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

项目总结 肠道病毒属包括许多具有医学和社会经济意义的人类病原体，例如 脊髓灰质炎病毒、柯萨奇病毒、EV-A71、EV-D68和鼻病毒。非脊髓灰质炎肠道病毒，如EV-A71 和EV-D68特别令人关注，因为它们是手足口病的病原体 (手足口病)，中度到严重的呼吸系统疾病，在极少数情况下，中枢神经系统(CNS)感染 人类。EV-A71和EV-D68都被列为NIH NIAID优先病原体。尽管有重大的 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 和EV-D68感染。因此，这项拟议的研究的目标是确定病毒2A型蛋白酶抑制剂 通过高通量筛选(HTS)作为有效的EV-A71和EV-D68抗病毒药物。 EV-A71和EV-D68 2A型蛋白水解酶通过裂解病毒在病毒复制中发挥重要作用 病毒转录后的多蛋白，启动病毒复制。EV-A71和EV-D68 2A型蛋白酶代表 未探索的新型抗病毒药物靶点-只有三种弱的或混杂的EV-A71 2Apro抑制剂被 文献中未见EV-D68 2APro抑制剂的报道。EV-A71和EV-D68 2Apro抑制剂的鉴定 通过HTS，我们开发了基于荧光共振能量转移(FRET)的两种酶分析方法 EV-A71和EV-D68 2Apro蛋白。随后使用EV-D68 2APro FRET试验进行 针对Selleckchem生物活性化合物文库(1902种化合物)的试点筛选，导致 TELAPREVR(VX-950)为第一个EV-D68 2APro抑制剂，二次细胞IC50为0.2微米 培养试验表明，替拉维韦对几种当代人类EV-D68具有亚到低的微摩尔效力 不同人类细胞系中的菌株。初步筛选的成功表明，鉴定2APro是可行的 通过基于FRET的HTS将抑制剂作为EV抗病毒药物。 由于EV-A71和EV-D68都会引起呼吸道感染，在极少数情况下还会引起神经系统感染，因此 需要血脑屏障(BBB)不通透性和BBB通透性的2APro抑制剂。BBB-不透水 2腺苷酶抑制剂是治疗呼吸道感染和预防病毒感染最需要的药物。 扩散到中枢神经系统。相反，神经疾病的治疗需要血脑屏障通透性的2APro抑制剂。 病毒通过病毒血症扩散到脊髓时的感染。此外，由于EV-A71和EV-D68 2A型 蛋白水解酶有~60%的序列相似性，我们希望找到针对EV-A71 2APro或EV-2的抑制剂。 D68 2Apro或两者兼而有之，进行两种基于FRET的酶促高温合成试验-一种是EV-A71 2Apro，一种是EV D68 2Apro. 该项目的预期结果是鉴定出一流的EV-A71和EV-D68 2Apro抑制剂 具有有效的作用机制和类似药物的特性。这项拟议的研究意义重大，因为它 代表着朝着解决未得到满足的EV-A71和EV-D68抗病毒药物的医疗需求迈进了一步。

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.