Understanding and targeting the Methionine-Aromatic motif in oxidized alpha-Synuclein

摘要 帕金森病（PD）的一个显著特征是神经毒性α-突触核蛋白（α-Syn）聚集成淀粉样蛋白 组件.尽管二十多年来一直在努力寻找直接干扰αSyn的抑制剂， 在聚集过程中，没有成功的化合物到达临床。帕金森病的分子基础研究 最近经历了一个戏剧性的转变，把重点放在有毒的寡聚α-Syn及其与 神经变性了解这一有前途的新治疗靶点， 纤维，现在需要生物物理学的洞察力的错误折叠的αSyn单体成有毒的寡聚体形式 蛋白质。最近的几项研究指出，氧化应激条件导致形成 高毒性α-Syn寡聚体。因此，我们的第一个目标是了解错误折叠的分子基础。 氧化α-Syn。我们将在过去两年中取得的几项重大发现的基础上再接再厉，这些发现突出了 C-末端酪氨酸残基在错误折叠过程中的重要性。我们将测试一个简单的假设 这是基于我们自己最近的发现（2016年发表在《自然化学生物学》上），即氧化 甲硫氨酸的存在导致与芳香族残基形成强的非共价相互作用，包括 酪氨酸。我们的方法将提供αSyn的化学和生物物理学的定量细节，包括状态- 最先进的NMR测量和计算建模。第二，我们将发现一套新颖的小 这些分子首次靶向毒性氧化αSyn寡聚体的形成和稳定性。我们 将利用一个强大的新平台进行高通量筛选，该平台基于精细的 灵敏的荧光寿命测量，并测试Hit化合物在神经元中的功能功效。 这些小分子最终将为我们的团队提供一个平台，在未来的R 01规模的提案中， 一个全面的药物化学药物发现运动;但在这个建议中，命中化合物将作为 探针，以进一步确定（通过NMR）与氧化甲硫氨酸驱动的特定侧链相互作用 αSyn的错误折叠 !

ABSTRACT A distinct feature of Parkinson’s Disease (PD) is the aggregation of neurotoxic α-Synuclein (αSyn) into amyloid assemblies. Despite over two decades of intense efforts to identify inhibitors that directly interrupt the αSyn aggregation process, no successful compounds have reached the clinic. Research on the molecular basis of PD has recently undergone a dramatic shift to focus on toxic oligomeric αSyn and its relation to neurodegeneration. Understanding this promising new therapeutic target, a departure from research on insoluble fibrils, now requires biophysical insight about the misfolding of αSyn monomers into the toxic oligomeric forms of the protein. Several recent studies have pointed to oxidative stress conditions as leading to the formation of highly toxic αSyn oligomers. Thus, our first goal is to understand the molecular basis for misfolding of oxidized αSyn. We will build on several high-impact discoveries made in the past two years that highlighted the importance of C-terminal tyrosine residues in the misfolding process. We will test a straightforward hypothesis that is based on our own recent discovery (published in Nature Chemical Biology in 2016), namely that oxidation of methionine leads to the formation of a strong non-covalent interaction with aromatic residues, including tyrosine. Our approach will provide quantitative details of the chemistry and biophysics of αSyn, including state- of-the-art NMR measurements and computational modeling. Second, we will discover a novel set of small molecules that, for the first time, target the formation and stability of toxic, oxidized αSyn oligomers. We will take advantage of a powerful new platform for high-throughput screening that is based on exquisitely sensitive fluorescence lifetime measurements, and test the functional efficacy of Hit compounds in neurons. These small molecules will ultimately provide a platform for our groups, in future R01-scale proposals, to launch a full-scale medicinal chemistry drug discovery campaign; but in this proposal, the Hit compounds will serve as probes to further determine (by NMR) which specific side-chain interactions with oxidized methionine drive misfolding of αSyn. !