ポスター発表
- 第2日 5月16日(水) ポスター会場
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2P-12 PDF
Combination of label-free DARTS and LC-MS/MS method reveals a target protein of small molecule inhibitor of autophagy
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.