High Throughput Screening of Inhibitors Targeting the Enterovirus A71 and D68 2A proteases

项目摘要 肠道病毒属包括许多医学和社会经济学上重要的人类病原体，例如 脊髓灰质炎病毒、柯萨奇病毒、EV-A71、EV-D 68和鼻病毒。非脊髓灰质炎肠道病毒（NPEV），如EV-A71 和EV-D 68是特别令人关注的，因为它们是手足口病的病原体 手足口病（HFMD）、中度至重度呼吸道疾病，以及在极少数情况下， 人类EV-A71和EV-D 68均被NIH NIAID列为优先病原体。尽管一个重要的 疾病负担，目前没有批准的抗病毒药物可用于预防和治疗EV-A71 EV-D 68感染。因此，拟议研究的目标是鉴定病毒2A蛋白酶抑制剂 作为有效的EV-A71和EV-D 68抗病毒药物通过高通量筛选（HTS）。 EV-A71和EV-D 68 2A蛋白酶在病毒复制中起重要作用，其通过切割病毒载体来实现。 病毒转录后的多聚蛋白，启动病毒复制。EV-A71和EV-D 68 2A蛋白酶代表 尚未探索的新型抗病毒药物靶点-只有三种弱或混杂的EV-A71 2Apro抑制剂已被 报道了EV-D 68 2Apro抑制剂，文献中没有EV-D 68 2Apro抑制剂。鉴定EV-A71和EV-D 68 2Apro抑制剂 通过HTS，我们开发了基于荧光共振能量转移（FRET）的酶测定法， EV-A71和EV-D 68 2Apro蛋白。随后使用EV-D 68 2Apro FRET测定进行免疫组化。 针对Selleckchem生物活性化合物库（1902种化合物）进行中试筛选， Telaprevir（VX-950）被鉴定为第一个EV-D 68 2Apro抑制剂，IC 50为0.2 μM。在二次电池中 在培养试验中，特拉匹韦显示出对几种当代人EV-D 68的亚微摩尔至低微摩尔效力 在不同的人类细胞系中。中试筛选的成功表明，鉴定2Apro是可行的 通过FRET为基础的HTS作为EV抗病毒药的抑制剂。 由于EV-A71和EV-D 68引起呼吸道感染，在极少数情况下，还引起神经系统感染， 需要血脑屏障（BBB）不可渗透的和BBB可渗透的2Apro抑制剂。BBB不渗透 2Apro抑制剂是治疗呼吸道感染以及预防病毒感染最需要的药物。 扩散到中枢神经系统相比之下，需要BBB可渗透的2Apro抑制剂用于治疗神经系统疾病。 当病毒通过病毒血症扩散到脊髓时的感染。此外，作为EV-A71和EV-D 68 2A 蛋白酶共享~60%的序列相似性，我们期望鉴定靶向EV-A71 2Apro或EV-A71 2Apro的抑制剂。 D 68 2Apro或两者，通过进行两种基于FRET的酶促HTS测定-一种用于EV-A71 2Apro，一种用于EV-A71 2Apro。 D68 2Apro. 该项目的预期成果是鉴定出一流的EV-A71和EV-D 68 2Apro抑制剂 具有经验证的作用机制和药物样性质。这项研究意义重大，因为它 这代表着朝着解决EV-A71和EV-D 68抗病毒药物未满足的医疗需求迈出了一步。

PROJECT SUMMARY The enterovirus genus includes many medically and socioeconomically important human pathogens such as poliovirus, coxsackievirus, EV-A71, EV-D68, and rhinoviruses. Non-polio enteroviruses (NPEV) such as EV-A71 and EV-D68 are of particular concern as they are causative pathogens for hand, foot, and mouth disease (HFMD), moderate to severe respiratory illness and, in rare cases, central nervous system (CNS) infections in humans. Both EV-A71 and EV-D68 are listed among the NIH NIAID priority pathogens. Despite a significant disease burden, no approved antiviral drugs are currently available for the prevention and treatment of EV-A71 and EV-D68 infection. Accordingly, the goal of the proposed research is to identify viral 2A protease inhibitors as potent EV-A71 and EV-D68 antivirals through high-throughput screening (HTS). The EV-A71 and EV-D68 2A proteases play an essential role in viral replication by cleaving the viral polyprotein after viral transcription, initiating viral replication. The EV-A71 and EV-D68 2A proteases represent unexplored novel antiviral drug targets—only three weak or promiscuous EV-A71 2Apro inhibitors have been reported and there is no EV-D68 2Apro inhibitor in the literature. To identify EV-A71 and EV-D68 2Apro inhibitors through HTS, we developed fluorescence resonance energy transfer (FRET)-based enzymatic assays for both the EV-A71 and EV–D68 2Apro proteins. The EV-D68 2Apro FRET assay was subsequently used to conduct a pilot screening against the Selleckchem Bioactive compound library (1902 compounds), leading to the identification of telaprevir (VX-950) as the first EV-D68 2Apro inhibitor with an IC50 of 0.2 µM. In secondary cell culture assays, telaprevir showed sub- to low micromolar potency against several contemporary human EV-D68 strains in different human cell lines. The success of the pilot screening suggests that it is feasible to identify 2Apro inhibitors as EV antivirals through FRET-based HTS. As EV-A71 and EV-D68 cause both respiratory infection and, in rare cases, neurological infection, there is a need for both blood-brain barrier (BBB)-impermeable and BBB-permeable 2Apro inhibitors. BBB-impermeable 2Apro inhibitors are the most needed for the treatment of respiratory infection as well as the prevention of viral spread to the CNS. In contrast, BBB-permeable 2Apro inhibitors are needed for the treatment of neurological infection when virus has spread to the spinal cord through viremia. In addition, as the EV-A71 and EV-D68 2A proteases share ~60% sequence similarity, we expect to identify inhibitors that target either EV-A71 2Apro or EV- D68 2Apro or both by conducting two FRET-based enzymatic HTS assays—one for EV-A71 2Apro and one for EV- D68 2Apro. The expected outcome of this project is the identification of first-in-class EV-A71 and EV-D68 2Apro inhibitors with validated mechanisms of action and drug-like properties. The proposed study is significant because it represents a step forward towards addressing the unmet medical need for EV-A71 and EV-D68 antivirals.