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Marine antifungal theonellamides target 3β-hydroxysterol to activate Rho1 signaling

Nature Chemical Biology volume 6, pages 519–526 (2010)Cite this article

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Abstract

Linking bioactive compounds to their cellular targets is a central challenge in chemical biology. Here we report the mode of action of theonellamides, bicyclic peptides derived from marine sponges. We generated a chemical-genomic profile of theonellamide F using a collection of fission yeast strains in which each open reading frame (ORF) is expressed under the control of an inducible promoter. Clustering analysis of the Gene Ontology (GO) terms associated with the genes that alter drug sensitivity suggested a mechanistic link between theonellamide and 1,3-β-D-glucan synthesis. Indeed, theonellamide F induced overproduction of 1,3-β-D-glucan in a Rho1-dependent manner. Subcellular localization and in vitro binding assays using a fluorescent theonellamide derivative revealed that theonellamides specifically bind to 3β-hydroxysterols, including ergosterol, and cause membrane damage. The biological activity of theonellamides was alleviated in mutants defective in ergosterol biosynthesis. Theonellamides thus represent a new class of sterol-binding molecules that induce membrane damage and activate Rho1-mediated 1,3-β-D-glucan synthesis.

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Figure 1: Generation of chemical-genomic profiles.
Figure 2: Cell wall abnormality predicted by GO term analysis.
Figure 3: Identification of 3β-hydroxysterols as the target of TNM-F.
Figure 4: Genetic interactions with ergosterol biosynthetic genes.
Figure 5: Disruption of plasma membrane integrity by TNM-F.

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Acknowledgements

We thank C. Boone (Univ. Toronto) for sharing unpublished results and discussions, A. Fujie (Astellas Pharma) for kind gifts of FK463 and cispentacin, K. Takegawa (Kyushu Univ.) for erg deletion strains, T. Kuno (Kobe Univ.) for the bgs1 mutant and pck deletion strains and K. Nakano (University of Tsukuba) for rho1-expression plasmids. We also thank J. Ishiguro (Konan Univ.) for the act1/cps8 mutant strain, which was provided through the Yeast Genetic Resource Center (YGRC), N. Kanoh, S. Shimizu and H. Osada (RIKEN Advanced Science Institute) for helping in the preparation of TNM affinity beads, J. Ochi (Kyoto Univ.) for technical assistance and J. Piotrowski (RIKEN Advanced Science Institute) for critical reading of the manuscript. We are grateful to the RIKEN Brain Science Institute's Research Resource Center for mass spectrometry. This work was supported in part by the New Energy and Industrial Technology Development Organization Project on Development of Basic Technology to Control Biological Systems Using Chemical Compounds, a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Chemical Genomics Research Project, RIKEN Advanced Science Institute.

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Authors and Affiliations

  1. Chemical Genomics Research Group, RIKEN Advanced Science Institute, Saitama, Japan

    Shinichi Nishimura, Hideaki Kakeya & Minoru Yoshida

  2. Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

    Shinichi Nishimura & Hideaki Kakeya

  3. Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Saitama, Japan

    Yuko Arita, Miyuki Honda, Akihisa Matsuyama, Atsuko Shirai & Minoru Yoshida

  4. Graduate School of Science and Engineering, Saitama University, Saitama, Japan

    Yuko Arita & Minoru Yoshida

  5. Department of Green and Sustainable Chemistry, Tokyo Denki University, Tokyo, Japan

    Miyuki Honda & Hisashi Kawasaki

  6. Lipid Biology Laboratory, RIKEN Advanced Science Institute, Saitama, Japan

    Kunihiko Iwamoto & Toshihide Kobayashi

  7. CREST Research Project, Japan Science and Technology Corporation, Saitama, Japan

    Akihisa Matsuyama & Minoru Yoshida

  8. Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

    Shigeki Matsunaga

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Contributions

M.Y. is responsible for project planning and experimental design, with support from K.I., H. Kawasaki, H. Kakeya and T.K.; S.N. performed most of the experiments; Y.A. assisted in vitro sterol binding experiments; M.H. assisted chemical-genomic screen; A.M. and A.S. prepared the yeast strain collection; S.M. prepared theonellamides.

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Correspondence to Minoru Yoshida.

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Supplementary Methods, Supplementary Figures 1–20, Supplementary Tables 1–3 and Supplementary Datasets 1–8 (PDF 2119 kb)

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Nishimura, S., Arita, Y., Honda, M. et al. Marine antifungal theonellamides target 3β-hydroxysterol to activate Rho1 signaling. Nat Chem Biol 6, 519–526 (2010). https://doi.org/10.1038/nchembio.387

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