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:

Developing Spindlin1 small-molecule inhibitors by using protein microarrays

Abstract

The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the 'druggability' of epigenetic effector molecules. The small-molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains and, with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs, we screened a protein-domain microarray of human methyllysine effector molecules to rapidly detect compounds with new binding profiles with either increased or decreased specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor-domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of SPIN1 inhibitors with increased selectivity (EML631–633), which engage SPIN1 in cells, block its ability to 'read' H3K4me3 marks and inhibit its transcriptional-coactivator activity. Protein microarrays can thus be used as a platform to 'target-hop' and identify small molecules that bind and compete with domain-motif interactions.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Protein microarrays identify domain-binding compounds.
Figure 2: Calorimetric and crystallographic studies of SPIN1–EML405.
Figure 3: Calorimetric and crystallographic studies of SPIN1–EML631.
Figure 4: Compounds EML631–633 block the reader ability of SPIN1 and are cell permeable.
Figure 5: RNA-seq identification of SPIN1-regulated transcripts and inhibition of this coactivator activity by EML631.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Huang, H., Lin, S., Garcia, B.A. & Zhao, Y. Quantitative proteomic analysis of histone modifications. Chem. Rev. 115, 2376–2418 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Strahl, B.D. & Allis, C.D. The language of covalent histone modifications. Nature 403, 41–45 (2000).

    CAS  PubMed  Google Scholar 

  3. Jenuwein, T. & Allis, C.D. Translating the histone code. Science 293, 1074–1080 (2001).

    CAS  PubMed  Google Scholar 

  4. Wozniak, G.G. & Strahl, B.D. Hitting the 'mark': interpreting lysine methylation in the context of active transcription. Biochim. Biophys. Acta 1839, 1353–1361 (2014).

    CAS  PubMed  Google Scholar 

  5. Su, Z. & Denu, J.M. Reading the combinatorial histone language. ACS Chem. Biol. 11, 564–574 (2016).

    CAS  PubMed  Google Scholar 

  6. McGrath, J. & Trojer, P. Targeting histone lysine methylation in cancer. Pharmacol. Ther. 150, 1–22 (2015).

    CAS  PubMed  Google Scholar 

  7. Watson, I.R., Takahashi, K., Futreal, P.A. & Chin, L. Emerging patterns of somatic mutations in cancer. Nat. Rev. Genet. 14, 703–718 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Yang, Y. & Bedford, M.T. Protein arginine methyltransferases and cancer. Nat. Rev. Cancer 13, 37–50 (2013).

    CAS  PubMed  Google Scholar 

  9. Helin, K. & Dhanak, D. Chromatin proteins and modifications as drug targets. Nature 502, 480–488 (2013).

    CAS  PubMed  Google Scholar 

  10. Beaver, J.E. & Waters, M.L. Molecular recognition of Lys and Arg methylation. ACS Chem. Biol. 11, 643–653 (2016).

    CAS  PubMed  Google Scholar 

  11. Musselman, C.A., Lalonde, M.-E., Côté, J. & Kutateladze, T.G. Perceiving the epigenetic landscape through histone readers. Nat. Struct. Mol. Biol. 19, 1218–1227 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Taverna, S.D., Li, H., Ruthenburg, A.J., Allis, C.D. & Patel, D.J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat. Struct. Mol. Biol. 14, 1025–1040 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Palmer, W.S. et al. Structure-guided design of IACS-9571, a selective high-affinity dual TRIM24-BRPF1 bromodomain inhibitor. J. Med. Chem. 59, 1440–1454 (2016).

    CAS  PubMed  Google Scholar 

  15. Musselman, C.A., Khorasanizadeh, S. & Kutateladze, T.G. Towards understanding methyllysine readout. Biochim. Biophys. Acta 1839, 686–693 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Herold, J.M., Ingerman, L.A., Gao, C. & Frye, S.V. Drug discovery toward antagonists of methyl-lysine binding proteins. Curr. Chem. Genomics 5, 51–61 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Santiago, C., Nguyen, K. & Schapira, M. Druggability of methyl-lysine binding sites. J. Comput. Aided Mol. Des. 25, 1171–1178 (2011).

    CAS  PubMed  Google Scholar 

  18. Miller, T.C. et al. Competitive binding of a benzimidazole to the histone-binding pocket of the Pygo PHD finger. ACS Chem. Biol. 9, 2864–2874 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Wagner, E.K., Nath, N., Flemming, R., Feltenberger, J.B. & Denu, J.M. Identification and characterization of small molecule inhibitors of a plant homeodomain finger. Biochemistry 51, 8293–8306 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Simhadri, C. et al. Chromodomain antagonists that target the polycomb-group methyllysine reader protein chromobox homolog 7 (CBX7). J. Med. Chem. 57, 2874–2883 (2014).

    CAS  PubMed  Google Scholar 

  21. Stuckey, J.I. et al. A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1. Nat. Chem. Biol. 12, 180–187 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Kireev, D. et al. Identification of non-peptide malignant brain tumor (MBT) repeat antagonists by virtual screening of commercially available compounds. J. Med. Chem. 53, 7625–7631 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Perfetti, M.T. et al. Identification of a fragment-like small molecule ligand for the methyl-lysine binding protein, 53BP1. ACS Chem. Biol. 10, 1072–1081 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Herold, J.M. et al. Small-molecule ligands of methyl-lysine binding proteins. J. Med. Chem. 54, 2504–2511 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. James, L.I. et al. Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain. Nat. Chem. Biol. 9, 184–191 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Milosevich, N. & Hof, F. Chemical inhibitors of epigenetic methyllysine reader proteins. Biochemistry 55, 1570–1583 (2016).

    CAS  PubMed  Google Scholar 

  27. Yun, M., Wu, J., Workman, J.L. & Li, B. Readers of histone modifications. Cell Res. 21, 564–578 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Su, X. et al. Molecular basis underlying histone H3 lysine-arginine methylation pattern readout by Spin/Ssty repeats of Spindlin1. Genes Dev. 28, 622–636 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Wang, W. et al. Nucleolar protein Spindlin1 recognizes H3K4 methylation and stimulates the expression of rRNA genes. EMBO Rep. 12, 1160–1166 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Wang, J.X. et al. SPINDLIN1 promotes cancer cell proliferation through activation of WNT/TCF-4 signaling. Mol. Cancer Res. 10, 326–335 (2012).

    CAS  PubMed  Google Scholar 

  31. Jafari, R. et al. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat. Protoc. 9, 2100–2122 (2014).

    CAS  PubMed  Google Scholar 

  32. Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).

    PubMed  Google Scholar 

  33. Espejo, A., Côté, J., Bednarek, A., Richard, S. & Bedford, M.T. A protein-domain microarray identifies novel protein-protein interactions. Biochem. J. 367, 697–702 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Espejo, A. & Bedford, M.T. Protein-domain microarrays. Methods Mol. Biol. 264, 173–181 (2004).

    CAS  PubMed  Google Scholar 

  35. Kim, J. et al. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 7, 397–403 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Barnes-Seeman, D., Park, S.B., Koehler, A.N. & Schreiber, S.L. Expanding the functional group compatibility of small-molecule microarrays: discovery of novel calmodulin ligands. Angew. Chem. Int. Edn Engl. 42, 2376–2379 (2003).

    CAS  Google Scholar 

  37. Koehler, A.N., Shamji, A.F. & Schreiber, S.L. Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis. J. Am. Chem. Soc. 125, 8420–8421 (2003).

    CAS  PubMed  Google Scholar 

  38. Yue, W., Sun, L.Y., Li, C.H., Zhang, L.X. & Pei, X.T. Screening and identification of ovarian carcinomas related genes. Ai Zheng 23, 141–145 (2004).

    CAS  PubMed  Google Scholar 

  39. Gao, Y. et al. Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells. Biochem. Biophys. Res. Commun. 335, 343–350 (2005).

    CAS  PubMed  Google Scholar 

  40. Franz, H. et al. The histone code reader SPIN1 controls RET signaling in liposarcoma. Oncotarget 6, 4773–4789 (2015).

    PubMed  PubMed Central  Google Scholar 

  41. Chew, T.G. et al. A tudor domain protein SPINDLIN1 interacts with the mRNA-binding protein SERBP1 and is involved in mouse oocyte meiotic resumption. PLoS One 8, e69764 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Wagner, T. et al. Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform. Nucleic Acids Res. 44, e88 (2016).

    PubMed  PubMed Central  Google Scholar 

  43. Sweis, R.F. et al. Discovery and development of potent and selective inhibitors of histone methyltransferase g9a. ACS Med. Chem. Lett. 5, 205–209 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Robaa, D. et al. Identification and structure-activity relationship studies of small-molecule inhibitors of the methyllysine reader protein Spindlin1. ChemMedChem 11, 2327–2338 (2016).

    CAS  PubMed  Google Scholar 

  45. Liang, M.D., Zhang, Y., McDevit, D., Marecki, S. & Nikolajczyk, B.S. The interleukin-1beta gene is transcribed from a poised promoter architecture in monocytes. J. Biol. Chem. 281, 9227–9237 (2006).

    CAS  PubMed  Google Scholar 

  46. O'Sullivan, A.C., Sullivan, G.J. & McStay, B. UBF binding in vivo is not restricted to regulatory sequences within the vertebrate ribosomal DNA repeat. Mol. Cell. Biol. 22, 657–668 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Laskowski, R.A., Moss, D.S. & Thornton, J.M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 231, 1049–1067 (1993).

    CAS  PubMed  Google Scholar 

  52. Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

M.T.B. was supported by an NIH grant (DK062248) and a CPRIT grant (RP130432) for the protein array analysis. The deep sequencing was supported by a CPRIT grant (RP120348) to J.S. The Center for Cancer Epigenetics at MDACC also supported this study. G.S. was supported by grants from the Italian Ministero dell'Istruzione, dell'Universita e della Ricerca (MIUR), Progetti di Ricerca di Interesse Nazionale (PRIN 2012ZHN9YH), the Universita di Salerno (Italy) and the European Cooperation in Science and Technology (COST Action CM1406). H.L. was supported by grants from the Major State Basic Research Development Program in China (2015CB910503 and 2016YFA0500700) and the Tsinghua University Initiative Scientific Research Program. N.B. was supported by the Odyssey Fellowship Program at the University of Texas MD Anderson Cancer Center. We thank the staff members at beamlines BL17U and BL18U of the Shanghai Synchrotron Radiation Facility for assistance in data collection.

Author information

Authors and Affiliations

Authors

Contributions

M.T.B. and G.S. conceived the project. N.B. and D.C. carried out the cell-based and competition experiments. C.S., C.J. and M.I.K. constructed, maintained and probed the protein-domain microarrays. M.V., S.C. and G.S. designed and synthesized the compounds used in this study. X.S., X.B. and H.L. performed the structural studies and ITC experiments. J.S. performed the deep sequencing. K.C. and J.L. performed the bioinformatics analysis of the RNA-seq data sets. M.T.B., G.S. and H.L. wrote the manuscript and supervised the work in their respective fields.

Corresponding authors

Correspondence to Haitao Li, Gianluca Sbardella or Mark T Bedford.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–4 and Supplementary Figures 1–12. (PDF 2455 kb)

Supplementary Note

Supplementary Schemes. (PDF 2713 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bae, N., Viviano, M., Su, X. et al. Developing Spindlin1 small-molecule inhibitors by using protein microarrays. Nat Chem Biol 13, 750–756 (2017). https://doi.org/10.1038/nchembio.2377

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2377

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing