Structural basis for influenza amantadine-resistance

描述（由申请人提供）：流感大流行长期以来一直是严重的全球健康威胁，在美国每年造成约20,000人死亡。甲型流感病毒和乙型流感病毒的小H+选择性通道M2和BM2在病毒复制中起关键作用，因此是抗流感治疗的有效靶点。金刚烷胺是一种获得许可的抗甲型流感药物，通过抑制M2通道的pH活化起作用。然而，许多M2变异体以及乙型流感的BM2通道可以逃脱金刚烷胺的作用，导致这些病毒对金刚烷胺产生耐药性。我们建议提供这些小通道的H+门控和药物相互作用的原子细节，以了解某些流感病毒株对金刚烷胺耐药的机制。首先我们将求解甲型流感病毒在pH 7.2封闭状态下M2 H+通道的高分辨率结构。本文设计了一种新的方法来促进溶液核磁共振结构测定，该方法结合了偶极耦合来获取取向参数和脂质/洗涤剂双层胶束，从而能够在近脂质双层环境中表征原子结构。我们还将求解pH值为5.2时开放状态下的通道结构，以描述与低pH活化相关的构象和动力学变化。为了了解金刚烷胺抑制的机制，我们将解决在药物结合存在下的通道结构，包括封闭和开放。还将确定通道中金刚烷胺相互作用的位点。与此同时，已知的抗金刚烷胺M2变异的结构将被直接比较，以阐明通道阻断的机制。BM2通道也将进行类似的研究，它的功能与M2相似，但对金刚烷胺具有抗性。不阻断通道的潜在金刚烷胺- bm2相互作用的结构细节也将被探索。这一结果将为我们寻找一种真正的用于抗流感治疗的H+通道阻滞剂的长期目标奠定坚实的基础。

DESCRIPTION (provided by applicant): The influenza pandemics have long been a serious global health threat responsible for approximately 20,000 deaths per year in the US. It has been established that small H+ selective channels M2 and BM2 from influenza A and B virus play a critical role in viral replication and thus are effective targets for anti-influenza therapy. Amantadine is a licensed anti-influenza A drug that functions by inhibiting the pH activation of the M2 channel. However a number of M2 variants, as well as the BM2 channel of influenza B can escape the effect of amantadine, leading to the amantadine-resistance of these viruses. We propose to provide the atomic details of H+ gating and drug interaction of these small channels for understanding the mechanisms by which some influenza strains are resistant to amantadine. Initially we will solve the high resolution structure of the M2 H+ channel of influenza A virus in the closed state at pH 7.2. A new method has been devised to facilitate structure determination by solution NMR, which incorporates the dipolar couplings for acquiring orientation parameters and lipid/detergent bilayered micelles to enable characterization of atomic structures in a near lipid-bilayer environment. We will also solve the channel structure in the open state at pH 5.2 to delineate changes in conformation and dynamics that are coupled to low-pH activation. To understand the mechanism of amantadine inhibition, we will solve the channel structures, both closed and open, in the presence of drug binding. The sites of amantadine interaction in the channel will also be located. In parallel, the structures of the known amantadine-resistant variants of M2 will be examined for direct comparison to elucidate mechanisms of channel blocking. Similar studies will be done for the BM2 channel, which functions similarly to M2 but is resistant to amantadine. Structural details of the potential amantadine-BM2 interaction that does not block the channel will also be explored. The results will lay a solid foundation for our long- range goal of finding a true blocker of H+ channels for anti-influenza treatment.