1. Minoru Yoshida Chemotherapy-2015 Valencia, Spain August 04- 06, 2015

2. Trichostatin A (TSA) JBC 1990 Trapoxin B (TPX) JBC 1993 PNAS 2001 O O O O OH OH CRM1/exportin 1 Leptomycin B (LMB) JBC 1994 Cell 1997 HDAC N H O O N OH NHN NH HN O O O O O O FK228 (romidepsin) ECR 1998 Cancer Res 2002 H N O O O S S O O O N N N H H H Spliceostatin A Nat Chem Biol 2007 Spliceosome Theonellamide Nat Chem Biol, 2010 3  -hydroxysterol O O N H O O HO O O O

3. Gene Protein Phenotype Chemical Physical interaction Genetic interaction Gene Protein Phenotype Chemical

4. Structure and activity of theonellamides, marine antifungals Isolation: Matsunaga, S., et al. (1989) J. Am. Chem. Soc. 111, 2582-2588. Matsunaga, S. & Fusetani, N. (1995) J. Org. Chem. 60, 1177-1181. R1R1 R2R2 R3R3 R4R4 R5R5 TNM-A:OHMeBrH  -D-Gal TNM-B:OHMeBr H TNM-C:HHHBrH TNM-D:HHBr  -D-Ara TNM-E:HHBr  -D-Gal TNM-F:HHBr H Theonella swinhoei TNM-A Miconazole A. fumigatas C. albicans C. neoformans S. pombe 3.131.56 6.25 3.13 0.20 0.500.25 MIC (  g/ml)

5. Colored scanning electron micrograph (SEM) of dividing Schizosaccharomyces pombe cells. Matsuyama et al. have cloned and partially characterized all protein- encoding open reading frames (the ORFeome) of fission yeast (p 841). Credit: Steve Gschmeissner/Science Photo Library. ORFeome and localizome data are available at http://www.riken.jp/SPD/index.html ORF clones are available from RIKEN BioResource Center http://www.brc.riken.jp/lab/dna/en/yoshida yeast_en.html

6. Strategy for target identification of theonellamide F Resistant Sensitive The fission yeast expression library Chr. II Chr. I Chr. III leu1 locus cen1 T ADH leu1 + ORF Pnmt1 FLAG 2 His 6 cen3 ORF FLAG 2 His 6 YFP ORF FLAG 2 His 6 Global analysis of localization （ Localizome ） TNM-BODIPY-FL

7. 212 GO categories x 11 compounds Log 2 of fold enrichment with p < 0.02, otherwise 0 Pearson correlation (centered), average linkage BFA Anisomycin Cycloheximide FCZ Cispentacin FK463 TNM-F Nystatin TBZ Tunicamycin TSA 2D-clustering analysis of GO terms of hit genes FK463: an inhibitor of 1,3-  -glucan synthase

8. FK463 (20  g/ml) (In YE + 1.2 M sorbitol) DMSO TNM-F (5  g/ml) DMSO 10  m Theonellamide F counteracts FK463 (Micafungin)

9. Rho1 WT control DMSO TNM-F Rho1 T20N Rho1 Q64L 10  m TNM-induced cell wall synthesis is mediated by Rho1

10. Subcellular localization of the target Localizome (Catalogue of protein localization) Target candidates Fluorescent compound A screen for the target protein using localizome

11. Subcellular localization of theonellamide target TNM-BF Pma1-YFP TNM-BODIPY-FL

12. Ergosterol biosynthesis squaleneSqualene-2,3-epoxide lanosterol zymosterolfecosterolepisterol 5,7,24(28)-ergostatrienol5,7,22,24(28)-ergostatetraenolergosterol Sts1/Erg4Erg5 Erg6Erg2Erg31, Erg32

13. Theonellamide recognizes ergosterol & cholesterol : TNM-BODIPY-FL Blocking reagent: 1% skim milk. : BODIPY-FL

14. 3  -hydroxyl group: structural feature required for TNM binding

15. TNM-BODIPY-FL (1.3  M, 30 min) & filipin (5  g/ml) DIC Filipin TNM-B 5  m Filipin III TNM-BF Co-localization with filipin, a probe for sterols

16. : ergosterol : upstream regulator of GS machinery GS Rho1  -1,3-glucan 細胞膜 細胞壁 Amphotericin B Theonellamide F FK463 : ions, etc. Vacuole fusion Nishimura et al. Nature Chem. Biol., 2010 Theonellamide as a novel class of sterol-binding molecules

17. relative fluorescence unit TNM-AMCAEGFP-Lysenin mergerhodamine-DOPE cholesterol (%) 0102030405060 20 min control TNM-A 0 min phase separation (%) controlTNM-A Theonellamides induce membrane phase separation Arita et al. Chem. Biol., 2015

18. 10 um theonellamide Liposome w/o Cell membraneCell morphology phase separationorder perturbation contraction w/ energy-dependent cell contraction dysregulation of cytoskeleton cell membrane cytoskeleton LoLd Theonellamides

19. Chemotherapy-2016 Website: chemotherapy.conferenceseries.com Meet the eminent gathering once again at Chemotherapy-2016 Dallas, USA June 06-07, 2016