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Splicing factor SF3b as a target of the antitumor natural product pladienolide

Nature Chemical Biology volume 3, pages 570–575 (2007)Cite this article

Abstract

Pladienolide is a naturally occurring antitumor macrolide that was discovered by using a cell-based reporter gene expression assay controlled by the human vascular endothelial growth factor promoter1,2. Despite the unique mechanisms of action3 and prominent antitumor activities of pladienolides B and D in diverse in vitro and in vivo systems3, their target protein has remained unclear. We used 3H-labeled, fluorescence-tagged and photoaffinity/biotin (PB)-tagged 'chemical probes' to identify a 140-kDa protein in splicing factor SF3b as the binding target of pladienolide. Immunoblotting of an enhanced green fluorescent protein fusion protein of SF3b subunit 3 (SAP130) revealed direct interaction between the PB probe and SAP130. The binding affinities of pladienolide derivatives to the SF3b complex were highly correlated with their inhibitory activities against reporter gene expression and cell proliferation. Furthermore, pladienolide B impaired in vivo splicing in a dose-dependent manner. Our results demonstrate that the SF3b complex is a pharmacologically relevant protein target of pladienolide and suggest that this splicing factor is a potential antitumor drug target.

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Figure 1: Structures and activities of pladienolides B and D, E7107 and the chemical probes.
Figure 2: Distribution and localization of chemical probes.
Figure 3: Identification of PB probe–crosslinked protein.
Figure 4: Target validation experiments of pladienolide.
Figure 5: Pladienolide B–induced inhibition of splicing in HeLa cells.

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Acknowledgements

We thank T. Tsuchida (Mercian Corporation Bioresource Laboratories) for supplying pladienolide B, T. Satoh for helpful technical advice, M. Date for experimental assistance and N. Asai and M. Gomibuchi for spectral analysis of the chemicals. We are grateful to K. Yoshimatsu, M. Asada, T. Nagasu and J. Kuromitsu for coordinating the research team.

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  1. Yasushi Ishihama

    Present address: Present address: Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan.,

Authors and Affiliations

  1. Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, 300-2635, Ibaraki, Japan

    Yoshihiko Kotake, Koji Sagane, Takashi Owa, Hajime Shimizu, Mai Uesugi, Yasushi Ishihama, Masao Iwata & Yoshiharu Mizui

  2. KAN Research Institute Inc., 6-7-3 Minatojimaminami-machi, Chuo-ku, Kobe, 650-0047, Hyogo, Japan

    Yuko Mimori-Kiyosue

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  1. Yoshihiko Kotake
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Contributions

Y.K. designed and performed research and wrote the manuscript; K.S. designed and performed research; T.O. designed and performed research; Y.M.-K. designed and performed research; H.S. performed research; M.U. performed research; Y.I. performed research; M.I. performed research; Y.M. designed and performed research and provided overall direction.

Corresponding author

Correspondence to Yoshiharu Mizui.

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Competing interests

Y.K., K.S., T.O., H.S., M.U., M.I. and Y.M. work for and have financial interests in Eisai Co. Ltd., a pharmaceutical company that is pursuing the clinical development of E7107. Y.M.K. works for and has financial interests in KAN Research Institute Inc., a subsidiary company of Eisai. Y.I. has no competing financial interests.

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Kotake, Y., Sagane, K., Owa, T. et al. Splicing factor SF3b as a target of the antitumor natural product pladienolide. Nat Chem Biol 3, 570–575 (2007). https://doi.org/10.1038/nchembio.2007.16

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