Drug target validation of the enterovirus D68 2A protease

肠道病毒D68(EV-D68)是一种与儿童中重度呼吸道疾病有关的病毒病原体，在极少数情况下也会感染神经系统，并导致神经并发症，如脊髓灰质炎样急性弛缓性脊髓炎(AFM)。尽管疾病负担很大，但没有获得批准的抗病毒药物或疫苗可用于EV-D68。由于其医学重要性，EV-D68目前被国家过敏和传染病研究所列为优先病原体。由于无法预测下一次EV-D68暴发的发生，迫切需要一种有效、作用广泛和安全的EV-D68抑制剂。这项建议的目的是探索新的病毒蛋白作为抗病毒药物的靶点，并开发相应的化学探针作为靶点验证的工具。这一建议是建立在我们最近的发现基础上的，即EV-D68 2A蛋白具有病毒多蛋白VP1-2A连接所特有的半胱氨酸蛋白酶活性，其蛋白酶活性可以被FDA批准的口服药物telaprevir抑制。我们进一步表明，在不同的人类细胞系中，包括人类神经细胞在内的不同人类细胞系中，telapvir对几种当代人类EV-D68株具有亚微摩尔到低微摩尔的效力。替拉维韦(IC50)对EV-D68的抗病毒效力与丙型肝炎病毒相似，表明将其重新用作EV-D68抗病毒药物具有临床意义。通过一系列病毒传代实验，独立地阐明了TELAPREVR抑制EV-D68病毒的机制，导致在EV-D68 2Apro蛋白活性部位附近选择了一个N84T突变，从而降低了TELAPREVR在酶和细胞抗病毒检测中的效力。为了验证EV-D68 2Apro在体外和体内对病毒复制是必不可少的假设，我们的目标是开发具有良好药代动力学特性的2Apro抑制剂，并将它们用作化学探针，以验证EV-D68 2Apro在细胞培养和EV-D68诱导的瘫痪疾病新生小鼠模型中作为抗病毒药物靶点的可行性。具体地说，我们建议通过对替拉维韦的构效关系研究，设计更有效和更具选择性的EV-D68 2Apro抑制剂。所有设计的化合物都将在体外、酶和细胞分析中进行系统测试，并分析药代动力学特性。将选择优先的先导化合物在EV-D68小鼠模型中进行测试。拟议研究的预期结果是验证EV-D68 2Apro作为可行的抗病毒药物靶点，并证明EV-D68 2Apro抑制剂的体内抗病毒效果。

Enterovirus D68 (EV-D68), a viral pathogen associated with moderate to severe respiratory illness in children, also infects the nervous system in rare cases and causes neurological complications such as polio-like acute flaccid myelitis (AFM). Despite a significant disease burden, no approved antiviral drugs or vaccines are available for EV-D68. Due to its medical importance, EV-D68 is currently listed as a priority pathogen by the National Institute of Allergy and Infectious Diseases. Because the occurrence of the next EV-D68 outbreak cannot be predicted, the need for a potent, broadly acting, and safe EV-D68 inhibitor is urgent. The objectives of this proposal are to explore novel viral proteins as antiviral drug targets and to develop corresponding chemical probes as tools for target validation. This proposal is built upon our recent discovery that the EV-D68 2A protein exerts cysteine protease activity that is specific for the viral polyprotein VP1-2A junction and its protease activity can be inhibited by an FDA-approved oral drug, telaprevir. We further showed that telaprevir had submicromolar to low micromolar potency against several contemporary human EV-D68 strains from clades A and B in different human cell lines, including human neuronal cells. The antiviral potency of telaprevir (IC50) against EV-D68 was similar to that of HCV, indicating clinical relevance of repurposing telaprevir as an EV-D68 antiviral. The mechanism underlying the inhibition of the EV-D68 virus by telaprevir was independently elucidated using a serial viral passage experiment, leading to the selection of an N84T mutation near the active site of EV-D68 2Apro protein that reduced the potency of telaprevir in enzymatic and cellular antiviral assays. To validate the hypothesis that EV-D68 2Apro is essential for viral replication both in vitro and in vivo, our goals are to develop 2Apro inhibitors with favorable pharmacokinetic properties and to use them as chemical probes to validate EV-D68 2Apro as a viable antiviral drug target in cell culture as well as in a neonatal mouse model of EV-D68-induced paralytic disease. Specifically, we propose to design more potent and selective EV- D68 2Apro inhibitors through structure-activity relationship studies of telaprevir. All designed compounds will be systematically tested in in vitro enzymatic and cellular assays and profiled for pharmacokinetic properties. Prioritized lead compounds will be selected to test in EV-D68 mouse models. The expected outcomes of the proposed research are to validate EV-D68 2Apro as a viable antiviral drug target and demonstrate the in vivo antiviral efficacy of EV-D68 2Apro inhibitors.