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，被列为B类病原体 NIAID，因为它很容易传播。在人类中，VEEV感染可导致炎症、急性退行性变 神经元变化、行为变化、记忆丧失和癫痫发作，严重情况下会导致死亡。那里 目前没有针对感染VEEV的患者的抗病毒治疗。TC83减毒活疫苗 可以预防VEEV感染，但由于副作用频率高，其使用仅限于 军方和处于危险中的实验室人员。因此，迫切需要发现新的治疗方法。Veev 能够通过阻止细胞核运输来抑制宿主转录，至少部分是由于其衣壳 与宿主蛋白Importinα/β(Impα/β1)和CRM1形成复合体的蛋白质。核的突变 衣壳的定位序列(NLS)导致病毒毒力的丧失，表明衣壳进入病毒的能力 核蛋白在VEEV的发病机制中起着关键作用，也是抗病毒治疗发展的有效靶点。我们 假设干扰衣壳-Impα蛋白质-蛋白质相互作用的小分子抑制剂将 预防VEEV诱发的病理改变。我们鉴定了两个新的小分子，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，减少 病毒复制和提高宿主细胞的存活率。在这里，我们建议设计和合成第二个- 新一代衣壳-ImpαPPI抑制剂具有更高的效力和生物利用度。为此，我们提出了4项 相关目标：目标1：第二代衣壳-Impα抑制剂的电子设计；目标2：合成和 第二代衣壳IMPα抑制剂的生化特征；目的3：体外测定 衣壳：IMPα抑制剂的选择性指数和生物利用度；以及目标4：测定PK/PD/TOX 衣壳类：IMPα抑制剂。我们从研究中获得的新知识将适用于广泛的应用 涉及衣壳与IMP的α相互作用，包括单纯疱疹病毒1型或东西方马脑炎病毒。

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.