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

In this work, we designed small molecules YG-1 and YG-2 derived from molecule Thioflavin-T (ThT) containing structural moieties targeting Cu(I). To realize the purpose of Cu(I) binding, we eliminate the methyl group of ThT 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 A beta 1-40 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 A beta 1-40-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 A beta 1-40 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.