阻断铜离子介导神经毒性的小分子抑制剂研究

Investigation on Small Molecules Targeting Cu(Ⅰ) Preventing Copper-Mediated Neurotoxicity

过量的铜离子介导淀粉样蛋白异常聚集被认为是导致淀粉样蛋白变性病的主要因素之一.基于淀粉样蛋白聚集机理及金属铜离子的氧化还原机理,我们选用β-淀粉样蛋白Aβ1-40作为蛋白模型,设计并合成出由硫代黄素T衍生出来的小分子抑制剂.该小分子抑制剂可以螯合淀粉样蛋白周围过量的一价铜离子,阻止有害的氧化还原循环过程,抑制活性氧物质的产生,从而达到保护细胞的目的.这为治疗铜离子介导的淀粉样蛋白错误折叠所造成的疾病提供了新的策略.

In this work, we designed small molecules YG-1 and YG-2 derived from molecule Thioflavin-T（Th T） containing structural moieties targeting Cu（Ⅰ）. To realize the purpose of Cu（Ⅰ） binding, we eliminate the methyl group of Th T molecule and incorporate a nitrogen atom instead of sulfur atom. The imine in benzimidole is expected to coordinate Cu（Ⅰ） effectively. This strategy is aiming at inhibiting the toxicity redox cycle and protecting cells. Using UV absorption spectroscopy, we find that YG-1 can interact with Cu（Ⅰ） effectively, but not with Cu（II）. ESI-MS further characterized the interactions between YG-1/2 and Cu（Ⅰ） in forms of YG-1/2-Cu（Ⅰ）-YG-1/2 and YG-1/2-Cu（Ⅰ）-YG-1/2-Cu（Ⅰ）-YG-1/2. Focusing on the peaks of complexes observed from the ESI-MS results, we calculate the binding motifs of Cu（Ⅰ） to YG-1/2 by MD simulation. In particular, we study all the five possible symmetric modes of YG-1 and YG-2 respectively, which are supposed to be more stable than asymmetric modes. The simulation results show that the mode with Cu（Ⅰ） binding to the imine in benzimidole of YG-1/2 in a linear geometry is the most stable structure with the lowest energy（-0.229 and-0.232 Hartree for YG-1 and YG-2 respectively）. As reducing the production of ROS is an important pathway to alleviate the copper-induced Aβ1-40 cytotoxicity, we test the effects of YG-1/2 on eliminating ROS production using 3-coumarin carboxylic acid（3-CCA） fluorescence spectroscopy, and find that YG-1/2 can block ROS production almost completely. Finally, we examine the cellular protective effects of YG-1 and YG-2 against the toxicity of Aβ1-40-Cu（Ⅰ） by 3-（4,5-dimethyl-2-thiaz-oyl）-2,5-diphenyl-tetrazolium bromide（MTT） assay. Experimental results show that YG-1/2 can alleviate the Cu（Ⅰ）-induced Aβ1-40 toxicity and increase cell viability. Up until now, researchers have devoted many efforts to prevent copper-mediated ROS toxicity. However, few attempts have been made to treat protein-misfolding diseases by targeting Cu（Ⅰ） and blocking the copper redox cycle. The designed molecules YG-1/2 in this work are able to capture Cu（Ⅰ） from participating further reactions in the protein-copper redox cycle and prohibit the generation of ROS. This finding may lead to a new therapeutic strategy for treating copper-mediated protein misfolding diseases.