Adamantane Drug Binding to Membrane-Bound Influenza A M2 Protein

描述(申请人提供)：这项研究的总体目标是了解金刚烷抗病毒药物如何与甲型流感M2蛋白结合。甲型流感病毒是一个重大的公共卫生问题，其每年的死亡人数和造成毁灭性大流行的可能性都是如此。尽管金刚烷胺药物作为抗病毒药物在临床上已经使用了40多年，但关于这些药物的作用方式仍然存在激烈的争论。甲型流感流行毒株现在基本上对金刚烷类药物产生了抗药性。然而，人们仍然对这些药物如何与非耐药株的M2蛋白结合，以及M2蛋白的结构信息是否可以促进下一代抗病毒药物的合理设计产生浓厚兴趣。以前发表的关于M2的生物物理工作大多集中在截短的M2多肽结构上，这些结构可溶解在洗涤剂或生理意义有限的双层中。为了消除由于使用M2片段而可能产生的伪影，以及由于非天然疏水环境造成的结构扭曲，本文提出的工作将使用重组成膜双层的全长蛋白质来反映甲型流感病毒粒子的脂组成。我们表征抗病毒结合位点的策略将依赖于位点定向自旋标签(SDSL)EPR实验，该实验旨在测量整个蛋白质中结合药物和自旋标记位点之间的距离。以最近发表的M2片段的高分辨率结构信息为指导，我们将制备一系列单位点自旋标记的M2蛋白。使用氚形式的抗病毒药物，将使用电子自旋回波包络调制(ESEEM)实验来测量药物上的氢核和M2蛋白上的自旋标记位置之间的距离。为了补充ESEEM的距离，将使用双重电子-电子共振(DER)实验来测量M2蛋白上自旋标记的位置和自旋标记的药物之间的距离。SDSL EPR研究的高灵敏度将允许测试一系列药物：肽：脂的比例，从而能够比以前更全面地表征金刚烷结合位点的位置(S)和亲和力(S)。一旦确定了结合位点位置，将收集一组SDSL EPR数据，并用于确定病毒模拟膜中全长M2蛋白的药物结合构象(S)。用于结构确定的SDSL EPR数据将包括自旋标记迁移率的模式、自旋-自旋耦合的距离限制以及对不同双层/水溶解度的顺磁性试剂的可及性。 公共卫生相关性：这项提案描述了旨在回答有关抗病毒药物如何与甲型流感M2蛋白结合的关键问题的实验。甲型流感病毒是一个重大的公共卫生问题，其每年的死亡人数和造成毁灭性大流行的可能性都是如此。对当前抗病毒药物日益增长的耐药性使充分了解药物靶点成为当务之急。

DESCRIPTION (provided by applicant): The overall goal of this research is to understand how adamantane antiviral drugs bind to the influenza A M2 protein. Influenza A virus is a major public health concern, both in its annual death toll and its potential to cause devastating pandemics. Although adamantane drugs have been in clinical use as antivirals for more than 40 years, there is still vigorous debate about how these drugs work. Circulating strains of influenza A are now largely resistant to the adamantane drugs. However, there is still intense interest is learning how these drugs bind to non-resistant strains of M2 protein and if structural information on the M2 protein can facilitate the rational design of the next generation of antiviral drugs. The majority of previously published biophysical work on M2 has focused on truncated M2 peptide constructs solubilixed in detergent or in bilayers of limited physiological relevance. To eliminate potential artifacts due to the use of fragments of M2 and distortion of the structure due to non-native hydrophobic environments, the work proposed here will use full-length protein reconstituted into membrane bilayers that reflect the lipid composition of influenza A virions. Our strategy for characterizing antiviral binding sites will rely on site-directed spin label (SDSL) EPR experiments designed to measure distances between bound-drug and spin-labeled sites throughout the protein. Using recently published high-resolution structural information on M2 fragments as a guide, we will prepare a series of single-site spin-labeled M2 proteins. Using deuterated forms of antiviral drugs, electron spin-echo envelope modulation (ESEEM) experiments will be used to measure distances between the deuterium nuclei on the drug and spin-labeled sites on the M2 protein. To complement the ESEEM distances, double electron-electron resonance (DEER) experiments will be used to measure distances between spin-labeled sites on the M2 protein and a spin-labeled drug. The high sensitivity of SDSL EPR studies will allow a range of drug:peptide:lipid ratios to be tested, enabling a fuller characterization of the location(s) and affinities of adamantane-binding site(s) than has been previously possible. Once binding site locations are determined, a battery of SDSL EPR data will be collected and used to determine drug-bound conformation(s) of the full length M2 protein in viral mimetic membranes. SDSL EPR data used in structure determination will include patterns of spin label mobilities, distance constraints from spin-spin couplings and accessibility to paramagnetic reagents of varying bilayer/aqueous solubility. PUBLIC HEALTH RELEVANCE: This proposal describes experiments designed to answer critical questions about how antiviral drugs bind to the influenza A M2 protein. Influenza A virus is a major public health concern, both in its annual death toll and its potential to cause devastating pandemics. Growing resistance to current antiviral drugs makes a full understanding of drug targets a priority.