Institute of Biostructures and Bioimages-Catania, National Research Council, Via Paolo Gaifami 8, 95126 Catania, Italy.
Department of Chemical Sciences, University of Napoli "Federico II" , Via Cintia 4, I-80126 Napoli, Italy.
ACS Chem Neurosci. 2017 Aug 16;8(8):1767-1778. doi: 10.1021/acschemneuro.7b00110. Epub 2017 Jun 9.
The self-assembling of the amyloid β (Aβ) peptide into neurotoxic aggregates is considered a central event in the pathogenesis of Alzheimer's disease (AD). Based on the "amyloid hypothesis", many efforts have been devoted to designing molecules able to halt disease progression by inhibiting Aβ self-assembly. Here, we combine biophysical (ThT assays, TEM and AFM imaging), biochemical (WB and ESI-MS), and computational (all-atom molecular dynamics) techniques to investigate the capacity of four optically pure components of the natural product silymarin (silybin A, silybin B, 2,3-dehydrosilybin A, 2,3-dehydrosilybin B) to inhibit Aβ aggregation. Despite TEM analysis demonstrated that all the four investigated flavonoids prevent the formation of mature fibrils, ThT assays, WB and AFM investigations showed that only silybin B was able to halt the growth of small-sized protofibrils thus promoting the formation of large, amorphous aggregates. Molecular dynamics (MD) simulations indicated that silybin B interacts mainly with the C-terminal hydrophobic segment MVGGVV of Aβ40. Consequently to silybin B binding, the peptide conformation remains predominantly unstructured along all the simulations. By contrast, silybin A interacts preferentially with the segments LVFF and NKGAII of Aβ40 which shows a high tendency to form bend, turn, and β-sheet conformation in and around these two domains. Both 2,3-dehydrosilybin enantiomers bind preferentially the segment LVFF but lead to the formation of different small-sized, ThT-positive Aβ aggregates. Finally, in vivo studies in a transgenic Caenorhabditis elegans strain expressing human Aβ indicated that silybin B is the most effective of the four compounds in counteracting Aβ proteotoxicity. This study underscores the pivotal role of stereochemistry in determining the neuroprotective potential of silybins and points to silybin B as a promising lead compound for further development in anti-AD therapeutics.
淀粉样β（Aβ）肽自组装成神经毒性聚集体被认为是阿尔茨海默病（AD）发病机制中的一个核心事件。基于“淀粉样蛋白假说”，人们已经投入了大量精力来设计能够通过抑制 Aβ 自组装来阻止疾病进展的分子。在这里，我们结合生物物理（ThT 测定、TEM 和 AFM 成像）、生化（WB 和 ESI-MS）和计算（全原子分子动力学）技术，研究了天然产物水飞蓟素的四个光学纯成分（水飞蓟宾 A、水飞蓟宾 B、2,3-脱水水飞蓟宾 A、2,3-脱水水飞蓟宾 B）抑制 Aβ 聚集的能力。尽管 TEM 分析表明，所有四种研究的类黄酮都能阻止成熟纤维的形成，但 ThT 测定、WB 和 AFM 研究表明，只有水飞蓟宾 B 能够阻止小尺寸原纤维的生长，从而促进大的无定形聚集体的形成。分子动力学（MD）模拟表明，水飞蓟宾 B 主要与 Aβ40 的 C 端疏水性片段 MVGGVV 相互作用。因此，在水飞蓟宾 B 结合后，肽构象在整个模拟过程中仍主要处于无结构状态。相比之下，水飞蓟宾 A 更倾向于与 Aβ40 的 LVFF 和 NKGAII 片段相互作用，这些片段在这两个结构域内和周围表现出形成弯曲、转角和β-折叠构象的高趋势。两种 2,3-脱水水飞蓟宾对映体都优先与 LVFF 片段结合，但导致形成不同的、ThT 阳性的 Aβ 聚集体。最后，在表达人 Aβ 的转基因秀丽隐杆线虫 Caenorhabditis elegans 菌株中的体内研究表明，水飞蓟宾 B 是这四种化合物中最有效地对抗 Aβ 蛋白毒性的化合物。这项研究强调了立体化学在确定水飞蓟素的神经保护潜力方面的关键作用，并指出水飞蓟宾 B 是进一步开发抗 AD 治疗药物的有前途的先导化合物。
