Developing capsid-importin alpha inhibitors for the treatment of VEEV infection

摘要 委内瑞拉马脑炎病毒（VEEV）感染人类，并被分类为B类病原体， 由于其易于传播，在人类中，VEEV感染可导致炎症、急性退行性变 神经元改变、行为改变、记忆丧失和癫痫发作，严重者导致死亡。那里 目前还没有针对感染VEEV的患者的抗病毒治疗。TC 83减毒活疫苗 可以防止VEEV感染，但由于不良副作用的频率很高，其使用仅限于 军事和危险实验室人员。因此，迫切需要发现新的治疗方法。VEEV 能够通过至少部分由于其衣壳而阻断细胞核运输来抑制宿主转录 与宿主蛋白输入蛋白α/β（Impα/β1）和CRM 1形成复合物的蛋白质。核突变 衣壳的定位序列（NLS）导致病毒毒力丧失，表明衣壳进入细胞的能力降低。 该细胞核对于VEEV的发病机理是关键的，并且是抗病毒治疗开发的可行靶点。我们 假设干扰capture-Imp α蛋白-蛋白相互作用（PPI）小分子抑制剂将 防止VEEV引起的病理。我们已经鉴定了两种新的小分子，1111684和G281-1485， 破坏VEEV衣壳与Impα相互作用的能力，导致衣壳定位改变， 病毒复制和宿主细胞的存活增加。在这里，我们建议设计和合成第二- 新一代capture-Imp α PPI抑制剂具有更高的效力和生物利用度。为此，我们提出了4 相互关联的目标：目标1：第二代capture-Imp α抑制剂的计算机设计;目标2：合成和 第二代capture-Imp α抑制剂的生化表征;目的3：测定体外 衣壳的选择性指数和生物利用度：Impα抑制剂;以及目的4：确定 衣壳：Impα抑制剂。从我们的研究中获得的新知识将适用于广泛的应用 涉及capture-Imp α相互作用，包括HSV-1或东部和西部马脑炎病毒。

ABSTRACT Venezuelan equine encephalitic virus (VEEV) infects humans and is classified as a Category B pathogen by NIAID due to its easy dissemination. In humans, VEEV infection can result in inflammation, acute degenerative neuronal changes, behavioral changes, memory loss and seizures, with severe cases resulting in death. There is currently no antiviral therapeutic treatment for patients infected with VEEV. The live attenuated vaccine TC83 can protect against VEEV infection, but due to a high frequency of adverse side effects, its use is limited to military and at-risk laboratory personnel. Therefore, the discovery of new therapeutics is urgently needed. VEEV is able to suppress host transcription by blocking cellular nuclear trafficking at least partially due to its capsid protein forming a complex with the host proteins importinα/β (Impα/β1) and CRM1. Mutation of the nuclear localization sequence (NLS) of capsid results in loss of viral virulence, indicating that the ability of capsid to enter the nucleus is critical for VEEV pathogenesis and a viable target for antiviral therapeutic development. We hypothesize that small molecule inhibitors that interfere with capsid-Impα protein-protein interaction (PPI) will prevent VEEV induced pathologies. We have identified two novel small molecules, 1111684 and G281-1485, which disrupt the ability of VEEV capsid to interact with Impα, leading to altered capsid localization, decreased viral replication and increased survival of the host cell. Here we propose to design and synthesize second- generation capsid-Impα PPI inhibitors with improved potency and bioavailability. To this end, we proposed 4 interlinked aims: Aim 1: In silico design of second-generation capsid-Impα inhibitors; Aim 2: Synthesis and biochemical characterization of second generation capsid-Impα inhibitors; Aim 3: Determine the in vitro selectivity index and bioavailability of capsid:Impα inhibitors; and Aim 4: Determine the PK/PD/Tox of capsid:Impα inhibitors. The new knowledge gained from our study will be applicable to wide-range applications involving capsid-Impα interactions including HSV-1 or eastern and western equine encephalitic viruses.