Target deconvolution using label-free method of autophagy inhibitor and its antitumor activity in glioblastoma Hui-Yun Hwang , Yoon Sun Cho , Jin Young Kim , Ki Na Yun , Jong Shin Yoo , Eunhyeong Lee , Injune Kim , György Marko-Varga 1,4 and Ho Jeong Kwon * 2 3 3 2 1 1 1 2 1 Chemical Genomics Global Research Laboratory, Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Korea 2 Biomedical Omics Group, Korea Basic Science Institute, Ochang, Chungbuk 28119, Korea 3 Biomedical Science and Engineering Interdisciplinary Program, Korea Advanced
Institute of Science and Technology, Daejeon, Korea 4 Clinical Protein Science & Imaging, Department of Biomedical Engineering, Lund University, BMC D13, SE-221 84 Lund, Sweden *Corresponding author: kwonhj@yonsei.ac.kr BACKGROUND AIM Manipulating autophagy is a promising strategy for treating cancer as To identify protein targets and uncover the unknown mechanisms several autophagy inhibitors are shown to induce autophagic cell death. of APZ, we used a label-free drug affinity responsive target stability (DARTS) approach with a liquid chromatography/tandem mass One of these, autophagonizer
(APZ), induces apoptosis-independent spectrometry (LC–MS/MS) readout. Collectively, our study cell death by binding an unknown target via an unknown mechanism in demonstrated that APZ, a new autophagy inhibitor, can be used as vitro & in vivo. We identified Hsp70 as a key target protein of unmodified a potent antitumor drug candidate to get over unassailable glioma APZ in autophagy. and revealed a novel function of Hsp70 in lysosomal integrity regulation of autophagy. METHODS Combined drug affinity responsive target stability (DARTS) and LC−MS/MS method identified a new protein target of APZ.
(1) Exposure of the small molecule to proteins in the cell lysate proteome pool, to allow for small molecule–protein target interactions, (2) pronase digestion of proteins unprotected by the small molecule, where proteins that bind resist protease treatment while proteins with little or no binding to the small molecule are degraded, (3) further proteolysis with trypsin and LC–MS/MS analysis of resulting peptides to determine sequence coverage, (4) identification of proteins protected by the small molecule based on its enhanced sequence coverage by more than 3% compared to controls, and (5)
selection of likely protein targets based on candidates with phenotypic relevancy to the small molecule. RESULTS Figure 1. APZ works not by inducing Figure 1 Electromicroscopy Double-tagged LC3 transfection autophagy, but by inhibiting autophagy Figure 2. Characterization of Hsp70 as a potential protein target of APZ Figure 3. APZ exhibits significant synergism with TMZ in orthotopic mouse xenograft model Red arrow: autophagosomes Flux (X) ★ Rapa: autophagy inducer Black arrow: autolysosomes Flux (O) ★ HeLa cells Figure 2 Peptide analysis detected in DARTS LC-MS DARTS biophysical
validation Figure 3 GFP-GL261 cells CONCLUSION REFERENCES ACKNOWLEDGEMENTS Supported by BK21 PLUS, NRF (MSIP; Collectively, our study showed that both APZ [1] Hwang HY, Cho YS, Kim JY, Yun KN, Yoo JS, Lee E, et al. 2015K1A1A2028365,2018M3A9C4076477, Autophagic Inhibition via Lysosomal Integrity Dysfunction treatment and orthogonal Hsp70 inhibition Leads to Antitumor Activity in Glioma Treatment. Cancers 2016K2A9A1A03904900) increased specific markers of autophagy to form 2020; 12. autophagosomes. In addition to the role of APZ as [2] Choi IK, Cho YS, Jung HJ, Kwon HJ. Autophagonizer, a a
specific inhibitor of Hsp70, we also revealed a novel synthetic small molecule, induces autophagic cell death. Contact information Biochemical and biophysical research communications 2010; new functional role of Hsp70 in autophagosome 393:849-854. formation and induction of incomplete lysosomes [3] Kim D, Hwang HY, Kwon HJ. Targeting Autophagy In • Presenter: Hui-Yun Hwang leading to autophagic cell death in cancer cells. Disease: Recent Advances In Drug Discovery. Expert opinion This novel role of Hsp70 likely involves the on drug discovery 2020; 15:1045-1064. ghkdgmldbs@naver.com
stabilization of lysosomes based on pH integrity • Coresspondence: Ho Jeong Kwon and suggests a mechanism for its action. . kwonhj@yonsei.ac.kr Presenter : Minjeong Ko [I. Chemical biology and drug discovery-1] Target deconvolution using label-free method of autophagy inhibitor and its antitumor activity in glioblastoma Hui-Yun Hwang¹˙#, Yoon Sun Cho¹, Eun Ji Gwak¹, Jin Young Kim², Ki Na Yun², Jong Shin Yoo², Eunhyeong Lee³, Injune Kim³, György Marko-Varga¹˙⁴, Ho Jeong Kwon¹˙* ¹Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Korea,
²Biomedical Omics Group, Korea Basic Science Institute, Chungbuk 28119, Korea, ³Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 34141, Korea, ⁴Clinical Protein Science & Imaging, Department of Biomedical Engineering, Lund University, Lund SE-221 84, Sweden Manipulating autophagy is a promising strategy for treating cancer as several autophagy inhibitors are shown to induce autophagic cell death. One of these, autophagonizer (APZ), induces apoptosis-independent cell death by binding an unknown target via an unknown mechanism. To
identify APZ targets, we used a label-free drug affinity responsive target stability (DARTS) approach with a liquid chromatography/tandem mass spectrometry (LC–MS/MS) readout. Of 35 protein interactors, we identified Hsp70 as a key target protein of unmodified APZ in autophagy. Either APZ treatment or Hsp70 inhibition attenuates integrity of lysosomes, which leads to autophagic cell death exhibiting an excellent synergism with a clinical drug, temozolomide, in vitro, in vivo, and orthotropic glioma xenograft model. These findings demonstrate the potential of APZ to induce autophagic cell death
and its development to combinational chemotherapeutic agent for glioma treatment. Collectively, our study demonstrated that APZ, a new autophagy inhibitor, can be used as a potent antitumor drug candidate to get over unassailable glioma and revealed a novel function of Hsp70 in lysosomal integrity regulation of autophagy. Target Identification of a natural compound that ameliorates atherosclerosis via autophagy-inducing activity 2 Minjeong Ko , Dongjin Lee , Ju Yeon Lee , Jin Young Kim , Jong Shin Yoo and Ho Jeong Kwon 1,* 1 1 2 2 1 Chemical Genomics Global Research Laboratory, Department of
Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Korea. 2 Korea Basic Science Institute, Cheongwon-Gun, Chungbuk 363-883, Korea. * Corresponding author: kwonhj@yonsei.ac.kr BACKGROUND AIM • Atherosclerosis is a life-threatening chronic inflammatory disease in which • This study provides new insights into the mechanism of an anti-atherosclerotic natural compound in linking with autophagy. blood vessels are blocked due to the accumulation of cholesterol and plaque formation. • To uncover the mode of action of compound YCGX, a combination of drug affinity
• Autophagy in endothelial cell is involved in vascular lipid homeostasis responsive target stability (DARTS) and LC-MS/MS method was applied to identify the target protein of compound YCGX. The target protein candidate was validated by which can protect the atherosclerotic lesion formation. However, the DARTS western blot analysis. deficiency of endothelial autophagy and malfunctions of macrophage • Further validation of interaction of compound with target protein was conducted with autophagy trigger the atherosclerotic plaque formation. knockdown of the target gene resulting in increase of
the autophagic features. METHODS Target identification using ‘DARTS (Drug Affinity Responsive Target Stability)-LC-MS/MS’ • DARTS is a new technology for protein target identification of non-labeled small molecules. • Target protein bound with drug of interest has high stability against pronase treatment and pronase can not easily degrade drug-bound proteins. RESULTS Figure 1 Figure 2 Figure 4 DMSO YCGX (5 µM) YCGX NT 24 48 72 24 48 72 (h) p62 LC3B Ⅰ Ⅱ β-actin Figure 3 Summary Figure1. Compound YCGX induced autophagic flux in HUVECs Figure 2. Compound YCGX inhibited foam cell formation via
increasing lysosomal activities in RAW 264.7 macrophages Figure 3. Target ID & VD through DARTS-LC-MS/MS Figure 4. Is target protein H responsible for autophagy activity? CONCLUSION REFERENCES ACKNOWLEDGEMENTS • Compound YCGX induces autophagy in Supported by BK21 PLUS, NRF (MSIP; HUVECs. • Lomenick et al. Target identification using drug 2015K1A1A2028365, 2018M3A9C4076477) • Compound YCGX inhibited foam cell formation affinity responsive target stability (DARTS). Curr Protoc Chem Biol. 2011 in oxLDL-induced RAW 264.7 macrophages via • Sergin et al. Exploiting macrophage autophagy- inducing
lysosome activity. lysosomal biogenesis as a therapy for atherosclerosis. Contact information • Target proteins of compound YCGX is Nat. Commun. 2017 identified by DARTS LC-MS/MS and validated • Guo et al. The role of the LncRNA-FA2H-2-MLKL via biological analysis. pathway in atherosclerosis by regulation of autophagy • Presenter : Minjeong Ko komj0714@yonsei.ac.kr • Target validation for protein H, the final target flux and inflammation through mTOR-dependent • Correspondence : Ho Jeong Kwon protein of compound YCGX is underway. signaling. Cell Death Differ. 2019 kwonhj@yonsei.ac.kr [I.
Chemical biology and drug discovery-2] Target identification of a natural compound that attenuates atherosclerosis via autophagy-inducing activity Minjeong Ko¹, Dongjin Lee¹, Ju Yeon Lee², Jin Young Kim², Jong Shin Yoo², Ho Jeong Kwon¹ ¹Chemical Genomics Global Research Laboratory, Department of Biotechnology, Yonsei University, Seoul 03722, Korea, ²Biomedical Omics Group, Korea Basic Science Institute, Cheongwon-Gun, Chungbuk 28119, Korea Traditional medicinal plants have long been widely used as pharmaceutical agents due to their fascinating biological activities. Among many medicinal herbs,
we recently identified a natural plant D is effective in ameliorating atherosclerotic plaque formation on ApoE-/- mice. In early stage of atherosclerosis, oxidized low-density lipoprotein (oxLDL) contributes to the endothelial cell dysfunction as well as macrophage foam cell formation and previous studies demonstrate that autophagy plays an important role in cardiovascular diseases. Herein, we revealed that compound YCGX, an active principle of plant D, induces autophagy in time dependent manner via the suppression of mTOR signaling and increasing TFEB nuclear translocation in HUVECs. Compound
YCGX inhibited ox-LDL-induced foam cell formation in Raw 264.7 macrophage and rescued the autophagy impaired by oxLDL in HUVECs. To uncover the mode of action of compound YCGX, a combination of DARTS and LC-MS/MS method was applied to identify the target protein of compound YCGX. Further validation of interaction of compound with target protein was conducted by DARTS analysis and knockdown study. Collectively, this study provides new insights into the mechanism of an anti-atherosclerotic natural compound in linking with autophagy. Inhibition of fatty acid binding protein 4 ameliorates impaired
ciliogenesis in gastric cancer cells 1 Yooju Jung , Jae-Ho Cheong , and Ho Jeong Kwon 1 2 1 Chemical Genomics Global Research Laboratory, Department of Biotechnology, College of Life Science & Biotechnology, Yonsei University, Seoul 03722, Korea 2 Department of Surgery, Yonsei University College of Medicine, Seoul, Republic of Korea BACKGROUND AIM ▶ FABP4 (Fatty Acid Binding Protein 4) in cancer ▶ Investigation on correlation between FABP4 and primary cilia in gastric • FABP4 is known as a carrier protein for fatty acids in adipocyte. cancer • Recently, FABP4 has been reported as a cancer
related protein in different kindsof cancer. • Altho
