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Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase

Nature Biotechnology volume 28, pages 738–742 (2010)Cite this article

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Abstract

The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control1. With prevalent hyperactivation of the mammalian TOR (mTOR) pathway in human cancers2, strategies to enhance TOR pathway inhibition are needed. We used a yeast-based screen to identify small-molecule enhancers of rapamycin (SMERs) and discovered an inhibitor (SMER3) of the Skp1-Cullin-F-box (SCF)Met30 ubiquitin ligase, a member of the SCF E3-ligase family, which regulates diverse cellular processes including transcription, cell-cycle control and immune response3. We show here that SMER3 inhibits SCFMet30 in vivo and in vitro, but not the closely related SCFCdc4. Furthermore, we demonstrate that SMER3 diminishes binding of the F-box subunit Met30 to the SCF core complex in vivo and show evidence for SMER3 directly binding to Met30. Our results show that there is no fundamental barrier to obtaining specific inhibitors to modulate function of individual SCF complexes.

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Figure 1: Two unsupervised data analyses classify five SMERs into three different groups based on their gene expression profiles.
Figure 2: SMER3 targets SCFMet30.
Figure 3: Molecular mechanism for the specificity of SCFMet30 inhibition by SMER3.

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Acknowledgements

We are grateful for grant support from the American Cancer Society and the U.S. National Institutes of Health and for traineeship support of M.A. and B.L. by the NIH UCLA Chemistry−Biology Interface Predoctoral Training Program. N.Z. and R.J.D. are investigators of the Howard Hughes Medical Institute. We thank D. Skowyra (Saint Louis University) and M. Tyers (University of Edinburgh, UK) for their generous gifts of bacculo virus constructs and anti-Met4 antibody, respectively. We also thank J. Salcedo (Roche Diagnostics Corporation) for support toward differential scanning fluorimetry experiments.

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Author notes

  1. Mariam Aghajan, Nao Jonai and Karin Flick: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, and the Molecular Biology Institute, University of California, Los Angeles, California, USA

    Mariam Aghajan, Nao Jonai, Fei Fu, Brett Lomenick, Rui Hao & Jing Huang

  2. Department of Biological Chemistry, School of Medicine, University of California, Irvine, California, USA

    Karin Flick, Ikram Ouni & Peter Kaiser

  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Manlin Luo

  4. Department of Chemistry and Biochemistry, University of California, Los Angeles, California, USA

    Xiaolu Cai, Ying Li, Kendall N Houk & Michael E Jung

  5. Department of Biology, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA

    Nathan Pierce, Rati Verma & Raymond J Deshaies

  6. Department of Pharmacology, Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA

    Xiaobo Tang & Ning Zheng

  7. Molecular Screening Shared Resource, University of California, Los Angeles, California, USA

    Robert Damoiseaux

  8. Roche Diagnostics Corporation, Roche Applied Science, Indianapolis, Indiana, USA

    Pierre M del Moral

  9. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Cheng Li

  10. Departments of Physiology & Biophysics and Developmental & Cell Biology, University of California, Irvine, California, USA

    Lan Huang

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  1. Mariam Aghajan
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Contributions

Figure 1a, M.A. and R.D.; 1b, F.F. and M.L.; 1c, C.L. and J.H.; 2a, N.J.; 2b, N.J. and R.H.; 2c, K.F.; 2d,e, I.O. and N.P.; 3a, K.F.; 3b, K.F. and L.H.; 3c, K.F.; 3d, N.J.; 3e, M.A.; Table 1, X.T., M.A. and P.M.d.M.; X.C., B.L., R.V., Y.L., K.N.H., M.E.J. and N.Z. contributed new reagents and analysis; all authors discussed data; M.A., F.F., M.E.J., R.J.D., P.K. and J.H. wrote the paper with input from all authors.

Corresponding authors

Correspondence to Peter Kaiser or Jing Huang.

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The authors declare no competing financial interests.

Supplementary information

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Supplementary Tables 1,2, Supplementary Figs. 1–8 and Supplmentary Note (PDF 1145 kb)

Supplementary Data Set 1 (XLS 57 kb)

Supplementary Data Set 2 (XLS 21 kb)

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Aghajan, M., Jonai, N., Flick, K. et al. Chemical genetics screen for enhancers of rapamycin identifies a specific inhibitor of an SCF family E3 ubiquitin ligase. Nat Biotechnol 28, 738–742 (2010). https://doi.org/10.1038/nbt.1645

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