Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Facile target validation in an animal model with intracellularly expressed monobodies

Nature Chemical Biology volume 14, pages 895–900 (2018)Cite this article

Subjects

Abstract

Rapidly determining the biological effect of perturbing a site within a potential drug target could guide drug discovery efforts, but it remains challenging. Here, we describe a facile target validation approach that exploits monobodies, small synthetic binding proteins that can be fully functionally expressed in cells. We developed a potent and selective monobody to WDR5, a core component of the mixed lineage leukemia (MLL) methyltransferase complex. The monobody bound to the MLL interaction site of WDR5, the same binding site for small-molecule inhibitors whose efficacy has been demonstrated in cells but not in animals. As a genetically encoded reagent, the monobody inhibited proliferation of an MLL-AF9 cell line in vitro, suppressed its leukemogenesis and conferred a survival benefit in an in vivo mouse leukemia model. The capacity of this approach to readily bridge biochemical, structural, cellular characterization and tests in animal models may accelerate discovery and validation of druggable sites.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Monobody Mb(S4) inhibits MLL1 enzyme activity and targets the Win motif-binding site on WDR5.
Fig. 2: The crystal structure of the WDR5–Mb(S4) complex.
Fig. 3: Mb(S4) inhibits HoxA9 expression and proliferation of MLL-AF9 cells.
Fig. 4: Expression of Mb(S4) in MLL-AF9 cells inhibits leukemia progression in a mouse model.

Similar content being viewed by others

References

  1. Arrowsmith, C. H. et al. The promise and peril of chemical probes. Nat. Chem. Biol. 11, 536–541 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Weiss, W. A., Taylor, S. S. & Shokat, K. M. Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat. Chem. Biol. 3, 739–744 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sha, F., Salzman, G., Gupta, A. & Koide, S. Monobodies and other synthetic binding proteins for expanding protein science. Protein Sci. 26, 910–924 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lawrence, H. J. et al. Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood 89, 1922–1930 (1997).

    CAS  PubMed  Google Scholar 

  5. Patel, A., Dharmarajan, V., Vought, V. E. & Cosgrove, M. S. On the mechanism of multiple lysine methylation by the human mixed lineage leukemia protein-1 (MLL1) core complex. J. Biol. Chem. 284, 24242–24256 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dou, Y. et al. Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat. Struct. Mol. Biol. 13, 713–719 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Cao, F. et al. An Ash2L/RbBP5 heterodimer stimulates the MLL1 methyltransferase activity through coordinated substrate interactions with the MLL1 SET domain. PLoS One 5, e14102 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Patel, A., Vought, V. E., Dharmarajan, V. & Cosgrove, M. S. A conserved arginine-containing motif crucial for the assembly and enzymatic activity of the mixed lineage leukemia protein-1 core complex. J. Biol. Chem. 283, 32162–32175 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Patel, A., Dharmarajan, V. & Cosgrove, M. S. Structure of WDR5 bound to mixed lineage leukemia protein-1 peptide. J. Biol. Chem. 283, 32158–32161 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Song, J. J. & Kingston, R. E. WDR5 interacts with mixed lineage leukemia (MLL) protein via the histone H3-binding pocket. J. Biol. Chem. 283, 35258–35264 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dharmarajan, V., Lee, J. H., Patel, A., Skalnik, D. G. & Cosgrove, M. S. Structural basis for WDR5 interaction (Win) motif recognition in human SET1 family histone methyltransferases. J. Biol. Chem. 287, 27275–27289 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Alicea-Velázquez, N. L. et al. Targeted disruption of the interaction between WD-40 repeat protein 5 (WDR5) and mixed lineage leukemia (MLL)/SET1 family proteins specifically inhibits MLL1 and SETd1A methyltransferase complexes. J. Biol. Chem. 291, 22357–22372 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Karatas, H. et al. High-affinity, small-molecule peptidomimetic inhibitors of MLL1/WDR5 protein-protein interaction. J. Am. Chem. Soc. 135, 669–682 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Senisterra, G. et al. Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5. Biochem. J. 449, 151–159 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. Cao, F. et al. Targeting MLL1 H3K4 methyltransferase activity in mixed-lineage leukemia. Mol. Cell 53, 247–261 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Getlik, M. et al. Structure-based optimization of a small molecule antagonist of the interaction between WD repeat-containing protein 5 (WDR5) and mixed-lineage leukemia 1 (MLL1). J. Med. Chem. 59, 2478–2496 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. Li, D. D. et al. High-affinity small molecular blockers of mixed lineage leukemia 1 (MLL1)-WDR5 interaction inhibit MLL1 complex H3K4 methyltransferase activity. Eur. J. Med. Chem. 124, 480–489 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Liu, H., Cheng, E. H. & Hsieh, J. J. MLL fusions: pathways to leukemia. Cancer Biol. Ther. 8, 1204–1211 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cox, M. C. et al. Chromosomal aberration of the 11q23 locus in acute leukemia and frequency of MLL gene translocation: results in 378 adult patients. Am. J. Clin. Pathol. 122, 298–306 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Sorensen, P. H. et al. Molecular rearrangements of the MLL gene are present in most cases of infant acute myeloid leukemia and are strongly correlated with monocytic or myelomonocytic phenotypes. J. Clin. Invest. 93, 429–437 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Thiel, A. T. et al. MLL-AF9-induced leukemogenesis requires coexpression of the wild-type Mll allele. Cancer Cell 17, 148–159 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Grebien, F. et al. Pharmacological targeting of the Wdr5-MLL interaction in C/EBPα N-terminal leukemia. Nat. Chem. Biol. 11, 571–578 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Koide, A., Wojcik, J., Gilbreth, R. N., Hoey, R. J. & Koide, S. Teaching an old scaffold new tricks: monobodies constructed using alternative surfaces of the FN3 scaffold. J. Mol. Biol. 415, 393–405 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Ruthenburg, A. J. et al. Histone H3 recognition and presentation by the WDR5 module of the MLL1 complex. Nat. Struct. Mol. Biol. 13, 704–712 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Avdic, V. et al. Structural and biochemical insights into MLL1 core complex assembly. Structure 19, 101–108 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Schuetz, A. et al. Structural basis for molecular recognition and presentation of histone H3 by WDR5. EMBO J. 25, 4245–4252 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Li, D. D. et al. Structure-based design and synthesis of small molecular inhibitors disturbing the interaction of MLL1-WDR5. Eur. J. Med. Chem. 118, 1–8 (2016).

    Article  CAS  PubMed  Google Scholar 

  28. Iwamoto, M., Björklund, T., Lundberg, C., Kirik, D. & Wandless, T. J. A general chemical method to regulate protein stability in the mammalian central nervous system. Chem. Biol. 17, 981–988 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Koide, S. Engineering of recombinant crystallization chaperones. Curr. Opin. Struct. Biol. 19, 449–457 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Spencer-Smith, R. et al. Inhibition of RAS function through targeting an allosteric regulatory site. Nat. Chem. Biol. 13, 62–68 (2017).

    Article  CAS  PubMed  Google Scholar 

  31. Sha, F. et al. Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains. Proc. Natl Acad. Sci. USA 110, 14924–14929 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Nady, N. et al. ETO family protein Mtgr1 mediates Prdm14 functions in stem cell maintenance and primordial germ cell formation. eLife 4, e10150 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Nishikori, S. et al. Broad ranges of affinity and specificity of anti-histone antibodies revealed by a quantitative peptide immunoprecipitation assay. J. Mol. Biol. 424, 391–399 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr. D. Biol. Crystallogr. 62, 859–866 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D. Biol. Crystallogr. 66, 22–25 (2010).

    Article  CAS  PubMed  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D. Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Laskowski, R. A., Macarthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  39. Davis, I. W., Murray, L. W., Richardson, J. S. & Richardson, D. C. MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res. 32, W615–W619 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dou, Y. et al. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltransferase MOF. Cell 121, 873–885 (2005).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank J. Andrade and B. Ueberheide (NYU Langone Medical Center) for proteomics analysis; S. Gräslund and C. Arrowsmith (Structural Genomics Consortium) for expression vectors; J. Schmollerl and F. Grebien (Ludwig Boltzmann Institute of Cancer Research) for a retroviral vector and guidance. Research reported in this publication was supported by the National Institutes of Health under award numbers R01 DA036887 and R01 CA194864 (to S.K.) and R01 GM082856 (to Y.D.). Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center (Beamline 19ID) at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. The mass spectrometric experiments were supported in part by the Laura and Isaac Perlmutter Cancer Center support grant P30CA016087 from the National Cancer Institute.

Author information

Authors and Affiliations

  1. Perlmutter Cancer Center, New York University Langone Medical Center, New York, NY, USA

    Ankit Gupta, Steven T. Tsai, Kohei Kurosawa, Akiko Koide & Shohei Koide

  2. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA

    Ankit Gupta, Kohei Kurosawa, Akiko Koide & Shohei Koide

  3. Department of Pathology, University of Michigan, Ann Arbor, MI, USA

    Jing Xu, Shirley Lee, Bo Zhou & Yali Dou

  4. Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL, USA

    Michael S. Werner & Alexander J. Ruthenburg

  5. Department of Medicine, New York University School of Medicine, New York, NY, USA

    Akiko Koide

  6. Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA

    Shohei Koide

Authors
  1. Ankit Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shirley Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Steven T. Tsai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bo Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kohei Kurosawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Michael S. Werner
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Akiko Koide
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alexander J. Ruthenburg
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yali Dou
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Shohei Koide
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.G., Y.D. and S.K. designed the study; A.G., A.K. and S.K. generated monobodies; A.G., S.T.T. and A.K. performed binding measurements; A.G. and S.L. tested monobody effects on the MLL1 complex; A.G. and S.K. determined and analyzed the X-ray crystal structure; A.G. and K.K. constructed degron-controlled vectors; A.G. performed immunoprecipitation experiments; A.G. and S.T.T. performed cell proliferation assays; A.G., J.X., B.Z., M.S.W., A.J.R. and Y.D. performed transcription and ChIP analyses; A.G., J.X. and B.Z. performed mouse experiments and histological analyses; A.G., Y.D. and S.K. wrote the manuscript, and all authors commented and approved the manuscript.

Corresponding authors

Correspondence to Yali Dou or Shohei Koide.

Ethics declarations

Competing interests

S.K. and A.K. are inventors on patent applications filed by the University of Chicago and Novartis Ag that cover monobody library designs (WO2012016245A3 and equivalent nationalized applications).

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–2, Supplementary Figures 1–11

Reporting Summary

Supplementary Dataset 1

Mass spectrometry analyses of proteins captured by Mb(S4) or Mb(S4mut) from the whole-cell lysate or nuclear extract of HEK293T cells

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gupta, A., Xu, J., Lee, S. et al. Facile target validation in an animal model with intracellularly expressed monobodies. Nat Chem Biol 14, 895–900 (2018). https://doi.org/10.1038/s41589-018-0099-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0099-z

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer