Figure 1.
Screening and Hit Prioritization Strategy.
Figure 2.
Shown are representative members of clusters along with results from the screen and prioritization experiments.
Figure 3.
Structure-activity relationships for substituted 8-hydroxyquinolines identified in the qHTS against JMJD2E (qHTS IC50 values derived from the FDH-coupled assay).
Some substitution patterns were chosen for further medicinal chemistry; IC50s for these synthesized compounds are indicated in italics.
Figure 4.
JMJD2A crystal structure complexed with 5-carboxy-8-HQ (SID 85736331).
(A) active site residues are in green sticks, 5-carboxy-8-hydroxyquinoline as cyan sticks, secondary structure helices in red and sheets in yellow. The Ni(II) ion (replacing Fe(II)), is a blue sphere and the Zn(II) is a grey sphere. (B) Binding mode of 5-carboxy-8-hydroxyquinoline in JMJD2A. The experimental 2Fo-Fc electron density is shown as grey mesh (contoured at 1σ). Hydrogen bonds to residues are shown in black. Hydrogen bonds involving waters (red spheres) are shown in red, distances in Å. (C) Overlay of JMJD2A crystal structure with the 5-carboxy-8-hydroxyquinoline (residues green, compound cyan, Ni(II) in blue) with the crystal structure with 2,4 PDCA (residues compound and Ni(II) ion are pink, PDB ID: 2VD7). The distances shown are for the 8-hydroxyquinoline structure between: 5-carboxy-8-hydroxyquinoline and His 276, Ni2+ and His 188 (in black), His 276 and Ni2+ (in red). Distances in Å. (D) Surface view of superimposition of 8-hydroxyquinoline 5-acid bound JMJD2A structure with that of JMJD2A bound to histone 3 lysine 9 trimethylated (H3K9me3) substrate peptide (grey and magenta sticks) and the 2-oxoglutarate (2OG) mimetic N-oxalylglycine (NOG, yellow sticks), PDB ID: 2OQ6. Binding of the 8-hydroxyquinoline 5-acid (cyan sticks) is likely to prevent binding of 2OG but not directly that of the H3K9me3 residue (in magenta) or other residues in the substrate peptide (in grey). Peptide sequence ARK(me3)STGGK(Ac). Ni2+ of structure 2OQ6, yellow sphere; Ni2+ of 8-hydroxyquinoline structure, green sphere. The triad of metal coordinating residues are in green sticks. Crystallographic data and refinement parameters are described in Table S2.
Figure 5.
5-Carboxy-8-HQ (SID 85736331) increases H3K9me3 levels in HeLa cells through inhibition of JMJD2A.
(A) Indirect immunofluorescence with anti-Flag (green), anti-H3K9me3 (red), and DAPI staining (blue) in HeLa cells overexpressing Flag-tagged JMJD2A. DMSO solvent treatment has no effect on JMJD2A demethylase activity (white arrows) while increasing concentrations of 5-carboxy-8-hydroxyquinoline treatment (100 µM to 300µM range shown) resulted in gradual increases in H3K9Me3 levels. The JMJD2A H188A enzymatic mutant does not affect H3K9Me3 levels when overexpressed. (B) Quantitation of H3K9me3 levels is shown as squares (▪). Standard deviations are derived from biological triplicates at inhibitor concentrations of 20 µM, 50 µM, 100 µM, 200 µM, 300 µM, and 400 µM treatments. Cytotoxicity (•) was assayed at 50 µM, 100 µM, 200 µM, 300 µM, 400 µM, 500 µM, 750 µM, 1 mM, 1,25 mM, 1.5 mM, 1.75 mM, 2 mM, 2.5 mM, and 3 mM.
Figure 6.
Inhibitor screening against JMJD2 and other human 2OG oxygenases.
IC50 values from MALDI-TOF MS assay. See Figure S4 for representative concentration-response curves and MALDI-TOF mass spectra.