日本質量分析学会 第66回質量分析総合討論会

Program

Poster Presentations

Day 2, May 16(Wed.)  Poster

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)
oHui-yun Hwang1, Yoon Sun Cho1, Jin Young Kim2, Ki Na Yun2, Jong Shin Yoo2, György Marko-varga1,3, Ho Jeong Kwon1

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.