RÉSUMÉ
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.
Sujet(s)
Inhibiteurs de désacétylase d'histone/pharmacologie , Acides hydroxamiques/pharmacologie , Indoles/pharmacologie , Phenylalanine 4-monooxygenase/antagonistes et inhibiteurs , Température , Relation dose-effet des médicaments , Cellules HepG2 , Inhibiteurs de désacétylase d'histone/composition chimique , Humains , Acides hydroxamiques/composition chimique , Indoles/composition chimique , Structure moléculaire , Panobinostat , Phenylalanine 4-monooxygenase/composition chimique , Phenylalanine 4-monooxygenase/métabolisme , Relation structure-activitéRÉSUMÉ
The 2-oxoglutarate-dependent dioxygenase target class comprises around 60 enzymes including several subfamilies with relevance to human disease, such as the prolyl hydroxylases and the Jumonji-type lysine demethylases. Current drug discovery approaches are largely based on small molecule inhibitors targeting the iron/2-oxoglutarate cofactor binding site. We have devised a chemoproteomics approach based on a combination of unselective active-site ligands tethered to beads, enabling affinity capturing of around 40 different dioxygenase enzymes from human cells. Mass-spectrometry-based quantification of bead-bound enzymes using a free-ligand competition-binding format enabled the comprehensive determination of affinities for the cosubstrate 2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate. We also profiled a set of representative drug-like inhibitor compounds. The results indicate that intracellular competition by endogenous cofactors and high active site similarity present substantial challenges for drug discovery for this target class.
Sujet(s)
Dioxygenases/métabolisme , Acides cétoglutariques/métabolisme , ProtéomiqueRÉSUMÉ
Optimization of KDM6B (JMJD3) HTS hit 12 led to the identification of 3-((furan-2-ylmethyl)amino)pyridine-4-carboxylic acid 34 and 3-(((3-methylthiophen-2-yl)methyl)amino)pyridine-4-carboxylic acid 39 that are inhibitors of the KDM4 (JMJD2) family of histone lysine demethylases. Compounds 34 and 39 possess activity, IC50 ≤ 100 nM, in KDM4 family biochemical (RFMS) assays with ≥ 50-fold selectivity against KDM6B and activity in a mechanistic KDM4C cell imaging assay (IC50 = 6-8 µM). Compounds 34 and 39 are also potent inhibitors of KDM5C (JARID1C) (RFMS IC50 = 100-125 nM).