Irwin Jacob A, Edward Wong H, Kwon Inchan
Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA.
Department of Chemical Engineering, University of Virginia, Charlottesville, VA 22904, USA; School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea.
Anal Biochem. 2015 Feb 1;470:61-70. doi: 10.1016/j.ab.2014.10.016. Epub 2014 Nov 1.
Numerous aromatic small molecule modulators of amyloid-beta peptide (Aβ) monomer aggregation and neurotoxicity have been identified with the ultimate goal of Alzheimer's disease (AD) treatment. Determining binding sites of these modulators on Aβ monomer is an important topic in the mechanistic understanding of AD pathology and drug development. However, Aβ monomer binding sites have been reported for only a very limited number of Aβ modulators. In this article, we present a convenient method for determining aggregation-modulating polycyclic aromatic small molecule ligand binding sites on Aβ monomer using immunostaining with a panel of Aβ sequence-specific antibodies. To validate our technique, we first examined one modulating aromatic ligand, Congo Red, with known binding sites, which yielded consistent results with previous findings. Then, using the same technique, binding sites on Aβ of four known Aβ monomer aggregation modulators, Erythrosin B, Eosin Y, Phloxine B, and Rose Bengal, were determined. The identified ligand binding sites were also confirmed by a separate fluorescence quenching-based assay using a panel of overlapping Aβ sub-fragments. The technique described here greatly increases researchers' ability to determine the Aβ monomer binding site(s) of aggregation-modulating aromatic small molecule ligands and to screen for new ligands that bind specific regions on Aβ.
为了治疗阿尔茨海默病（AD），人们已经鉴定出许多能够调节淀粉样β肽（Aβ）单体聚集和神经毒性的芳香族小分子调节剂。确定这些调节剂在Aβ单体上的结合位点是理解AD病理机制和药物开发的重要课题。然而，仅报道了极少数Aβ调节剂的Aβ单体结合位点。在本文中，我们提出了一种便捷的方法，通过使用一组Aβ序列特异性抗体进行免疫染色，来确定调节聚集的多环芳香族小分子配体在Aβ单体上的结合位点。为了验证我们的技术，我们首先检测了一种具有已知结合位点的调节性芳香族配体刚果红，其结果与先前的发现一致。然后，使用相同的技术，确定了四种已知的Aβ单体聚集调节剂赤藓红B、伊红Y、焰红B和孟加拉玫瑰红在Aβ上的结合位点。通过使用一组重叠的Aβ亚片段进行单独的基于荧光猝灭的测定，也证实了所确定的配体结合位点。本文所述技术大大提高了研究人员确定调节聚集的芳香族小分子配体的Aβ单体结合位点以及筛选与Aβ特定区域结合的新配体的能力。
