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

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.

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.

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

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

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

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.

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

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

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

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

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

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

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

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

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

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