HCV NS4B: Translating molecular virology into novel antiviral therapies

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是导致肝脏疾病的重要原因。目前的治疗方法是不够的。多种药物的鸡尾酒，每种药物都针对一种独立的病毒功能，为有效的药物控制提供了最好的机会。我们的长期目标是更好地了解丙型肝炎病毒的分子病毒学，增加新靶点的库，这些新靶点可以转化为新的药物类别，以便包括在未来的抗丙型肝炎鸡尾酒中。我们已经在NS4B中发现并在基因上证实了两个新的靶点：一个富含精氨酸的基序介导的RNA结合活性，它特异地与病毒负RNA链的3‘末端(3’项(-))结合；一个两亲性螺旋，称为4BAH2，具有显著的促进脂泡聚集的能力，是创建膜网络的理想候选生化活性，即丙型肝炎病毒复制平台。然后，我们确定了这两个靶点的小分子抑制剂，具有显著的抗丙型肝炎病毒活性：第一代H1抗组胺-克列咪唑-有效地抑制NS4B RNA结合，并在体外与丙型肝炎病毒NS3蛋白酶抑制剂SCH503034显示出显著的协同作用。两种不同的化合物-A2和C4-分别被发现是4BAH2介导的脂泡聚集的有效特异性抑制物。最后，我们最近开发了一种快速定位RNA二级结构的新方法，为研究NS4B结合靶标等RNA元件提供了新的途径。我们的总体假设是，NS4B内的两种不同的新描述的功能活动各自对于调节NS4B在丙型肝炎病毒复制中的作用是必不可少的。因此，旨在破坏这些结构域功能的方法可能被用于抑制患者体内的丙型肝炎病毒复制。更具体地说，我们假设：1)克莱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对这些化合物的耐药突变体和衍生物的分析进一步验证；3)虽然A2和C4都针对相同的4BAH2，但它们通过不同的机制发挥作用，其中一个靶点是4BAH2与膜的S相互作用，一个靶点是4BAH2与自身的S相互作用；4)NS4BRNA结合抑制剂与NS3蛋白酶抑制剂具有广泛的协同作用；5)NS4BRNA结合抑制剂和4BAH2抑制剂都是未来抗丙型肝炎治疗鸡尾酒的有吸引力的潜在药物类别，旨在最大限度地提高疗效和减少耐药性；6)4BAH2‘S脂泡聚集活性反映了一种对膜网形成很重要的生化活性，它对遗传和药物破坏都是适用的，对这种活性至关重要的特定氨基酸可以通过突变分析来鉴定；7)丙型肝炎病毒3’端(-)的二级结构在与NS4B或克霉唑相互作用时改变，或在突变时改变以逃避克霉唑的抑制；8)野生型和耐药形式的NS4B的表达可以使关键的三维结构测定成为可能。为了验证这些假说，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丙型肝炎鸡尾酒的关键成分的潜力，并更好地了解这些化合物在NS4B和丙型肝炎病毒生命周期中的靶标的结构和功能。 公共卫生相关性：丙型肝炎病毒仍然是全球肝病的重要原因，目前的治疗方法不足以治疗这些肝病。我们在丙型肝炎病毒NS4B蛋白中发现了两个新靶点，并确定了这些靶点的小分子抑制物。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和丙型肝炎病毒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丙型肝炎鸡尾酒的关键成分的潜力。

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.