Structure of M2 Proton Channel from Influenza A Virus

甲型流感病毒 M2 质子通道的结构

• 批准号: 8725174
• 负责人: WILLIAM DEGRADO
• 金额: $ 36.13万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8725174
• 关键词: Address; Amantadine; Amantadine resistance; Amines; Ammonium; Antibodies; Bacteriophages; Binding; Biological Warfare; Cell surface; Charge; Chemical Structure; Complex; Diffuse; Drug Design; Drug Targeting; Drug resistance; Environment; Funding; Future; Generations; Health; Homo; Human; Influenza A virus; Influenza B virus; Investigation; Label; Length; Membrane; Membrane Proteins; Micelles; Modeling; Molecular Conformation; Motion; Mutation; Nature; Neuraminidase; Oseltamivir; Pharmaceutical Preparations; Phase; Phospholipids; Process; Protein Dynamics; Proteins; Protons; Resistance; Resolution; Rimantadine; Risk; Site; Solutions; Spin Labels; Structure; Testing; Therapeutic antibodies; Transmembrane Domain; Variant; Vesicle; Viral; Viral Proteins; Virus; Water; Work; anti-influenza; anti-influenza drug; aqueous; base; design; hydronium ion; inhibitor/antagonist; insight; interfacial; mutant; nanodisk; pathogen; protein structure; protonation; public health relevance; response; small molecule

DESCRIPTION (provided by applicant): The M2 proton channel protein of the influenza A virus is the target of the anti-influenza drug, amantadine. M2 contains a single transmembrane domain that forms the homo-tetrameric pore of this channel. M2's small size and simple structure makes it an attractive model for understanding the mechanism of charge-stabilization and proton conduction through membrane proteins. In the previous period, high-resolution NMR and crystal structures were solved to elucidate the mechanism of proton conduction. The channel has a long, water-filled pore that leads to a selectivity filter defined by His37 and Trp41 Protons diffuse through this aqueous pore to bind at the His37 tetrad and open the Trp41 gate. To understand the structural basis for this process, structures will be solved in the next funding period. Influenza A virus is a major threat to human health. There are two different classes of approved anti-influenza drugs: amantadine and rimantadine target the M2 proton channel, while Tamiflu (oseltamivir) and related compounds target neuraminidase. Resistance to both classes of drugs poses a major problem, and most isolates of influenza A virus are now amantadine-resistant. We therefore are solving structures of amantadine and rimantadine complexes with M2, and drug-resistant mutants. Drug binds to M2 by targeting its ammonium to one of the three low energy hotspot sites aligned along the channel axis. Only a handful of mutations are tolerated at these sites in transmissible viruses. Here, hypothesis-directed and structure-based approaches are used to design new inhibitors of this set of resistant mutants. This endeavor will not only provide new leads for anti-influenza medications, but it should also advance structure-based design of drugs targeting membrane proteins - an increasingly important endeavor as the number of medicinally important membrane protein structures grows. In Aim 1, hypothesis-directed structural approaches are used to discover small molecules that inhibit relevant mutants of M2. In Aim 2, EPR, crystallographic, and NMR investigations will probe the mechanism of conduction and drug inhibition. Site-directed spin labeling will focus on the full-length protein in phospholipid vesicles, and evaluate conformational changes due to variations in pH and drug-binding. These studies will facilitate understanding of more high-resolution crystal structures and NMR structures. Crystallographic structures of M2 mutants will be solved with and without bound drugs to inform drug design in Aim 1 and also probe the mechanism of proton conduction. Our early crystallographic work was conducted with the isolated transmembrane domain in micelles; the membrane environment as well as missing domains might influence the structure. Thus, we are now crystallizing longer constructs from both bicelles and lipidic cubic phases. To help stabilize these constructs in their native conformations, we are solving structures with known therapeutic antibodies and antibodies selected on phage. Finally we will structurally characterize the proton channel of influenza B virus, BM2, which shows no sequence similarity to M2 aside from having a His-X3-Trp motif. These studies will also enable future structure-based drug design of BM2 inhibitors. In parallel we will conduct solution NMR studies in micelles, bicelles, and nanodisks. We combine biosynthetic and synthetic labeling strategies to facilitate structure determination and to increase the resolution of solution NMR structures. These studies will provide new insight into the mechanism of proton conduction through M2 and lay the groundwork for the design of new inhibitors.

描述（由申请方提供）：甲型流感病毒的M2质子通道蛋白是抗流感药物金刚烷胺的靶点。M2含有形成该通道的同源四聚体孔的单个跨膜结构域。M2的小尺寸和简单的结构使其成为理解电荷稳定和质子传导通过膜蛋白的机制的有吸引力的模型。在前一阶段，高分辨率NMR和晶体结构被解决，以阐明质子传导的机制。该通道具有长的充满水的孔，其导致由His 37和Trp 41限定的选择性过滤器。质子通过该水性孔扩散以结合His 37四分体并打开Trp 41门。为了了解这一进程的结构基础，将在下一个供资期解决结构问题。甲型流感病毒是对人类健康的主要威胁。有两种不同类别的批准抗流感药物：金刚烷胺和金刚乙胺靶向M2质子通道，而达菲（奥司他韦）和相关化合物靶向神经氨酸酶。对这两类药物的耐药性是一个主要问题，大多数甲型流感病毒分离株现在都对金刚烷胺有耐药性。因此，我们正在解决金刚烷胺和金刚乙胺与M2和耐药突变体的复合物的结构。药物通过将其铵靶向于沿通道轴沿着排列的三个低能热点位点之一来与M2结合。在可传播病毒的这些位点上，只有少数突变是可以容忍的。在这里，假设导向和基于结构的方法被用来设计这组耐药突变体的新抑制剂。这一奋进不仅将为抗流感药物提供新的线索，而且还应该推进靶向膜蛋白的药物的基于结构的设计-随着医学上重要的膜蛋白结构数量的增加，这一奋进越来越重要。在目标1中，假设导向的结构方法用于发现抑制M2相关突变体的小分子。在目标2中，EPR，晶体学和NMR研究将探索传导和药物抑制的机制。定点自旋标记将集中在磷脂囊泡中的全长蛋白质上，并评估由于pH和药物结合的变化而引起的构象变化。这些研究将有助于理解更高分辨率的晶体结构和NMR结构。M2突变体的晶体结构将在有和没有结合药物的情况下得到解决，以告知Aim 1中的药物设计，并探索质子传导的机制。我们早期的晶体学工作是用胶束中分离的跨膜结构域进行的;膜环境以及缺失的结构域可能会影响结构。因此，我们现在从双胞和双胞立方相结晶更长的结构。为了帮助稳定这些构建体在其天然构象，我们正在解决与已知的治疗抗体和噬菌体上选择的抗体的结构。最后，我们将在结构上表征流感B病毒BM 2的质子通道，其除了具有His-X3-Trp基序之外与M2没有序列相似性。这些研究还将使未来基于结构的BM 2抑制剂药物设计成为可能。同时，我们将在胶束，bicelles和nanodisks进行溶液NMR研究。我们将联合收割机生物合成和合成标记策略相结合，以促进结构测定并提高溶液NMR结构的分辨率。这些研究将为M2质子传导机制提供新的见解，并为设计新的抑制剂奠定基础。