Discovery of Enterovirus D68 2A protease inhibitors for antiviral therapy

摘要 肠道病毒D68(EV-D68)是一种高传染性的非脊髓灰质炎肠道病毒，导致2014年爆发，影响 数以千计的人来自多个国家，包括美国、加拿大和欧洲。从历史上看，EV的症状- D68感染包括鼻漏、肌肉疼痛和咳嗽。然而，在2014年疫情期间，有 涉及更严重呼吸道症状和住院的病例越来越多。另外，因为EV-D68 感染与急性弛缓性脊髓炎(AFM)病例增加同时发生，EV-D68被怀疑与 尽管需要更多的数据来了解它们之间的关系，但使用AFM。EV-D68目前 被列为NIAID的优先病原体之一。不幸的是，目前还没有FDA批准的药物或疫苗来治疗 肠道病毒D68感染的治疗。因此，医学上迫切需要更有效的疗法。 为EV-D68量身定做。该项目的总体目标是识别和开发小分子蛋白酶抑制剂。 EV-D68 2APro作为预防和/或治疗EV-D68感染的药物，最好也用于 高度相关的EV-A71病毒，它是导致手足口病的病毒，它的2APro共享 EV-D68与2APro的保守序列和催化三联体。我们的战略是解决未满足的医疗问题 需要寻找针对新鉴定的EV-D68酶活性的小分子抑制剂 蛋白水解酶，2Apro.方法是利用我们在2Apro和同质生化筛查方面的经验 利用FRET鉴定抑制2APro酶活性的小分子。在初步研究中，我们 建立了EV-D68 2Apro的FRET检测方法，并将其应用于中低通量EV-D68 2Apro的筛选 ~2000个Z‘因子≥为0.7的化合物。Pilot Screen确定替拉维韦为EV-D68 2APro的抑制剂 证实了对一组当代EV-D68毒株的抗病毒活性。在第一阶段，对于AIM 1，FRET HTS 将被优化并应用于不同的化学库，以识别抑制 2APro的酶活性。在目标2中，FRET二次检测、热位移检测和细胞CPE检测 将被优化并用于验证确认的命中，以确定结合亲和力，并在体外测量 对D68和A71血清型菌株的抗病毒活性。在目标3中，验证的命中根据他们的药物确定优先顺序- 将评估它们的ADME特性的相似性和抗病毒光谱。我们还将初步探索 按优先级排序的已验证命中的SAR。在第二阶段，我们将在化学上优化优先抑制剂的效力和 选择性、体外和体内药代动力学特性，并在动物感染模型中进行评估。

Abstract Enterovirus D68 (EV-D68), a highly contagious non-polio enterovirus, caused the 2014 outbreak affecting thousands of people in multiple countries, including the US, Canada and Europe. Historically, symptoms of EV- D68 infections include rhinorrhea, muscle aches and cough. However, during the 2014 outbreak, there were increasing cases involving more severe respiratory symptoms and hospitalization. Also, because the EV-D68 infections coincided with an increase of acute flaccid myelitis (AFM) cases, EV-D68 is suspected to be associated with AFM even though more data are needed to understand the relationship between them. EV-D68 is currently listed as one of NIAID’s priority pathogens. Unfortunately, there are no FDA-approved drugs or vaccines for the treatments of the EV-D68 infection. Therefore, there is an urgent medical need for more potent therapeutics tailored for EV-D68. The overall goal of this project is to identify and develop small molecule protease inhibitors of EV-D68 2Apro as prophylactics and/or therapeutics for EV-D68 infections and preferably also for infections of the highly related virus EV-A71, which is responsible for hand-foot-and-mouth disease and whose 2Apro shares a conserved sequence and catalytic triad with 2Apro of EV-D68. Our strategy is to address the unmet medical need by identifying small molecule inhibitors targeting the enzymatic activity of a newly characterized EV-D68 protease, 2Apro. The approach is to leverage our experience with 2Apro and homogeneous biochemical screens utilizing FRET to identify small molecules that inhibit the enzymatic activity of 2Apro. In Preliminary Studies, we developed a FRET assay for EV-D68 2Apro and have applied the assay in a low/medium-throughput screen of ~2000 compounds with Z’-factors ≥0.7. The pilot screen identified telaprevir as an inhibitor of EV-D68 2Apro with confirmed antiviral activity against a panel of contemporary EV-D68 strains. In Phase I, for Aim 1, an FRET HTS will be optimized and applied to diverse chemical libraries for the identification of small molecules that inhibit the enzymatic activity of 2Apro. In Aim 2, FRET secondary assays, a thermal shift assay and a cell-based CPE assay will be optimized and used to validate confirmed hits, to determine the binding affinity, and to measure in vitro antiviral activity against strains of serotypes D68 and A71. In Aim 3, validated hits prioritized based on their drug- likeness and antiviral spectrum will be evaluated for their ADME properties. We will also explore preliminary SAR of prioritized validated hits. In Phase II, we will chemically optimize priority inhibitors for potency and selectivity, in vitro and in vivo pharmacokinetic properties, and evaluate them in animal infection models.