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ポスター発表

第2日　5月16日（水）　　ポスター会場

2P-12　　PDF

Combination of label-free DARTS and LC-MS/MS method reveals a target protein of small molecule inhibitor of autophagy

(¹Yonsei University・ ²Korea Basic Science・ ³Lund University)
^oHwang, Hui-yun¹・ Cho, Yoon Sun¹・ Kim, Jin Young²・ Yun, Ki Na²・ Yoo, Jong Shin²・ Marko-varga, György^1,3・ Kwon, Ho Jeong¹

Autophagy inhibitors that induce autophagic cell death have unrealized potential as anti-cancer drugs. Previously, we identified a novel autophagy regulating small molecule based on its ability to induce autophagosome (AP)-like vesicles. The small molecule induces apoptosis-independent cell death by binding an unknown target via an unknown mechanism. Using a combination of label-free DARTS (drug affinity response target stability) and liquid chromatography/tandem mass spectrometry (LC-MS/MS), Hsp70 was identified as a candidate target protein of unmodified small molecule. Target protein inhibition through siRNA knockdown or a specific inhibitor, inhibited autophagy in HeLa cells, whereas Hsp70 overexpression prevented inhibition. Moreover, Hsp70 inhibition affects fusion of lysosomes with autophagosomes, as demonstrated by GFP-RFP double-tagged LC3 transfection. These findings demonstrate the potential small molecule to induce autophagic cell death and warrant its development as an anticancer drug. Additionally, we showed combined DARTS and LC-MS/MS-based target identification to be effective for novel protein target identification.