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Selective degradation of splicing factor CAPERα by anticancer sulfonamides

Nature Chemical Biology volume 13, pages 675–680 (2017)Cite this article

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Abstract

Target-protein degradation is an emerging field in drug discovery and development. In particular, the substrate-receptor proteins of the cullin–ubiquitin ligase system play a key role in selective protein degradation, which is an essential component of the anti-myeloma activity of immunomodulatory drugs (IMiDs), such as lenalidomide. Here, we demonstrate that a series of anticancer sulfonamides NSC 719239 (E7820), indisulam, and NSC 339004 (chloroquinoxaline sulfonamide, CQS) induce proteasomal degradation of the U2AF-related splicing factor coactivator of activating protein-1 and estrogen receptors (CAPERα) via CRL4DCAF15 mediated ubiquitination in human cancer cell lines. Both CRISPR–Cas9-based knockout of DCAF15 and a single amino acid substitution of CAPERα conferred resistance against sulfonamide-induced CAPERα degradation and cell-growth inhibition. Thus, these sulfonamides represent selective chemical probes for disrupting CAPERα function and designate DCAFs as promising drug targets for promoting selective protein degradation in cancer therapy.

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Figure 1: Downregulation of CAPERα by E7820, indisulam, and CQS.
Figure 2: E7820, indisulam, and CQS promote CRL4DCAF15 mediated CAPERα degradation.
Figure 3: DCAF15 is the primary target of E7820, indisulam, and CQS in the ubiquitination of CAPERα.
Figure 4: CAPERα degradation is a key biochemical activity that underlies the anticancer properties of E7820, indisulam, and CQS.
Figure 5: Comparison of mRNA splicing modulation and gene expression between the E7820-induced degradation and siRNA-mediated knockdown of CAPERα.

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Acknowledgements

T.U. thanks B.F. Cravatt, A. Fukamizu, and B.A. Littlefield for helpful advice and comments in preparing this manuscript. We thank A. Ishii and K. Tomita for technical support of the qPCR assay, M. Fukuda for support of informatics work, M. Kato for NMR analyses, and K. Kubara and M. Nakao for support of amplicon sequencing.

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  1. takashi_owa@91.alumni.u-tokyo.ac.jp

Authors and Affiliations

  1. Eisai Co., Ltd., Tokodai, Tsukuba-shi, Ibaraki, Japan

    Taisuke Uehara, Yukinori Minoshima, Koji Sagane, Naoko Hata Sugi, Kaoru Ogawa Mitsuhashi, Noboru Yamamoto, Hiroshi Kamiyama, Kentaro Takahashi, Yoshihiko Kotake, Mai Uesugi, Akira Yokoi, Atsushi Inoue & Taku Yoshida

  2. School of Pharmacy, Hyogo University of Health Sciences, Minatojima, Chuo-ku, Kobe-shi, Hyogo, Japan

    Miyuki Mabuchi & Akito Tanaka

  3. Eisai Inc., Woodcliff Lake, New Jersey, USA

    Takashi Owa

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Contributions

T.U., Y.M., Y.K., T.Y., A.Y., A.T., and T.O. designed the study and experiments. T.U. performed biochemical and cell biology experiments including LC-MS/MS analysis and identified CRL4DCAF15-mediated CAPERα degradation. Y.M. and K.S. performed the gene sequencing and gene editing experiments and prepared the plasmid vectors. N.H.S., K.O.M., and H.K. performed biochemical and cell biology experiments. N.Y. and Y.K. prepared the chemicals. K.T. analyzed the exome sequencing data and A.I. analyzed the protein structure in PDB. M.U., T.Y., and K.O.M. performed the analyses of gene expression and RNA splicing. M.M. and A.T. established the photoaffinity labeling procedure. T.U. and T.O. interpreted experiments, directed study, and wrote the manuscript. All authors discussed results and contributed to the writing and editing of the manuscript.

Corresponding authors

Correspondence to Taisuke Uehara or Takashi Owa.

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Competing interests

Two of the three sulfonamide small molecules in this article, NSC 719239 (E7820) and indisulam, are pharmaceutical candidate compounds of Eisai Co., Ltd. to which the majority of the authors belong.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–23. (PDF 2164 kb)

Supplementary Note

Synthetic Procedures. (PDF 552 kb)

Supplementary Dataset 1

Whole cell proteome analysis (Data Independent Acquisition). (XLSX 2080 kb)

Supplementary Dataset 2

Co-immunoprecipitation followed by LC-MS/MS analysis (Data Independent Acquisition). (XLSX 1239 kb)

Supplementary Dataset 3

DNA microarray. (XLSX 814 kb)

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Uehara, T., Minoshima, Y., Sagane, K. et al. Selective degradation of splicing factor CAPERα by anticancer sulfonamides. Nat Chem Biol 13, 675–680 (2017). https://doi.org/10.1038/nchembio.2363

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