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.

  • Brief Communication
  • Published:

Thermal profiling reveals phenylalanine hydroxylase as an off-target of panobinostat

Nature Chemical Biology volume 12, pages 908–910 (2016)Cite this article

Subjects

Abstract

We describe a two-dimensional thermal proteome profiling strategy that can be combined with an orthogonal chemoproteomics approach to enable comprehensive target profiling of the marketed histone deacetylase inhibitor panobinostat. The N-hydroxycinnamide moiety is identified as critical for potent and tetrahydrobiopterin-competitive inhibition of phenylalanine hydroxylase leading to increases in phenylalanine and decreases in tyrosine levels. These findings provide a rationale for adverse clinical observations and suggest repurposing of the drug for treatment of tyrosinemia.

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

Figure 1: Proteins showing changes in thermal stability in 2D-TPP experiments and analyzed in the chemoproteomics experiment with panobinostat.
Figure 2: Panobinostat inhibits PAH activity in a tetrahydrobiopterin-competitive manner and requires the N-hydroxycinnamide moiety.

Similar content being viewed by others

References

  1. Simon, G.M., Niphakis, M.J. & Cravatt, B.F. Nat. Chem. Biol. 9, 200–205 (2013).

    Article  CAS  Google Scholar 

  2. Martinez Molina, D. et al. Science 341, 84–87 (2013).

    Article  Google Scholar 

  3. Martinez Molina, D. & Nordlund, P. Annu. Rev. Pharmacol. Toxicol. 56, 141–161 (2016).

    Article  CAS  Google Scholar 

  4. Savitski, M.M. et al. Science 346, 1255784 (2014).

    Article  Google Scholar 

  5. Huber, K.V. et al. Nat. Methods 12, 1055–1057 (2015).

    Article  CAS  Google Scholar 

  6. Bantscheff, M. & Drewes, G. Bioorg. Med. Chem. 20, 1973–1978 (2012).

    Article  CAS  Google Scholar 

  7. Schenone, M., Dančík, V., Wagner, B.K. & Clemons, P.A. Nat. Chem. Biol. 9, 232–240 (2013).

    Article  CAS  Google Scholar 

  8. Ziegler, S., Pries, V., Hedberg, C. & Waldmann, H. Angew. Chem. Int. Edn Engl. 52, 2744–2792 (2013).

    Article  CAS  Google Scholar 

  9. Atadja, P. Cancer Lett. 280, 233–241 (2009).

    Article  CAS  Google Scholar 

  10. Bailey, H., Stenehjem, D.D. & Sharma, S. J. Blood Med. 6, 269–276 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Younes, A. et al. J. Clin. Oncol. 30, 2197–2203 (2012).

    Article  CAS  Google Scholar 

  12. Werner, T. et al. Anal. Chem. 84, 7188–7194 (2012).

    Article  CAS  Google Scholar 

  13. Franken, H. et al. Nat. Protoc. 10, 1567–1593 (2015).

    Article  CAS  Google Scholar 

  14. Robers, M.B. et al. Nat. Commun. 6, 10091 (2015).

    Article  CAS  Google Scholar 

  15. Pootoolal, J. et al. Proc. Natl. Acad. Sci. USA 99, 8962–8967 (2002).

    Article  CAS  Google Scholar 

  16. Reinhard, F.B. et al. Nat. Methods 12, 1129–1131 (2015).

    Article  CAS  Google Scholar 

  17. Bantscheff, M. et al. Nat. Biotechnol. 29, 255–265 (2011).

    Article  CAS  Google Scholar 

  18. Diamond, A. Acta Paediatr. Suppl. 407, 89–91 (1994).

    Article  CAS  Google Scholar 

  19. Degroot, L.J. & Niepomniszcze, H. Metabolism 26, 665–718 (1977).

    Article  CAS  Google Scholar 

  20. Novartis. Farydak prescribing information, revised 02/2016. (2016).

  21. Uhlén, M. et al. Mol. Cell. Proteomics 4, 1920–1932 (2005).

    Article  Google Scholar 

  22. Hosoda, F. et al. Oncogene 34, 1196–1206 (2015).

    Article  CAS  Google Scholar 

  23. van Vliet, D. et al. JIMD Rep. 18, 117–124 (2015).

    Article  Google Scholar 

  24. Dittmann, A., Ghidelli-Disse, S., Hopf, C. & Bantscheff, M. Methods Mol. Biol. 1156, 279–291 (2014).

    Article  CAS  Google Scholar 

  25. Becher, I. et al. ACS Chem. Biol. 8, 599–607 (2013).

    Article  CAS  Google Scholar 

  26. Drewes, G. et al. US patent 20090238808 (2009).

  27. Savitski, M.M. et al. J. Am. Soc. Mass Spectrom. 21, 1668–1679 (2010).

    Article  CAS  Google Scholar 

  28. Savitski, M.M. et al. J. Proteome Res. 12, 3586–3598 (2013).

    Article  CAS  Google Scholar 

  29. Roberts, K.M., Khan, C.A., Hinck, C.S. & Fitzpatrick, P.F. Biochemistry 53, 7846–7853 (2014).

    Article  CAS  Google Scholar 

  30. Daubner, S.C. & Fitzpatrick, P.F. Biochemistry 38, 4448–4454 (1999).

    Article  CAS  Google Scholar 

  31. Zhang, S., Hinck, A.P. & Fitzpatrick, P.F. Biochemistry 54, 5167–5174 (2015).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Stuhlfauth for cell culture; M. Jundt, K. Kammerer, M. Klös-Hudak, M. Paulmann and T. Rudi for expert technical assistance; and G. Drewes for helpful suggestions. We also thank W.F. Mueller for providing cells for metabolomics experiments. P.F.F. acknowledges support from the Welch Foundation (grant AQ-1245). C.R.B. was supported by a VENI grant (project 722.013.009) from the Netherlands Organization for Scientific Research.

Author information

Authors and Affiliations

  1. Cellzome GmbH, Heidelberg, Germany

    Isabelle Becher, Thilo Werner, Carola Doce, Ina Tögel, Anne Rueger, Marcel Muelbaier, Elsa Salzer, Marcus Bantscheff & Mikhail M Savitski

  2. Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Isabelle Becher & Mikhail M Savitski

  3. Biomolecular Mass Spectrometry and Proteomics, Utrecht University, Utrecht, the Netherlands

    Esther A Zaal & Celia R Berkers

  4. Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, USA

    Crystal A Khan & Paul F Fitzpatrick

Authors
  1. Isabelle Becher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thilo Werner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carola Doce
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Esther A Zaal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ina Tögel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Crystal A Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Anne Rueger
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marcel Muelbaier
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Elsa Salzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Celia R Berkers
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Paul F Fitzpatrick
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Marcus Bantscheff
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Mikhail M Savitski
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

I.B., M.B. and M.M.S. conceived the project; I.B., T.W., M.B. and M.M.S. designed the biochemical, cell biological and MS experiments; I.B. and T.W. performed MS experiments; E.A.Z. and C.R.B. designed and performed metabolomics experiments; A.R. and M.M. synthesized the panobinostat-amide and gave advice; P.F.F. and C.A.K. designed and performed enzyme activity experiments; I.B., I.T. and E.S. performed biochemical and cell biological experiments; I.B., T.W., C.D., M.B. and M.M.S. contributed to data analysis; E.A.Z., C.R.B. and P.F.F. contributed to the manuscript; I.B., M.B. and M.M.S. wrote the manuscript.

Corresponding authors

Correspondence to Marcus Bantscheff or Mikhail M Savitski.

Ethics declarations

Competing interests

I.B., T.W., C.D., I.T., A.R., M.M., E.S., M.B. and M.M.S. are employees and/or shareholders of Cellzome GmbH and GlaxoSmithKline, which funded this work.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–6 and Supplementary Figures 1–17. (PDF 4463 kb)

Supplementary Note

Synthetic procedures (PDF 156 kb)

Supplementary Data Set 1

2D-TPP in HepG2 cells. (XLSX 23511 kb)

Supplementary Data Set 2

2D-TPP in HepG2 cell extract. (XLSX 16105 kb)

Supplementary Data Set 3

HepG2 TPP TR (reference for melting temperature). (XLSX 3614 kb)

Supplementary Data Set 4

TTC38 immunoprecipitation. (XLSX 347 kb)

Supplementary Data Set 5

TPP CCR vorinostat in HepG2 cells. (XLSX 5219 kb)

Supplementary Data Set 6

HepG2 TPP TR with vehicle and panobinostat. (XLSX 11588 kb)

Supplementary Data Set 7

Affinity-based pulldowns using panobinostat beads, with and without detergent. (XLSX 3584 kb)

Supplementary Data Set 8

Affinity-based pulldowns using panobinostat beads, competition with panobinostat in SH-SY5Y cell extract. (XLSX 1919 kb)

Supplementary Data Set 9

Metabolomics data for amino acids intracellular and in cell medium after HepG2 treatment with panobinostat. (XLSX 43 kb)

Supplementary Data Set 10

Metabolomics data for intracellular and in cell medium amino acid levels after treatment with panobinostat (SH-SY5Y cells). (XLSX 40 kb)

Supplementary Data Set 11

Affinity-based pulldowns using panobinostat beads, competition with tetrahydrobiopterin. (XLSX 1921 kb)

Supplementary Data Set 12

Affinity-based pulldowns using panobinostat beads, competition with panobinostat-amide. (XLSX 1331 kb)

Supplementary Data Set 13

Affinity-based pulldowns using panobinostat beads, competition with belinostat. (XLSX 1932 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Becher, I., Werner, T., Doce, C. et al. Thermal profiling reveals phenylalanine hydroxylase as an off-target of panobinostat. Nat Chem Biol 12, 908–910 (2016). https://doi.org/10.1038/nchembio.2185

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2185

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research