双功能抑制剂SK10对Cu2+存在下Aβ聚集的抑制和解聚作用

Bifunctional Inhibitory Effect of SK10 on Aβ Aggregation and Depolymerization in the Presence of Cu2+

由于β-淀粉样蛋白(amyloidβ-protein,Aβ)的聚集是阿尔茨海默症(Alzheimer’sdisease,AD)发病机制中的关键过程,而金属离子Cu2+存在下的Aβ的聚集速率加快,且Cu2+本身会促进活性氧(ROS)的产生,增加神经毒性,因此开发既能螯合Cu2+又能抑制Aβ聚集的双功能抑制剂非常重要.本文将金属螯合三肽(SSH)和Aβ聚集七肽抑制剂Ac-LVFFARK-NH2(LK7)相组合,设计合成了新的双功能Aβ聚集十肽抑制剂SSHLVFFARK-NH2(SK10).通过硫代黄素T(ThT)荧光实验、聚集动力学实验、原子力显微镜检测(AFM)、等温滴定量热和MTT细胞毒性测定等研究了SK10对Cu2+存在下Aβ聚集的抑制和解聚作用以及细胞毒性的抑制作用.实验结果表明:SK10不仅可以抑制Cu2+存在下的Aβ40聚集(ThT荧光强度降低了60%),随着SK10浓度的提高使Aβ40纤维逐渐减少,还能降低Cu2+存在下Aβ40聚集产生的细胞毒性,使细胞活性恢复至90%以上;同样,SK10对Cu2+存在下的Aβ42聚集也有抑制作用.由于SK10对Cu2+具有较强的特异性亲和力(Kd=0.03μmol/L),能够螯合Cu2+-Aβ40复合物中的Cu2+,因此能抑制由Cu2+催化产生的活性氧(ROS)对细胞的毒性.进一步研究表明SK10还可解聚形成的Cu2+-Aβ40聚集体,使已形成的Cu2+-Aβ40聚集体消失,缓解其产生的细胞毒性,使细胞活性提高到90%.以上研究结果不但体现了SK10的药用潜力,也为今后设计和开发金属螯合Aβ聚集双功能抑制剂提供了思路.

The aggregation of amyloid β-protein (Aβ) is an important process in the pathogenesis of Alzheimer’s disease(AD). The aggregation rate of Aβ in the presence of metal ions such as Cu2+ gets accelerated,and Cu2+ itself promotes the production of reactive oxygen species (ROS) and increases neurotoxicity. Therefore,it is important to develop dual-function inhibitors that simultaneously chelate copper ions and inhibit Aβ aggregation. In this study,we designed a novel bifunctional decapeptide SSHLVFFARK-NH2 (SK10) by combining a metal chelating tripeptide (SSH) and an Aβ aggregation inhibitor Ac-LVFFARK-NH2(LK7). We conducted thioflavin T(ThT) fluorescence and aggregation kinetics experiments and performed atomic force microscopy(AFM),isothermal titration calorimetry, and MTT cytotoxicity assays to investigate the inhibition and depolymerization of SK10 on the aggregation and cytotoxicity of Aβ in the presence of Cu2+. The experimental results show that SK10 can inhibit Aβ40 aggregation in the presence of Cu2+(ThT fluorescence decreased by 60%) and the Aβ40 fiber gradually decreased with increase in the SK10 concentration and reduce the cytotoxicity induced by Aβ40 aggregation in the presence of Cu2+ such that the cellular activity was restored to >90%. Similarly, SK10 inhibited the aggregation of Aβ42 in the presence of Cu2+. Because SK10 has a specific affinity for Cu2+(Kd=0.03 μmol/L) and is capable of sequestering Cu2+ from Cu2+-Aβ40 species,it can inhibit the toxicity of ROS catalyzed by copper ions with respect to cells. Additional studies revealed that the formed Cu2+-Aβ40 aggregates can be depolymerized by SK10,whereby the formed Cu2+-Aβ40 aggregates disappear and the cell viability is increased to 90%. The above results reflect both the medicinal potential of SK10 and provide ideas for the design and development of bifunctional inhibitors that can chelate metal ions and inhibit Aβ aggregation.