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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 for treatment of tyrosinemia.

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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.

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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.

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Authors

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.

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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)

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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

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