Selective Inhibitors of the JMJD2 Histone Demethylases: Combined Nondenaturing Mass Spectrometric Screening and Crystallographic Approaches

Article abstract of DOI:10.1021/jm901680b

Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of N^e-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.

Full text of DOI:10.1021/jm901680b

1810 J. Med. Chem. 2010, 53, 1810–1818
DOI: 10.1021/jm901680b
Selective Inhibitors of the JMJD2 Histone Demethylases: Combined Nondenaturing
Mass Spectrometric Screening and Crystallographic Approaches†
‡
Nathan R. Rose, Esther C. Y. Woon, Guy L. Kingham, Oliver N. F. King, Jasmin Mecinovic, Ian J. Clifton,
‡
‡
‡
‡
ꢀ ‡
Stanley S. Ng,§ Jobina Talib-Hardy,‡ Udo Oppermann,§ Michael A. McDonough,‡ and Christopher J. Schofield*,‡
‡The Department of Chemistry and the Oxford Centre for Integrative Systems Biology, Chemistry Research Laboratory, University of Oxford,
12 Mansfield Road, Oxford, OX1 3TA, United Kingdom, and Structural Genomics Consortium, University of Oxford, Old Road Campus
§
Roosevelt Drive, Headington OX3 7DQ, United Kingdom, The Botnar Research Centre, Oxford Biomedical Research Unit, Oxford OX3 7LD,
United Kingdom
Received November 13, 2009
Ferrous ion and 2-oxoglutarate (2OG) oxygenases catalyze the demethylation of Nε-methylated lysine resi-
dues 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.
Introduction
JMJD2E has been classified as a pseudogene,8 recombinant
JMJD2E has HDM activity, and is amenable to inhibition
studies.9 JMJD2F has not been characterized.
Histone lysine methylation is a dynamic post-translational
modification, with sequence-specific N-methyltransferases
and demethylases acting to add and remove methyl groups
(Figure 1).1 The methylation (or demethylation) of specific
lysine residues can enable both euchromatic or heterochro-
matic states, thus transcriptionally activating or silencing
genes. Histone demethylases (HDMsa) are implicated in dis-
eases including leukemia, prostate cancer, squamous cell
carcinoma, and X-linked mental retardation.2 The largest
identified family of HDMs is the JmjC-domain-containing
family, the members of which are Fe(II) and 2-oxoglutarate
(2OG) dependent oxygenases.3,4
The human JMJD2 HDM subfamily has six members
(JMJD2A, 2B, 2C, 2D, 2E, and 2F), of which JMJD2A-C
have been shown to catalyze demethylation of the methylated
forms of histone 3 lysine 9 (H3K9) and histone 3 lysine 36
(H3K36) in vitro and in cells, while JMJD2D is selective for
the demethylation of H3K9.4-7 JMJD2C has been implicated
in prostate cancer, and inhibition of JMJD2C expression has
been shown to decrease cancer cell proliferation.4 Although
The precise in vivo roles of individual HDMs in the
regulation of gene expression are unclear or only beginning
to emerge. A degree of redundancy likely exists, with different
demethylases targeting the same histone methyllysines. Small
molecule chemical probes may be useful for investigating the
in vivo role of these demethylases,10 and the therapeutic
potential of HDM inhibitors is being considered.11 To be
useful as probes, HDM inhibitors should not inhibit at least
some of the other Fe(II)/2OG dependent oxygenases, e.g.,
those involved in the hypoxic response pathway, including the
hypoxia-inducible factor (HIF) asparaginyl hydroxylase,
factor-inhibiting HIF (FIH), and the human HIF prolyl
hydroxylase domain 2 (PHD2), because the activity of these
enzymes regulates a large array of human genes.12-14
Recently, wereported screening methods and several HDM
inhibitor “scaffolds” for the JMJD2 HDM subfamily.9 Com-
pounds analyzed included the known histone deacetylase
inhibitors suberoylanilide hydroxamic acid (SAHA), trichos-
tatin A (TSA), pyridine-dicarboxylic acids, and various other
2OG analogues. The latter included N-oxalyl-D-phenylala-
nine 1d, which has previously been shown to be selective for
the inhibition of FIH with respect to PHD2;15 this selectivity
was achieved by exploiting a hydrophobic subpocket adjacent
to the 2OG binding site present in FIH but not in PHD2. FIH
has been regarded as part of the Jmj subfamily of 2OG
dependent oxygenases6,16,17 and is more similar in sequence
and structure to the JMJD demethylases than it is to other
human 2OG oxygenase subfamilies.12
†PDB ID 2WWJ.
*To whom correspondence should be addressed. Phone: þ44 (0) 1865
275 625. Fax: þ44(0)1865 285 022. E-mail: Christopher.schofield@
chem.ox.ac.uk.
aAbbreviations: ESI-MS, electrospray ionization-mass spectrometry;
2OG, 2-oxoglutarate; siRNA, small-interfering ribonucleic acid; JMJD,
Jumonji domain containing protein; PHD, prolyl hydroxylase domain
containing protein; FIH, factor-inhibiting hypoxia-inducible factor;
HIF-1R, hypoxia inducible factor-1R; SAHA, suberoylanilide hydro-
xamic acid; DCMS, dynamic combinatorial mass spectrometry; FDH,
formaldehyde dehydrogenase; CODD, C-terminal oxygen degradation
domain; CAD, C-terminal transactivation domain; NOG, N-oxalylgly-
cine; TCA, tricarboxylic acid; HDM, histone demethylase.
Crystallographic studies and modeling imply that the
JMJD2s possess a similar subpocket to that observed for FIH,
r
pubs.acs.org/jmc
Published on Web 01/20/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1811
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.
which is adjacent to the active site.15,18 However, the crystal-
lographic analyses imply that the subpocket of the JMJD2s
is significantly larger and more open than that of FIH
and extends into the substrate binding groove. We set out
to explore this subpocket by derivatization of N-oxalyl-
D-phenylalanine 1d as a possible means of achieving selectivity
toward the JMJD2 subfamily. We used a combined approach
employing binding assays using nondenaturing mass spectro-
metry, disulfide exchange based dynamic combinatorial
chemistry, inhibition studies, and crystallographic analyses,
which led to the identification of N-oxalyl-D-tyrosine deriva-
tives as potent inhibitors of JMJD2E.
Results
Initially, we used nondenaturing electrospray ionization
mass spectrometry (ESI-MS) to assess binding of potential
inhibitors to JMJD2E.19 When the target protein is amenable
Figure 2. Structures of potential inhibitors used in this study.
to such assays, the stoichiometry of interaction and binding
strength can be rapidly assessed. This method has been used in
the study of metallo-enzyme inhibition (e.g., refs 20, 21) and
enzyme-metal ion interactions.22 In these ESI-MS analyses,
it is important to appreciate that different types of noncova-
lent interaction survive the transition from solution to gas-
phase differently (for review, see ref 6). However, in some cases,
including metallo-enzymes,20-25 good agreement between results
from nondenaturing ESI-MS binding data and solution-phase data
can occur.
Our starting points for the binding assay were the 2OG
analogues that we had identified in the initial study aimed at
identifying “template” inhibitors of JMJD2E suitable for deri-
vatization, in particular N-oxalylglycine 1a (IC50=24 μM) and
N-oxalyl-D-homophenylalanine 1h (IC50 =39 μM).9 A set of
N-oxalyl-D-amino acids 1b-g and their corresponding L-iso-
mers 1i-n were assessed for potential binding to the JMJD2E
3
Fe(II) Zn(II) complex (hereafter JMJD2E) (Figure 2) by non-
3
denaturing ESI-MS screening. The results revealed that of
the 13 compounds tested, N-oxalylglycine 1a and N-oxalyl-
D-cysteine 1b bound to JMJD2E (41256 Da), with formation
of new peaks at 41403 and 41448 Da, corresponding to
JMJD2E 1a and JMJD2E 1b complexes, respectively. Rela-
3
3
tively weak binding was observed for N-oxalyl-D-phenylalanine
1d (JMJD2E 1d: 41493 Da), N-oxalyl-D-valine 1f (JMJD2E 1f:
41445 Da), and N-oxalyl-D-alanine 1g (JMJD2E 1g: 41417 Da).
3
3
Figure 3. Dynamic combinatorial mass spectrometry (DCMS) ap-
proach. (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.
3
These observations were in general agreement with the
formaldehyde dehydrogenase (FDH) coupled assay inhibition
results (measuring JMJD2E turnover with a histone H3 Lys9
trimethylated (H3K9me3) peptide fragment substrate) (Table
S1, Supporting Information). It is notable that while some of the
N-oxalyl-D-amino acids are active, none of the L-compounds
to N-oxalyl-L-cysteine 1i (IC50>1 mM) for inhibition of
JMJD2E.
1i-n bind to JMJD2E, and all, with the exception of 1n (IC50
=
285 μM), are inactive as inhibitors in FDH coupled assays.
The importance of the D-stereochemistry in the N-oxalyl
amino acid series is demonstrated by the significantly greater
potency of N-oxalyl-D-cysteine 1b (IC50 = 73 μM) compared
To explore the extent of the JMJD2E 2OG-substrate bind-
ing pocket that is accessible to the N-oxalyl amino acid series,
a disulfide exchange based dynamic combinatorial-mass spec-
trometry (DCMS) screen21,24,26 was then performed. The

1812 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Rose et al.
Figure 4. Structures of the N-oxalylamino acids investigated in this study.
DCMS screen exploited the thiol side chain of N-oxalyl-
D-cysteine 1b, which is predicted to project into the subpocket
of JMJD2E as a “support” ligand (Figure 3). In this method, a
compound that binds to the active site (the support ligand)
which contains a thiol is allowed to react (either on the enzyme
in the active site, or in solution) with a set of thiols to form a
mixture of disulfides. Nondenaturing ESI-MS is then used to
analyze which disulfides bind preferentially to the enzyme.
A mixture of five thiols, each thiol at 15 μM, including
thiophenol, 3-bromothiophenol, 3-methoxythiophenol, pro-
panethiol, and 2-naphthalenethiol, was mixed with an equi-
molar amount of 1b (15 μM) and JMJD2E (15 μM) in
ammonium acetate (15 mM, pH 7.5) under aerobic condi-
tions. The mixture was then analyzed for binding with
JMJD2E (mass 41264 Da) by nondenaturing ESI-MS. New
complexes with peaks at 41587 and 41557 Da were observed
(Figure S1, Supporting Information); these peaks correspond
to the additionof disulfides 1b 3-methoxythiophenol (2a) and
3
1b thiophenol (2b) to JMJD2E, respectively. This result was
3
confirmed by deletion experiments in which specific thiols
were removed from the mixture. Because disulfides are not
necessarily stable under the FDH coupled assay conditions,
the carbon analogues, 3a and 3b, respectively, wherein the
disulfide bond is replaced with a C-S bond, were then
synthesized (Figure 2). Both 3a and 3b bind to JMJD2E, as
shown by nondenaturing ESI-MS. The FDH coupled
assay results indicate that both carbon analogues 3a (IC50 =
204 μM) and 3b (IC50 = 300 μM) are less potent as JMJD2E

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1813
inhibitors compared to the support compound 1b (IC50
=
73 μM). This difference could be due to the differences in
interactions in solution and in nondenaturing ESI-MS condi-
tions or the inability of the carbon compounds to mimic the
S-S bond in disulfides. Nonetheless, the DCMS method
revealed that larger side-chains than those of N-oxalyl-
D-cysteine could likely be accommodated in subpocket of
the JMJD2E active site.
It was noted that disulfide 2a binds slightly more strongly
to JMJD2E than 2b, indicating that the presence of the
methoxy group may improve inhibitory activity. This led us
to consider using N-oxalyl-D-tyrosine 1c as a lead for further
optimization of the aromatic ring. Consequently, a set of
N-oxalyl-D-tyrosine derivatives (5a-12, Figure 4) substituted
at the tyrosinyl-OH position with a variety of substituents,
including benzoate esters (5a-j), other aliphatic/aromatic
esters (6a-k), sulfonate esters (7a-j), aliphatic ethers (9a-i),
β-ketoethers (8a-m, 11a-c, 12) and benzyl ethers (10a-q)
were analyzed for binding to, and inhibition of, JMJD2E.
The N-oxalyl-D-tyrosine derivatives (5a-12, Figure 4) were
initially screened for binding affinity to JMJD2E by the
nondenaturing ESI-MS method. The binding affinity of each
compound was classified into one of five rank classes. Com-
petition experiments (data not shown) showed that the top
11 compounds all bound more tightly than either N-oxalylgly-
cine 1a or N-oxalyl-D-phenylalanine 1d.
The N-oxalyl-D-tyrosinyl derivatives were then tested (at
100 μM) for activity against JMJD2E using the FDH based
coupled assay (Figure 6),9 and the compounds were ranked by
inhibitory potency based on the reduction in initial demethy-
lation rate observed. IC50s were then determined for nine of
the most potent compounds 7f, 7c, 7e, 10k, 7d, 8m, 6d, 10e,
10a, and three of the least potent compounds 7i, 8b, and 9h
as a control (Table 1). The most potent compound in the
FDH coupled assay was 7f, with an IC50 of 5.4 μM; other
potent compounds also had IC50s in the low micromolar
range (8-40 μM). This represents an improvement of potency
with respect to N-oxalylglycine 1a (IC50 = 24 μM), N-oxalyl-
D-phenylalanine 1d (IC50 = 320 μM), and N-oxalyl-D-homo-
phenylalanine 1h (IC50 = 39 μM).9
The rankings of binding affinity as determined by nonde-
naturing ESI-MS and inhibitory potency by the FDH coupled
assay were compared using Kendall’s rank correlation (τB =
0.58, p<0.0001) and Spearman’s rank correlation (F=0.72,
p<0.0001). These results indicate a statistically significant
positive correlation between the inhibitor rankings by non-
denaturing ESI-MS and by FDH coupled assay activity
(Figure 5b). Because there are differences in the physical
interactions between ligand and enzyme in the gas phase as
opposed tosolution phase (ingas phase, ionicinteractions and
hydrogen bonding interactions are believed to be preserved
more efficiently than hydrophobic interactions),19 inhibitors
which rely on nonionic interactions for binding to the enzyme
in solution may show relatively weaker binding affinity in the
mass spectrometric screen. Nonetheless the results reveal that
screening for binding by nondenaturing ESI-MS is an efficient
and useful method for ranking affinity, which is amenable to
medium-throughput analyses and displays a strong correla-
tion to the FDH coupled assay inhibition data.
Figure 5. Correlation of nondenaturing ESI-MS analyses and in-
hibition 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 un-
3
3
bound:bound; rank 2: ∼1:2 unbound:bound; rank 3: ∼1:1 un-
bound: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
τ
B = 0.58 (p < 0.0001), Spearman’s F = 0.72 (p < 0.0001).
PHD2, analyzed by MALDI-TOF MS27). No inhibitory
activity toward PHD2 was observed in the biochemical assay
for the three N-oxalyl-D-tyrosine derivatives 7f, 7c, and 7e or
for 1d; N-oxalylglycine 1a, however, was a potent inhibitor of
PHD2 as previously reported.28-31 Some binding was ob-
served between these inhibitors and PHD2 by nondenaturing
ESI-MS. However, nondenaturing ESI-MS binding assays in
the presence of the known potent inhibitor 1a showed binding
of both 1a and either 7f, 7c, and 7e simultaneously, suggesting
that binding of these N-oxalylamino acids was not occurring in
the 2OG binding site of the enzyme. Thus, these inhibitors
are selective for the JMJD2 HDMs over the human prolyl
hydroxylase PHD2. This result is consistent with the lack of a
large pocket adjacent to the 2OG side chain binding site in
PHD2 as opposed to that observed for JMJD2A.18,32,33
The most potent inhibitors identified by these screens
(7f, 7c, and 7e, as well as previously described inhibitors 1a
and 1d) were also screened against PHD2 by nondenaturing
ESI-MS binding affinity assays and biochemical activity
assays (hydroxylation of CODD peptide substrate by
Compounds 7f and 7c were also screened against FIH
using a MALDI-TOF assay as reported.34 Dose-response
curves representing the extent of hydroxylation as a function
of inhibitor concentration were used to determine IC50s. Both of

1814 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Rose et al.
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.
Table 1. IC50 Values for the Most Potent Compounds Tested (7f, 7c,
these compounds, 7f and 7c (IC50 values for JMJD2E of 5.4
7e, 10k, 7d, 8m, 10e, 6d, 10a), and for Three Less Potent Compounds
(7i, 8b, 9h) (JMJD2E Concentration = 2 μM)
and 8.6 μM, respectively), inhibited FIH with IC50 values of
60.2 and 37.2 μM, respectively. Note that direct comparisons of
IC50 values determined for JMJD2E and FIH should be treated
ratio of JMJD2E-inhibitor
complex to free JMJD2E
(by ESI-MS)
IC50
(μM)
with caution because the assay used for FIH relies on measure-
ment of hydroxylation after a significantly longer incubation
period rather than those used in JMJD2E FDH coupled assay.
However, the results suggest that, at least for compounds 7f
and 7c, selectivity of inhibition with regard to FIH was
relatively low as might be expected based on the similarity of
its 2OG binding subpocket to that of the JMJD2s (Figure S2,
Supporting Information).
compd
7f
7c
5.4
8.6
4.3
poor spectrum
poor spectrum
7e
11.7
14.7
14.7
14.8
16.6
20.1
37.1
82.9
185.1
326.5
10k
7d
3.1
5.7
2.0
3.6
3.5
1.7
1.0
0.9
0.9
8m
10e
6d
Crystallization of the most potent N-oxalyl-D-tyrosine
inhibitors (Table 1) together with JMJD2A was then attemp-
ted, and a crystal structure was obtained for compound 10a in
10a
7i
˚
8b
9h
complex with JMJD2A to 2.4 A resolution (Figure S3 and
Table S2, Supporting Information), confirming the predicted
binding mode for N-oxalyl-D-tyrosinyl derivatives. 10a was
observed to be bound to the active site of both the JMJD2A
molecules that are present in the asymmetric unit. The oxalyl
carbonyloxygen and one ofthe carboxylicacid oxygensof10a
chelate the active site metal in a manner analogous to 2OG
superimposed on the JMJD2A:10a structure (Figure 9),
no overlap is observed between the inhibitor side chain and
the substrate K9me3 side chain (Supporting Information
Figure S3). However, the benzyl ether moiety of 10a is
observed to partially occupy the same space as Thr11 and
Gly12 of the H3K9me3 substrate, indicating that the binding
of 10a (and probably the other potent inhibitors) to JMJD2A
likely interferes with binding of the H3K9me3 substrate in
addition to competing with 2OG.
To investigate the rationale for the increased potency of
the sulfonates 7c-f relative to the other derivatives, inhibitors
7f, 10k, 6d, and 8m were manually modeled into the active
site of the JMJD2A:10a structure by superimposing their
structures with 10a and altering the position of each tyrosine
substituent to reduce steric clashes and to maximize hydrogen
bonding interactions (Figure S4 and Table S3, Supporting
Information). All compounds were constrained to form a
hydrogen bond between the tyrosinyl oxygen and the backbone
amide nitrogen of Ala186, as observed in the JMJD2A:10a
structure (Figure S4). In all of these compounds, additional
hydrogen bonds were predicted to occur compared to those
observed in the JMJD2A:10a crystal structure. Modeling
studies also suggest reasons why 7i, 8b, and 9h are less potent
˚
and N-oxalylglycine 1a (avg. 2.2 A) (Figures 7, 8). The other
carboxylic acid of 10a forms a salt bridge with Lys206 Nε
˚
˚
(2.8 A) and a hydrogen bond with Tyr132 OH (2.4 A).
Apparent hydrophobic interactions between the tyrosinyl side
chain of 10a and JMJD2A include side chains of residues
Ile71, Tyr132, Tyr177, Phe185, and Lys241 (methylenes). The
tyrosinyl side-chain oxygen of 10a is positioned to make a
˚
weak hydrogen bond (3.7 A) with the backbone amide nitro-
gen of Ala186. All residues involved in binding 10a are
conserved between JMJD2A (used for crystallographic ana-
lysis) and JMJD2E (presently more amenable to biochemical
inhibition assays), and the most potent JMJD2E inhibitors
displayed activity against JMJD2A in a MS-based turnover
assay (Table 2).
Comparison of the JMJD2A:10a crystal structure with the
JMJD2A-H3K9me3 substrate complex (PDB ID 2OQ6)
revealed little difference in the conformation of the active
site residues (Supporting Information Table S5). When
the JMJD2A:H3K9me3 substrate complex structure is

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1815
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. Electron density map (stereoview) showing compound 10a (yellow sticks) in the active site of JMJD2A (green sticks). The
experimental 2Fo - Fc electron density, displayed as blue mesh, is shown for the metal (gray sphere) coordinating residues and ligands
(contoured to 1σ).
inhibitors: steric interaction in the case of 7i and 8b, and
conformational flexibility in the case of 9h.
that binding strength under noncatalytic conditions does not
necessarily reflect inhibitory potency. One advantage of a
binding-type assay is that it can be used to identify (potential)
inhibitors in the absence of a catalytic turnover assay which
requires knowledge of the substrate (substrates are not yet
known for many of the 2OG oxygenases).35
Our work has also identified inhibitors selective for inhibi-
tion of the JMJD2 histone demethylase subfamily members
over the HIF hydroxylase PHD2, in that the most potent
inhibitors of JMJD2E did not inhibit PHD2 (within the
limits of detection), as predicted from structural comparisons
Taken together, our results indicate that a combined ap-
proach involving nondenaturing ESI-MS binding assays to
identify hits followed by biochemical assays provides an
efficient approach to lead identification of inhibitors for
metallo-enzymes. The statistical correlation between the bind-
ing strength of compounds to the enzyme under nondenatur-
ing ESI-MS conditions and the ability of compounds to
inhibit enzyme activity in biochemical assays validates the
nondenaturing ESI-MS method although it is appreciated

1816 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Rose et al.
Table 2. Inhibition Data for JMJD2A with Selected Compounds by the
MALDI-TOF Assaya
aluminum-backed plates (Merck, silica 60 F254). Melting points
were determined using a Leica Galen III hot-stage melting point
apparatus and microscope. Infrared spectra were recorded from
Nujol mulls between sodium chloride discs on a Bruker Tensor
27 FT-IR spectrometer. NMR spectra were acquired using a
Bruker DPX500 NMR spectrometer. Chemical shifts (δ) are
giveninppm, andthe multiplicitiesare givenas singlet (s), doublet
(d), triplet (t), quartet (q), multiplet (m), andbroad (br). Coupling
constants J are given in Hz to the nearest 0.1 Hz. High resolution
mass spectra (HRMS) were recorded using Bruker MicroTOF.
The purity of all compound synthesized were >95% as deter-
mined by analytical reverse-phase HPLC (Ultimate 3000).
The chemical synthesis and purity of 3a and 3b are described in
the Supporting Information. The 73 N-oxalyl-D-tyrosine deriva-
tives were purchased from ChemOvation (Horsham, UK). The
identity and purity of the lead compounds were confirmed by 1H
NMR, HRMS, and HPLC (for all compounds), and these data
(for compounds 6d, 7c, 7d, 7e, 7f, 7i, 8b, 8m, 9h, 10a, 10e, and 10k)
are given in the Supporting Information.
JMJD2E IC50
by FDH
JMJD2E IC50 by
MALDI-TOF
(μM)
JMJD2A IC50 by
MALDI-TOF
(μM)
compd
assay (μM)
7f
7c
5.4
8.6
21
61
12
13
7e
11.7
14.7
14.7
14.8
16.6
37.1
82.9
185.1
326.5
38
47
20
14
10k
7d
43
59
17
13
8m
10e
10a
7i
25
76
10
33
ND
ND
ND
ND
ND
ND
8b
9h
a JMJD2A was used at 2 μM, 2OG at 10 μM and ARKme3STGGK
peptide at 10 μM. JMJD2E IC50s were also determined by MALDI-TOF
MS for comparison. ND = not determined.
The catalytic domain of human JMJD2E (residues 1-337) was
produced as N-terminally His6-tagged protein in Escherichia coli
and purified by Ni-affinity chromatography as reported.18
JMJD2E inhibition was assessed using a FDH coupled assay,
as reported.9 All compounds were initially tested at 100 μM, and
the initial rates of demethylation measured by measuring NADH
production using an Envision multilabel reader (Perkin-Elmer,
Waltham, MA).
For JMJD2A, a MALDI-TOF MS based assay was used
becuase JMJD2A was not optimized for analysis in our current
FDH assay. JMJD2A 2 μM, Fe(II) 10 μM, and ascorbate 100 μM
in 50 mM HEPES pH 7.5 with inhibitor stock DMSO solutions
where final inhibitor concentrations varied but final DMSO
concentration was always 5% of assay mix were incubated for
15 min at 25 °C, after which time reactions were initiated by
addition of 2OG (10 μM) and peptide (10 μM), followed by 30
min incubation at 37 °C. Reactions were quenched with methanol
1:1 (v/v) followed by addition of four volumes of 20 mM
triammonium citrate. The diluted assay mixture (1 μL) was then
mixed with 1 μL of R-cyano-4-hydroxycinnamic acid (the MAL-
DI-TOF-MS matrix) and spotted onto a MALDI-TOF-MS
plate.18 The relative intensities of different methylation states
observed in the mass spectra were then used to calculate percen-
tage demethylation. IC50s were calculated from the variation in
percentage demethylation at different inhibitor concentrations.
FIH and PHD2 assays were carried out as reported.27,34 The
binding of compounds to JMJD2E was evaluated by nondena-
turing ESI-MS as described.9 His-tagged JMJD2E was desalted
using a Bio-Spin 6 Column (Bio-Rad, Hemel Hempstead, UK)
in 15 mM ammonium acetate pH 7.5. The stock solution was
diluted with the same buffer to a final concentration of 100 μM.
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 sub-
strate binding cleft. It may interfere with binding of the Thr11 side
chain of the H3K9 substrate.
3
3
FeSO4 7H2O was dissolved in 20 mM HCl at a concentration of
of the binding pockets. Binding within the identified JMJD2
subpocket may be a way of achieving more potent inhibition
with other series of “template” inhibitors that bind to the
active site iron. These compounds may be useful as chemical
tools to probe the in vivo roles of the JMJD2 demethylases.
However, those inhibitors screened against FIH did inhibit
HIF asparaginyl hydroxylation, demonstrating that further
expansion of this, or related series of compounds that chelate
the active site iron, is required to achieve higher selectivity
within the JmjC subfamily. In this regard, it is notable that
although both FIH and the JMJD2 demethylases contain a
relatively large subpocket, there are significant differences in
the residues that form these subpockets.
3
100 mM. This was then diluted with Milli-Q water to give final
working concentrations of 100 μM. The protein (15 μM) was
mixed with 1 equiv of Fe(II) and 1 equiv of inhibitor and
incubated for 30 min at 37 °C prior to nondenaturing ESI-MS
analysis. For competition experiments, the protein was mixed
with equimolar amounts of Fe(II) and two inhibitors each at
concentration of 15 μM and incubated for 30 min at 37 °C prior
to nondenaturing ESI-MS analysis. Data were acquired on a
Q-TOF mass spectrometer (Q-TOF micro, Micromass, Altrinc-
ham, UK) interfaced with a Nanomate (Advion Biosciences,
Ithaca, NY) with a chip voltage of 1.70 kV and a delivery pressure
0.25 psi (1 psi = 6.81 kPa). The sample cone voltage was typically
80 V, with a source temperature of 40 °C and with an acquisition/
scan time of 10 s/1 s. Calibration and sample acquisition were
performed in the positive ion mode in the range of 500-5000 m/z.
The pressure at the interface between the atmospheric source and
the high vacuum region was fixed at 6.60 mbar. External instru-
ment calibration was achieved by using sodium iodide. Data were
processed with MASSLYNX 4.0 (Waters). The limit of detection
Experimental Section
All reagents and solvents were purchased from Aldrich or
Alfa Aesar and used without further purification. Reactions
were monitored by TLC, which was performed on precoated

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1817
by this assay is an IC50 of ∼1 μM because the JMJD2E enzyme
concentration required to achieve sufficient sensitivity in the
current assay conditions is 2 μM.
Statistical analyses on correlation between MS and % inhibi-
tion rankings were carried out using Kendall’s τB and Spearman’s
rank correlation tests, using StatsDirect, http://www.statsdirect.
com.
(8) Katoh, M.; Katoh, M. Identification and characterization of
JMJD2 family genes in silico. Int. J. Oncol. 2004, 24 (6), 1623–1628.
ꢀ
ꢀ
(9) Rose, N. R.; Ng, S. S.; Mecinovic, J.; Lienard, B. M. R.; Bello, S. H.;
Sun, Z.; McDonough, M. A.; Oppermann, U.; Schofield, C. J.
Inhibitor Scaffolds for 2-Oxoglutarate-Dependent Histone Lysine
Demethylases. J. Med. Chem. 2008, 51 (22), 7053–7056.
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Chem. Biol. 2009, 5 (7), 436–440.
Crystals of JMJD2A:10a were grown by vapor diffusion at
4 °C in 200 nL sitting drops, with a 1:1 protein to reservoir
solution ratio. The JMJD2A protein solution for crystalliza-
tion was composed of 11.0 mg/mL protein, 10 mM HEPES pH
7.5, 500 mM NaCl, 5% v/v glycerol, and 0.75 mM 10a. The
reservoir solution included 0.1 M citrate pH 5.5, 18.5% w/v
polyethylene glycol 3350, 4 mM NiCl2. The crystals were cryo-
protected by transferring to 25% v/v glycerol in well solution and
flash frozen by rapidly plunging in liquid nitrogen. Data were
collected from a single crystal at 100 K at the Diamond Light
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E. S.; Lienard, B. M. R.; Bray, J. E.; Savitsky, P.; Gileadi, O.; von
Delft, F.; Rose, N. R.; Offer, J.; Scheinost, J. C.; Borowski, T.;
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Combined Mass Spectrometry and Dynamic Chemistry Approach
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˚
Source beamline I03 using 0.97860 A wavelength beam. The data
were processed with MOSFLM and SCALA.36 Iterative rounds
of model building in COOT37 and refinement using REFMAC36
resulted in the final model with R = 18.1% and Rfree = 24.8%.
All residues were in acceptable regions of a Ramachandran plot
as calculated by MolProbity.38 Refinement details are shown in
Table S2 of the Supporting Information.
Acknowledgment. This work was supported by the Com-
monwealth Scholarship Commission (N.R.), the Newton-
Abraham Fund (J.M.), the Biotechnology and Biological
Sciences Research Council, Cancer Research UK (G.K.), the
Wellcome Trust, and the European Union, The Structural
Genomics Consortium is a registered charity (no. 1097737) that
receives funds from multiple sources, details of which are found
in the Supporting Information. The project was supported by
the Oxford NIHR Musculoskeletal Biomedical Research Unit.
We thank the staff at Diamond Light Source for their help and
support and for providing access to the beamline to carry out this
research. Conflict of interest statement: C.J.S. is a cofounder of
Re-Ox Ltd., a company that aims to exploit research about the
hypoxic response for therapeutic benefit.
(21) Lienard, B. M.; Huting, R.; Lassaux, P.; Galleni, M.; Frere, J. M.;
Schofield, C. J. Dynamic combinatorial mass spectrometry leads to
metallo-beta-lactamase inhibitors. J. Med. Chem. 2008, 51 (3), 684–
688.
(22) Mecinovic, J.; Chowdhury, R.; Lienard, B. M.; Flashman, E.;
Buck, M. R.; Oldham, N. J.; Schofield, C. J. ESI-MS studies on
prolyl hydroxylase domain 2 reveal a new metal binding site.
ChemMedChem 2008, 3 (4), 569–572.
Supporting Information Available: Compound quality ana-
lyses, inhibition assay methods, mass spectrometric data, and
crystallization and structure solution methods. This material is
available free of charge via the Internet at http://pubs.acs.org.
(23) Mecinovic, J.; Loenarz, C.; Chowdhury, R.; Schofield, C. J.
2-Oxoglutarate analogue inhibitors of prolyl hydroxylase domain
2. Bioorg. Med. Chem. Lett. 2009, 19 (21), 6192–6195.
(24) Poulsen, S. A. Direct screening of a dynamic combinatorial library
using mass spectrometry. J. Am. Soc. Mass Spectrom. 2006, 17 (8),
1074–1080.
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