Genome; Hepatitis C virus; Image; In Vitro; Interferons; Internet; Knowledge; Length; Life Cycle Stages; Light; Lipids; Liver diseases; Maps; Mediating; Membrane; Methods; Microfluidics; Microscopic; Modification; Molecular; Molecular Virology; Mutation; New Agents; Patients; Pharmaceutical Preparations; Protease Inhibitor; Proteins; Quartz; RNA; RNA Binding; Reagent; Resistance; Resolution; Role; Structure; Technology; Testing; Therapeutic; Time; Toxic effect; Translating; Vesicle; Viral; Viral Physiology; Virus; Virus Replication; anti-hepatitis C; base; chronic liver disease; design; hepatitis C virus NS3 protein; high throughput screening; inhibitor/antagonist; member; mutant; novel; public health relevance; small molecule; tertiary amine; three dimensional structure

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is an important cause of liver disease. Current therapies are inadequate. A cocktail of multiple drugs, each targeting an independent viral function offers the best chance for effective pharmacologic control. Our long-term objectives are to better understand the molecular virology of HCV, increase the repertoire of new targets that can be translated into novel drug classes for inclusion in future anti-HCV cocktails. We have identified and genetically validated two new targets within NS4B: an arginine-rich-like motif mediated RNA binding activity that is specific for the 3' terminus of the viral negative RNA strand (3'term(-)); and an amphipathic helix, termed 4BAH2, with a dramatic ability to promote lipid vesicle aggregation, an ideal candidate biochemical activity for creating the membranous web, the HCV replication platform. We then identified small molecule inhibitors of these two targets with significant anti-HCV activity: a first generation H1 antihistamine-clemizole-potently inhibits NS4B RNA binding and exhibits dramatic in vitro synergy with the HCV NS3 protease inhibitor SCH503034. Two distinct compounds-A2 and C4-were each found to be potent specific inhibitors of 4BAH2-mediated lipid vesicle aggregation. Finally, we recently developed a new method for rapidly mapping RNA secondary structure, affording new ways to study RNA elements such as the target of NS4B binding. Our overall hypothesis is that two different newly described functional activities within NS4B are each essential for mediating NS4B's role in HCV replication. Thus approaches designed to disrupt the function of these domains may be potentially used to inhibit HCV replication in patients. More specifically, we hypothesize: 1) clemizole's antiviral activity is distinct from its antihistamine activity; 2) the mechanism of action of A2 and C4 can be further validated by analysis of resistant mutants and derivatives of these compounds; 3) while A2 and C4 both target the same 4BAH2, they do so via distinct mechanisms, where one targets 4BAH2's interaction with membranes, and one targets 4BAH2's interaction with itself; 4) NS4B RNA binding inhibitors are broadly synergistic with NS3 protease inhibitors; 5) both NS4B RNA binding inhibitors and 4BAH2 inhibitors represent attractive potential new drug classes for future anti-HCV therapeutic cocktails designed to maximize efficacy and minimize resistance; 6) 4BAH2's lipid vesicle aggregating activity reflects a biochemical activity important for membranous web formation that is amenable to both genetic and pharmacologic disruption, and the specific amino acids critical for this activity can be identified by mutational analysis; 7) the secondary structure of the HCV 3'term(-) is altered upon interaction with NS4B or clemizole, or upon mutation to escape clemizole inhibition; 8) expression of wild-type and drug-resistant forms of NS4B can enable critically important 3D structure determinations. To test these hypotheses, we propose to further study these compounds' mechanism of action, determine their potential as critical components of future anti-HCV cocktails, and better understand the structure and function of these compounds' targets within NS4B and the HCV life cycle.

描述（由申请人提供）：丙型肝炎病毒（HCV）是肝脏疾病的重要原因。目前的治疗是不够的。多种药物的混合物，每种药物都针对独立的病毒功能，为有效的药理控制提供了最佳机会。我们的长期目标是更好地了解HCV的分子病毒学，增加新靶点的库，这些靶点可以转化为新的药物类别，用于未来的抗HCV鸡尾酒。我们已经在NS 4 B中鉴定并遗传验证了两个新的靶点：富含精氨酸的样基序介导的RNA结合活性，其对病毒负RNA链的3'末端具有特异性（3'末端（-））;和两亲性螺旋，称为4 BAH 2，具有促进脂质囊泡聚集的显著能力，这是用于创建膜网（HCV复制平台）的理想候选生物化学活性。然后，我们鉴定了这两个靶点的具有显著抗HCV活性的小分子抑制剂：第一代H1抗组胺药-克立咪唑-有效地抑制NS 4 B RNA结合，并与HCV NS 3蛋白酶抑制剂SCH 503034表现出显著的体外协同作用。两种不同的化合物-A2和C4-分别被发现是有效的4 BAH 2介导的脂质囊泡聚集的特异性抑制剂。最后，我们最近开发了一种快速绘制RNA二级结构的新方法，为研究RNA元件（如NS 4 B结合靶点）提供了新的方法。我们的总体假设是，NS 4 B内两种不同的新描述的功能活动都是介导NS 4 B在HCV复制中的作用所必需的。因此，旨在破坏这些结构域的功能的方法可能被用于抑制患者中的HCV复制。更具体地说，我们假设：1）克立咪唑的抗病毒活性与其抗组胺活性不同; 2）A2和C4的作用机制可以通过分析这些化合物的抗性突变体和衍生物来进一步验证; 3）虽然A2和C4都靶向相同的4 BAH 2，但它们通过不同的机制这样做，其中一个靶向4 BAH 2与膜的相互作用，一种靶向4 BAH 2与其自身的相互作用; 4）NS 4 B RNA结合抑制剂与NS 3蛋白酶抑制剂具有广泛的协同作用; 5）NS 4 B RNA结合抑制剂和4 BAH 2抑制剂都代表了未来抗HCV治疗鸡尾酒的有吸引力的潜在新药类别，其设计为使功效最大化并使抗性最小化; 6）4 BAH 2的脂质囊泡聚集活性反映了对膜网形成重要的生物化学活性，其易于遗传和药理学破坏，并且可以通过突变分析鉴定对该活性至关重要的特定氨基酸; 7）HCV 3 '端（-）的二级结构在与NS 4 B或克立咪唑相互作用时改变，或在突变以逃避克立咪唑抑制时改变; 8）NS 4 B的野生型和耐药形式的表达可以实现至关重要的3D结构确定。为了验证这些假设，我们建议进一步研究这些化合物的作用机制，确定它们作为未来抗HCV鸡尾酒关键成分的潜力，并更好地了解这些化合物在NS 4 B和HCV生命周期中的结构和功能。