Targeting Viroporins and Coronavirus M Protein

项目3：靶向VIROPORINS和冠状病毒M蛋白 摘要 冠状病毒表达四种结构蛋白：棘蛋白(S)、膜蛋白(M)、包膜蛋白(E)和核衣壳蛋白(N)， 它们对于有效的病毒颗粒形成是必不可少的。我们试图理解和描述E的结构特征 和M，并利用这些信息设计抗病毒药物。M是一种分子支架，它将 结构蛋白，E是病毒孔蛋白家族的成员，它们作为离子通道来控制 寄主和病毒中的离子组成。此前，洪和德格拉多解决了病毒孔蛋白的结构 来自A型流感病毒AM2，并利用这一信息设计了解决以下问题的新药 抵抗。我们现在把注意力转向SARS-CoV-2的E蛋白。此外，我们将确定 其他多种冠状病毒和Togaviridae家族甲病毒的病毒孔蛋白的结构 基于结构的蛋白质设计。M是病毒颗粒组装所必需的。我们将从结构上描述M 单独和与病毒相互作用的伙伴在囊泡和病毒样颗粒(VLP)中。这项工作将阐明 它们在稳定病毒和产生发芽所需的膜曲率方面起到了作用。我们将使用这个 信息加上高通量筛选(HTS)，以发现针对M。 更具体地说，在目标1中，我们将使用X射线对E蛋白和药物复合体进行结构表征 结晶学、低温电子显微镜和固体核磁共振。我们还将确定E蛋白的结构从多个 感染人类的甲型冠状病毒和乙型冠状病毒谱系。在目标2中，我们将确定冷冻-EM和 在脂质体、VLP和病毒粒子中，M单独的晶体结构和与病毒蛋白伙伴相关的晶体结构。这个 在这一目标中确定的高分辨率结构将使基于结构的药物设计成为可能。同时，我们将使用 一种简便的VLP分析方法，用于定位M和E中对包装和进入至关重要的残留物。VLP测定法 也将针对HTS进行配置。在目标3中，我们将把我们的研究扩展到甲型病毒的病毒孔蛋白。 Togaviridae科，包括基孔肯雅(CHIKV)、辛比斯(SINV)、塞姆利基森林(SFV)和罗斯 河(RRV)病毒。这些病毒含有病毒孔蛋白，这是在体内有效复制所必需的。vbl.使用 方法在目标1中，我们将探索它们的结构和开发抑制剂，以促进药物发现。 在目标4中，我们将开发针对E和M的优化先导化合物，以便转移到罗氏。当前 对E有微弱抑制作用的化合物，包括金刚烷胺和六亚甲基阿米洛利(HMA)， 为设计更高特异性和亲和力的化合物提供起点。同时，我们将使用HTS，基于 对VLP和酵母菌的通道功能进行筛选，为药物先导化合物的优化奠定基础。以年为单位 2.5到5，我们将设计针对M的小分子。总体而言，我们的目标是开发优化的口服生物利用度。 在细胞检测和体内活性测定中，以50 mg/kg的剂量以IC50和100 nM领先靶向E和M。

PROJECT 3: TARGETING VIROPORINS AND CORONAVIRUS M PROTEIN SUMMARY Coronaviruses express four structural proteins; spike (S), membrane (M), envelope (E) and nucleocapsid (N), which are essential for efficient viral particle formation. We seek to understand and structurally characterize E and M, and to use this information to design antiviral drugs. M is a molecular scaffold that brings together the structural proteins, and E is a member of the viroporin family of proteins, which act as ion channels to control ionic composition in the host and virus. Previously, Hong and DeGrado solved the structures of the viroporins from influenza A virus, AM2, and used this information to design novel drugs that address the problem of resistance. We now have turned our attention to the E protein of SARS-CoV-2. Furthermore, we will determine structures of viroporins from a variety of other coronaviruses and alphaviruses of the Togaviridae family to enable structure-based protein design. M is essential for virus particle assembly. We will structurally characterize M alone and with its viral interacting partners in vesicles and virus-like particles (VLPs). This work will elucidate their role in stabilizing the virus and generating membrane curvature required for budding. We will use this information plus high-throughput screening (HTS) to discover drugs targeting M. More specifically, in Aim 1, we will structurally characterize the E protein and drug complexes using X-ray crystallography, Cryo-EM and solid-state NMR. We will also determine structures of E proteins from multiple alphacoronavirus and betacoronavirus lineages that infect humans. In Aim 2, we will determine Cryo-EM and crystal structures of M alone and in association with viral protein partners, in liposomes, VLPs and virions. The high-resolution structures determined in this aim will enable structure-based drug design. In parallel, we will use a convenient VLP assay to map residues in M and E that are essential for packaging and entry. The VLP assay will also be configured for HTS. In Aim 3, we will expand our studies to viroporins of alphaviruses of the Togaviridae family, which include the chikungunya (CHIKV), Sindbis (SINV), Semliki Forest (SFV), and Ross River (RRV) viruses. These viruses have viroporins, which are essential for effective replication in vivo. Using methods in Aim 1, we will explore their structures and develop inhibitors to facilitate drug discovery. In Aim 4, we will develop Optimized Lead compounds targeting E and M for transfer to Roche. Current compounds that show weak inhibition of E, which include amantadine and hexamethylene amiloride (HMA), provide starting points for design of higher specificity and affinity compounds. In parallel, we will use HTS, based on VLPs and a yeast screen of channel function to develop starting points for optimization of drug leads. In years 2.5 to 5, we will design small molecules to target M. Overall, we aim to develop orally bioavailable Optimized Leads that target E and M with IC50 < 100 nM in cellular assays and in vivo activity at dose <50 mg/kg.