Inhibiting protein-primed replication of human enteroviruses.

描述(申请人提供)：持续感染人类肠道病毒(HEV)是扩张型心肌病的主要原因，并与1型糖尿病、胃肠道和神经系统疾病有因果关系。HEV是耐酸的，可以在胃肠道和其他器官中长时间存活。目前还没有被批准用于治疗HEV感染的药物。这个高风险/高回报项目的目标是确定一种小蛋白VPG尿苷基化的抑制剂，并表明这些化合物为治疗复制缓慢的HEV毒株的慢性感染提供了一种替代治疗途径。由于尿苷基化是病毒特异性的，这些抑制剂对哺乳动物细胞的副作用应该较少。这项翻译工作将结合计算、结构和高通量筛选(HTS)方法来选择干扰尿苷基化和HEV复制的抑制剂。在R21阶段选择的尿酰基化抑制剂将在R33阶段测试它们在哺乳动物细胞和慢性HEV感染的小鼠模型中抑制HEV的效果。目标1是开发使用共识VPG的尿苷基化分析，并筛选干扰这一反应的化合物的多样性集合。HTS中确定的活性化合物将在目标2中进行表征，以推导结构-活性关系和 确定用于筛选基于网络的大型化合物文库的子结构。基于VPG结构的药效团将用于进一步的电子筛选这些文库。选定的化合物将被购买和检测，或在内部生产。假设已经确定了3-10种尿苷基化抑制剂，R33阶段的工作将开始。目的3是根据药物性质(毒性低、溶解性好、在细胞培养液中稳定性高)，进一步表征尿苷酰化抑制剂的活性，并选择最适合生物检测的药物。化合物可制成纳米颗粒，以增强细胞摄取。将进一步测试最有效、生物可接受的抑制剂是否有能力单独或与其他活性化合物和抗病毒药物联合使用，以减少HEV毒株在感染细胞中的复制。使用核磁共振和天然凝胶电泳的溶液研究，以及聚合酶与最好的抗病毒化合物的共晶结构将被用来确定它们的活性模式。最后，将测试这些抗病毒药物在培养的心肌细胞和柯萨奇B3病毒慢性感染小鼠模型中阻止HEV复制的能力。成功的抑制剂将成为治疗HEV感染的潜在新疗法，可以单独使用，也可以与作为全球根除脊髓灰质炎倡议一部分开发的其他化合物联合使用，包括抑制细胞进入的衣壳结合化合物。因此，以尿苷酰化为靶点的抗HEV化合物也可能在确保根除脊髓灰质炎方面发挥作用。

DESCRIPTION (provided by applicant): Persistent infections with Human enteroviruses (HEV) are a major cause of dilated cardiomyopathies, and have been causally implicated in type 1 diabetes, gastrointestinal and nervous system disorders. HEVs are acid-resistant, and can survive in the gastrointestinal tract and other organs for long periods of time. There are currentl no drugs approved to treat HEV infections. The goal of this high-risk/high-reward project is to identify inhibitors of uridylylation of a small protein, VPg, and show that these compounds provide an alternative treatment route for treating chronic infections with slow replicating HEV strains. As uridylylation is virus-specific, the inhibitors should have fewer side effects on mammalian cells. This translational work will combine computational, structural and high throughput screening (HTS) methods to select inhibitors that interfere with uridylylation, and HEV replication. Uridylylation inhibitors selected, in the R21 phase, will be tested, in the R33 phase, for their effects on inhibiting HEV in mammalian cells and in a murine model of chronic HEV infection. Aim 1 is to develop uridylylation assays using a consensus VPg, and screen a diversity set of compounds for those that interfere with this reaction. Active compounds identified in the HTS will be characterized in Aim 2 to derive structure-activity relationships and identify substructures for screening large, web-based compound libraries. A pharmacophore based on VPg structures will be used for further in silico screening of these libraries. Selected compounds will be purchased and assayed, or produced in-house. Assuming that 3-10 uridylylation inhibitors have been identified, the R33 phase of the work will begin. Aim 3 is to further characterize the activity of the uridylylation inhibitors and select those most suitable fo biological assays according to their pharmaceutical properties (low toxicity, good solubility and high stability in cell culture media). Compounds may be formulated in nanoparticles to enhance cell uptake. The most potent, biologically acceptable inhibitors will be further tested for their ability to diminish the replication of HEV strains in infected cells, alone or in combination with other active compounds and antiviral agents. Solution studies using NMR and native gel electrophoresis, and co-crystal structures of the polymerase with the best antiviral compounds will be used to establish their mode of activity. Finally, the antivirals will be tested for their bility to halt HEV replication in cultured cardiomyocytes and a murine model of chronic infection with Coxsackie virus B3. Successful inhibitors will be potential new therapies for HEV infections, that can be used alone or in combination with other compounds developed as part of the Global Polio Eradication Initiative, including capsid binding compounds that inhibit cell entry. Anti-HEV compounds that target uridylylation may thus also play a role in securing polio eradication.