Selective inhibitors of the JMJD2 histone demethylases: combined nondenaturing mass spectrometric screening and crystallographic approaches - PubMed (original) (raw)

. 2010 Feb 25;53(4):1810-8.

doi: 10.1021/jm901680b.

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Selective inhibitors of the JMJD2 histone demethylases: combined nondenaturing mass spectrometric screening and crystallographic approaches

Nathan R Rose et al. J Med Chem. 2010.

Free PMC article

Abstract

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of N(epsilon)-methylated lysine residues in histones. Here we report studies on the inhibition of the JMJD2 subfamily of histone demethylases, employing binding analyses by nondenaturing mass spectrometry (MS), dynamic combinatorial chemistry coupled to MS, turnover assays, and crystallography. The results of initial binding and inhibition assays directed the production and analysis of a set of N-oxalyl-d-tyrosine derivatives to explore the extent of a subpocket at the JMJD2 active site. Some of the inhibitors were shown to be selective for JMJD2 over the hypoxia-inducible factor prolyl hydroxylase PHD2. A crystal structure of JMJD2A in complex with one of the potent inhibitors was obtained; modeling other inhibitors based on this structure predicts interactions that enable improved inhibition for some compounds.

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Figures

Figure 1

Figure 1

Reactions catalyzed by the JMJD2 histone demethylases. Each step is coupled to the conversion of O2 and 2-oxoglutarate to succinate and CO2. 2OG = 2-oxoglutarate, succ = succinate.

Figure 2

Figure 2

Structures of potential inhibitors used in this study.

Figure 3

Figure 3

Dynamic combinatorial mass spectrometry (DCMS) approach. (A) The _N_-oxalyl group of support ligand 1b anchors the molecule into the active site of JMJD2E via interaction with the Fe(II) ion (magenta), leaving the thiol side chain free for disulfide formation with the thiol library in solution. (B) Selective formation of a JMJD2E−disulfide complex with the thiol member that fits best into the active site.

Figure 4

Figure 4

Structures of the _N_-oxalylamino acids investigated in this study.

Figure 5

Figure 5

Correlation of nondenaturing ESI-MS analyses and inhibition results for JMJD2E inhibitors. (a) Compounds were grouped in five ranking sets reflecting the strength of binding to the JMJD2E·Fe(II)·Zn(II) complex (E). Rank 1: ∼1:4 ratio unbound:bound; rank 2: ∼1:2 unbound:bound; rank 3: ∼1:1 unbound:bound; rank 4: ∼4:1 unbound:bound, and rank 5: ∼10:1 unbound:bound. The MS spectra show examples of data for representative compounds from each ranking set. Some samples (11 of the 73 compounds tested) were not considered to produce spectra of sufficient quality for classification and were thus excluded from the ranking. (b) Initial rates of all compounds tested as JMJD2E inhibitors (100 μM) binding rank as determined by ESI-MS, demonstrating correlation between the two data sets. Kendall’s τB = 0.58 (p < 0.0001), Spearman’s ρ = 0.72 (p < 0.0001).

Figure 6

Figure 6

Screening for JMJD2E inhibitors. Compounds were tested as JMJD2E inhibitors (100 μM) using the FDH assay, and the percentage activities relative to the DMSO control are shown. Measurements were made in duplicate and shown as averages with standard errors of the mean.

Figure 7

Figure 7

Stereoviews from the JMJD2A:10a crystal structure. (a) Stereoview of the crystal structure of JMJD2A (transparent ribbons and green sticks) in complex with compound 10a (yellow sticks). Ni(II) (orange sphere) replaces Fe(II) in the active site; the structural Zn(II) is shown as a blue sphere. Highlighted residues (green) chelate active site metal and form part of the substrate/cosubstrate binding pocket. (b) Close-up stereoview of substrate/cosubstrate binding site with compound 10a, showing details of hydrogen bonding interactions between 10a, Lys206 and Tyr132 side chains, and the backbone amide nitrogen of Ala186.

Figure 8

Figure 8

Electron density map (stereoview) showing compound 10a (yellow sticks) in the active site of JMJD2A (green sticks). The experimental 2_F_o − _F_c electron density, displayed as blue mesh, is shown for the metal (gray sphere) coordinating residues and ligands (contoured to 1σ).

Figure 9

Figure 9

Binding of 10a to JMJD2A likely interferes with both 2OG and histone substrate binding. Superimposition of H3K9me3 substrate (orange and pink sticks) with JMJD2A·Ni(II)·Zn(II).10a structure (PDB ID 2OQ6) is shown. The surface of JMJD2A (blue) illustrates separate K9me3 (pink) and cosubstrate (NOG, cyan, replaces 2OG in this structure) subpockets. Compound 10a (yellow sticks) occupies a large hydrophobic pocket adjacent to the substrate binding cleft. It may interfere with binding of the Thr11 side chain of the H3K9 substrate.

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