Targeting Viroporins and Coronavirus M Protein

靶向病毒孔蛋白和冠状病毒 M 蛋白

• 批准号: 10512629
• 负责人: WILLIAM DEGRADO
• 金额: $ 384.67万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10512629
• 关键词: 2019-nCoV; Address; Affinity; Alphavirus; Amantadine; Amiloride; Antiviral Agents; Attention; Biochemistry; Biological Assay; Cellular Assay; Chikungunya virus; Collaborations; Communicable Diseases; Complex; Coronavirus; Cryoelectron Microscopy; Crystallization; Dose; Drug Design; Drug Targeting; Electrophysiology (science); Family; Hand; Head; Human; In Vitro; Influenza A virus; Ion Channel; Letters; Liposomes; Maps; Mass Spectrum Analysis; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Nucleocapsid; Nucleocapsid Proteins; Oral; Pathogenicity; Pathology; Pharmaceutical Chemistry; Pharmaceutical Preparations; Production; Protein Engineering; Protein Family; Proteins; Proteomics; Resistance; Resolution; Role; Ross river virus; Specificity; Structural Protein; Structure; Therapeutic Index; Time; Togaviridae; Vesicle; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus-like particle; Work; X-Ray Crystallography; Yeasts; analytical ultracentrifugation; base; betacoronavirus; chikungunya; conformer; crosslink; design; drug development; drug discovery; env Gene Products; forest; high throughput screening; in vitro Assay; in vivo; inhibitor; lead optimization; member; novel therapeutics; pandemic disease; particle; programs; rational design; response; scaffold; screening; simulation; small molecule; solid state nuclear magnetic resonance; structural biology; virology

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.

项目 3：针对病毒孔蛋白和冠状病毒 M 蛋白 概括 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)， 这对于有效的病毒颗粒形成至关重要。我们寻求理解 E 并从结构上表征 E 和M，并利用这些信息来设计抗病毒药物。 M是一个分子支架，将 结构蛋白，E 是病毒孔蛋白家族的成员，充当离子通道来控制 宿主和病毒中的离子组成。此前，Hong 和 DeGrado 解析了病毒孔蛋白的结构 来自甲型流感病毒 AM2，并利用这些信息设计了解决以下问题的新药： 反抗。现在我们将注意力转向 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，使 基于结构的蛋白质设计。 M 对于病毒颗粒组装至关重要。我们将从结构上表征 M 单独存在以及与囊泡和病毒样颗粒（VLP）中的病毒相互作用伙伴一起存在。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）来发现针对支原体的药物。 更具体地说，在目标 1 中，我们将使用 X 射线对 E 蛋白和药物复合物进行结构表征 晶体学、冷冻电镜和固态核磁共振。我们还将确定多种 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 和 脂质体、VLP 和病毒粒子中单独的 M 以及与病毒蛋白伴侣相关的 M 的晶体结构。这 为此目的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 一种方便的 VLP 测定，可绘制 M 和 E 中对于包装和输入至关重要的残基。 VLP 检测 还将针对 HTS 配置。在目标 3 中，我们将把我们的研究扩展到甲病毒的病毒孔蛋白。 披膜病毒科，包括基孔肯雅病毒 (CHIKV)、辛德比病毒 (SINV)、塞姆利基森林病毒 (SFV) 和罗斯病毒 河流 (RRV) 病毒。这些病毒具有病毒孔蛋白，这对于体内有效复制至关重要。使用 按照目标 1 中的方法，我们将探索其结构并开发抑制剂以促进药物发现。 在目标 4 中，我们将开发针对 E 和 M 的优化先导化合物，并转让给罗氏。当前的 对 E 表现出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 和通道功能的酵母筛选进行研究，以开发优化药物先导化合物的起点。几年内 2.5到5，我们将设计针对M的小分子。总体而言，我们的目标是开发口服生物利用度优化的 在细胞测定中和剂量 <50 mg/kg 的体内活性中，靶向 E 和 M 的先导化合物的 IC50 < 100 nM。