Target identification of small-molecule compounds by affinity elution in tandem hydrophobic interaction chromatography (AETHIC)

• Main Authors: Wei-Chieh Huang, 黃偉杰
• Other Authors: 張震東
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/65389622126866640167

博士 === 國立臺灣大學 === 生化科學研究所 === 104 === Target identification is essential to understanding the complicated actions of bioactive small compounds. However, available techniques constrained by processing time and specific equipment do not meet the growing demands. Here, we have developed a novel method combining hydrophobic interaction chromatography and affinity elution, named affinity elution in tandem hydrophobic interaction chromatography (AETHIC) that enables systematic and efficient identification of small molecule-protein interactions. The essence of this strategy is using a series of HIC matrices encompassing aliphatic chains of different length which provide a wide range of hydrophobicity for interactions with most proteins. After samples are applied to the tandem columns, target proteins are selectively eluted with a specific ligand. As our first proof-of-principle of AETHIC, we demonstrated that AETHIC was able to enrich ATP-binding proteins from mouse brain extract as near nineteen percent of total protein identifications are previously assigned ATP binders. In addition, we have revealed that raf kinase inhibitory protein (RKIP) is an ATP binding protein. ATP attenuates the interaction between RKIP and Raf-1. As a result, short-term energy depletion augments interaction between RKIP and Raf-1 resulting in decreased activation of the downstream ERK signaling in cultured HEK293 cells. Therefore, the ATP-binding function renders RKIP’s inhibition on Raf-1 modulated by cellular ATP concentrations. These data shed light on how energy levels link to cellular signaling. In light of these results, we envisaged the potential of AETHIC to be applied in the target identification of bioactive compounds such as metabolites and therapeutic small-molecule drugs.