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

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

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our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS REVERSIBLE HISTONE DEACETYLASE ACTIVITY CATALYZES LYSINE ACYLATION Article 26 March 2025 TARGET DECONVOLUTION OF

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_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 * Cellzome GmbH, Heidelberg, Germany Isabelle Becher, Thilo Werner, Carola Doce, Ina Tögel, Anne Rueger, Marcel Muelbaier, Elsa Salzer, Marcus Bantscheff & 

Mikhail M Savitski * Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Isabelle Becher & Mikhail M Savitski * Biomolecular Mass Spectrometry and Proteomics,

Utrecht University, Utrecht, the Netherlands Esther A Zaal & Celia R Berkers * Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas, USA Crystal A

Khan & Paul F Fitzpatrick Authors * Isabelle Becher View author publications You can also search for this author inPubMed Google Scholar * Thilo Werner View author publications You can

also search for this author inPubMed Google Scholar * Carola Doce View author publications You can also search for this author inPubMed Google Scholar * Esther A Zaal View author

publications You can also search for this author inPubMed Google Scholar * Ina Tögel View author publications You can also search for this author inPubMed Google Scholar * Crystal A Khan

View author publications You can also search for this author inPubMed Google Scholar * Anne Rueger View author publications You can also search for this author inPubMed Google Scholar *

Marcel Muelbaier View author publications You can also search for this author inPubMed Google Scholar * Elsa Salzer View author publications You can also search for this author inPubMed 

Google Scholar * Celia R Berkers View author publications You can also search for this author inPubMed Google Scholar * Paul F Fitzpatrick View author publications You can also search for

this author inPubMed Google Scholar * Marcus Bantscheff View author publications You can also search for this author inPubMed Google Scholar * Mikhail M Savitski View author publications You

can also search for this author inPubMed 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

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: 15 January 2016 * Accepted: 20 July 2016 * Published: 26 September 2016 * Issue Date: November 2016 * DOI:

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