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: 8013309
• 负责人: JEFFREY S GLENN
• 金额: $ 41.68万
• 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/8013309
• 关键词: Amino Acids; Antihistamines; Antiviral Agents; Antiviral Therapy; Arginine; Atomic Force Microscopy; Binding; Biochemical; Cells; Characteristics; Chemicals; Chronic; 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; 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. PUBLIC HEALTH RELEVANCE: HCV remains an important cause of worldwide liver disease for which current therapies are inadequate. We have discovered two new targets within the HCV NS4B protein and identified small molecule inhibitors of these targets. We propose to further study these compounds' mechanism of action, better understand the structure and function of these compounds' targets within NS4B and the HCV life cycle, and determine these compounds' potential as critical components of future anti-HCV cocktails.

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。目前的治疗方法还不够。多种药物的混合物，每种药物针对独立的病毒功能，提供了有效药物控制的最佳机会。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，这些新靶点可以转化为新的药物类别，以纳入未来的抗 HCV 鸡尾酒。我们在 NS4B 中鉴定并进行了两个新靶标的基因验证：富含精氨酸的基序介导的 RNA 结合活性，该活性对病毒负 RNA 链 (3'term(-)) 的 3' 末端具有特异性；和称为 4BAH2 的两亲性螺旋，具有促进脂质囊泡聚集的显着能力，这是创建膜网（HCV 复制平台）的理想候选生化活性。然后，我们鉴定了这两个靶标的具有显着抗 HCV 活性的小分子抑制剂：第一代 H1 抗组胺药克立咪唑可有效抑制 NS4B RNA 结合，并与 HCV NS3 蛋白酶抑制剂 SCH503034 表现出显着的体外协同作用。两种不同的化合物 - A2 和 C4 - 均被发现是 4BAH2 介导的脂质囊泡聚集的有效特异性抑制剂。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 NS4B 结合靶点等 RNA 元件提供了新方法。我们的总体假设是，NS4B 内新描述的两种不同的功能活动对于介导 NS4B 在 HCV 复制中的作用至关重要。因此，旨在破坏这些结构域功能的方法可能有可能用于抑制患者体内的 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) 通过分析这些化合物的耐药突变体和衍生物，可以进一步验证A2和C4的作用机制； 3) 虽然 A2 和 C4 都靶向相同的 4BAH2，但它们通过不同的机制实现这一目标，其中一种靶向 4BAH2 与膜的相互作用，另一种靶向 4BAH2 与其自身的相互作用； 4) NS4B RNA结合抑制剂与NS3蛋白酶抑制剂具有广泛的协同作用； 5) NS4B RNA 结合抑制剂和 4BAH2 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，旨在最大限度地提高疗效并最大限度地减少耐药性； 6) 4BAH2的脂质囊泡聚集活性反映了对膜网形成很重要的生化活性，该活性容易受到遗传和药理破坏，并且可以通过突变分析来鉴定对该活性至关重要的特定氨基酸； 7)HCV 3'末端(-)的二级结构在与NS4B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) NS4B 野生型和耐药形式的表达可以实现至关重要的 3D 结构测定。为了检验这些假设，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 NS4B 和 HCV 生命周期中靶点的结构和功能。 公共卫生相关性：HCV 仍然是世界范围内肝病的一个重要原因，而目前的治疗方法对此还不够。我们在 HCV NS4B 蛋白中发现了两个新靶标，并鉴定了这些靶标的小分子抑制剂。我们建议进一步研究这些化合物的作用机制，更好地了解这些化合物在 NS4B 和 HCV 生命周期中靶标的结构和功能，并确定这些化合物作为未来抗 HCV 鸡尾酒关键成分的潜力。