Targeting Viroporins and Coronavirus M Protein

项目3：靶向病毒孔蛋白和冠状病毒M蛋白 总结 冠状病毒表达四种结构蛋白：刺突（S）、膜（M）、包膜（E）和核衣壳（N）， 其对于有效的病毒颗粒形成是必需的。我们试图理解和结构特征E 和M，并利用这些信息来设计抗病毒药物。M是一种分子支架， 结构蛋白，E是蛋白质的病毒孔蛋白家族的成员，其作为离子通道来控制 离子组成在宿主和病毒。在此之前，Hong和DeGrado解决了病毒孔蛋白的结构 从A型流感病毒AM 2中分离，并利用这些信息设计新的药物， 阻力我们现在把注意力转向SARS-CoV-2的E蛋白。此外，我们将确定 来自披膜病毒科的多种其他冠状病毒和甲病毒的病毒孔蛋白的结构， 基于结构蛋白质设计M是病毒颗粒组装所必需的。我们将从结构上描述M 单独以及与其在囊泡和病毒样颗粒（VLP）中的病毒相互作用伴侣。这项工作将阐明 它们在稳定病毒和产生出芽所需的膜曲率方面的作用。我们将使用这个 信息加上高通量筛选（HTS），以发现靶向M的药物。 更具体地说，在目标1中，我们将使用X-射线结构表征E蛋白和药物复合物 晶体学，冷冻电镜和固态核磁共振。我们还将确定E蛋白的结构， 感染人类的α冠状病毒和β冠状病毒谱系。在目标2中，我们将确定Cryo-EM， 在脂质体、VLP和病毒体中，单独的M和与病毒蛋白伴侣结合的M的晶体结构。的 在此目的中确定的高分辨率结构将使得能够进行基于结构的药物设计。同时，我们将使用 一种方便的VLP测定法，用于绘制包装和进入所必需的M和E中的残基。VLP测定 也将为HTS配置。在目标3中，我们将扩大我们的研究，以病毒孔蛋白的甲病毒， 披膜病毒科家族，包括基孔肯雅病毒（CHIKV）、辛德毕斯病毒（SINV）、塞姆利基森林病毒（SFV）和罗斯病毒（Ross 河（RRV）病毒。这些病毒具有病毒孔蛋白，其对于体内有效复制是必需的。使用 方法，我们将探索它们的结构并开发抑制剂以促进药物发现。 在目标4中，我们将开发靶向E和M的优化先导化合物，以转移给罗氏。电流 显示出对E弱抑制的化合物，包括金刚烷胺和氨氯地平（HMA）， 为设计更高特异性和亲和力的化合物提供了起点。同时，我们将使用HTS，基于 对VLP和酵母筛选通道功能，以开发药物先导物优化的起点。多年来 2.5到5，我们将设计小分子来靶向M。总的来说，我们的目标是开发口服生物利用度优化 在细胞测定中靶向E和M的先导化合物的IC 50 < 100 nM，在剂量<50 mg/kg时具有体内活性。

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.