Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, United States.
Department of Medicinal Chemistry, Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, Minnesota 55414, United States.
ACS Chem Neurosci. 2022 Sep 7;13(17):2646-2657. doi: 10.1021/acschemneuro.2c00327. Epub 2022 Aug 24.
Recent high-resolution structures of alpha-synuclein (aSyn) fibrils offer promise for rational approaches to drug discovery for Parkinson's disease and Lewy body dementia. Harnessing the first such structures, we previously used molecular dynamics and free energy calculations to suggest that threonines 72 and 75─which line water-filled cavities within the fibril stacks─may be of central importance in stabilizing fibrils. Here, we used experimental mutagenesis of both wild-type and A53T aSyn to show that both threonine residues play important but surprisingly disparate roles in fibril nucleation and elongation. The T72A mutant, but not T75A, resulted in a large increase in the extent of fibrillization during primary nucleation, leading us to posit that T72 acts as a "brake" on run-away aggregation. An expanded set of simulations of five recent high-resolution fibril structures suggests that confinement of cavity waters around T72 correlates with this finding. In contrast, the T75A mutation led to a modest decrease in the extent of fibrillization. Furthermore, both T72A and T75A completely blocked the initial fibril elongation in seeded fibrillization. To test whether these threonine-lined cavities are druggable targets, we used computational docking to identify potential small-molecule binders. We show that the top-scoring hit, aprepitant, strongly promotes fibril growth while specifically interacting with aSyn fibrils and not monomer, and we offer speculation as to how such compounds could be used therapeutically.
最近解析出的α-突触核蛋白（aSyn）纤维的高分辨率结构为帕金森病和路易体痴呆的药物研发提供了合理的方法。我们利用首批此类结构，以前曾使用分子动力学和自由能计算来表明，位于纤维堆内充满水的腔中的苏氨酸 72 和 75 可能对稳定纤维具有重要作用。在这里，我们通过对野生型和 A53T aSyn 的实验诱变表明，这两个苏氨酸残基在纤维成核和伸长中都起着重要但令人惊讶的不同作用。T72A 突变体，但不是 T75A，导致在初级成核过程中纤维形成程度大大增加，这使我们假设 T72 作为“刹车”来阻止失控聚集。对五个最近的高分辨率纤维结构的扩展模拟集表明，T72 周围腔水的限制与这一发现相关。相比之下，T75A 突变导致纤维形成程度略有下降。此外，T72A 和 T75A 完全阻止了种子化纤维形成中的初始纤维伸长。为了测试这些苏氨酸排列的腔是否可作为药物靶点，我们使用计算对接来鉴定潜在的小分子结合物。我们表明，得分最高的阿瑞匹坦与 aSyn 纤维而不是单体强烈促进纤维生长，并对其结合方式进行了推测，以及这些化合物如何在治疗上使用。
