Inhibitor scaffolds for 2-oxoglutarate-dependent histone lysine demethylases

Article abstract of DOI:10.1021/jm800936s

The dynamic methylation of histone lysyl residues plays an important role in biology by regulating transcription, maintaining genomic integrity, and by contributing to epigenetic effects. Here we describe a variety of inhibitor scaffolds that inhibit the human 2-oxoglutarate-dependent JMJD2 subfamily of histone demethylases. Combined with structural data, these chemical starting points will be useful to generate small-molecule probes to analyze the physiological roles of these enzymes in epigenetic signaling.

Full text of DOI:10.1021/jm800936s

J. Med. Chem. 2008, 51, 7053–7056
7053
Modulation of histone modifications has significant medicinal
potential. At present, the histone deacetylase (HDAC) inhibitor
suberoylanilide hydroxamic acid (SAHA) is used clinically in
cancer treatment2 and monoamine oxidase inhibitors used in
psychotherapy inhibit lysine-specific demethylase 1, LSD1, a
flavin adenine dinucleotide-dependent demethylase that catalyzes
demethylation of di- and monomethyl lysine residues.3 Small
molecules also have potential as tools to dissect the roles of
epigenetic modifications of both proteins and nucleic acids, not
only in cell-based studies but particularly in studying intergen-
erational effects. Here we report assays for inhibitors of the
JMJD2 histone demethylase subfamily, with the objective of
identifying different scaffolds for histone demethylase inhibition.
Inhibitor Scaffolds for
2-Oxoglutarate-Dependent Histone Lysine
Demethylases
Nathan R. Rose,† Stanley S. Ng,‡ Jasmin Mecinovic´,†,§
Benoˆıt M.R. Lie´nard,†,§ Simon H. Bello,† Zhe Sun,‡
Michael A. McDonough,† Udo Oppermann,*,‡ 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,
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
To identify a suitable JMJD2 enzyme for inhibition assays,
we produced the recombinant forms of the catalytic domains
(Supporting Information, Figure S1) of five of the six predicted
human JMJD2 demethylase genes (JMJD2A, B, C, D, and E)4
in Escherichia coli and purified them to >95% purity (by SDS-
PAGE analyses, Supporting Information, Figure S2). Of the five
enzymes, JMJD2E was chosen as the most suitable for inhibition
work because of its stability, activity levels, and substrate
selectivity profile. Kinetic assays employing MALDI-TOF
(matrix-assisted laser desorption ionization-time-of-flight) mass
spectrometry (MS) led to the selection of an 8-residue
(ARKme3STGGK) histone H3 fragment substrate (H3K9me3).
A coupled-assay for JMJD2E activity employing formaldehyde
dehydrogenase (FDH) from Pseudomonas putida was developed
based on that reported for JMJD2A.5 Formaldehyde release by
demethylation of the histone peptide substrate was monitored
by its oxidation to give formate as catalyzed by FDH, which is
carried out concomitantly with the reduction of nicotinamide
adenine dinucleotide (NAD+). The production of NADH was
monitored by fluorescence spectroscopy (Table 1).
ReceiVed July 25, 2008
Abstract: The dynamic methylation of histone lysyl residues plays
an important role in biology by regulating transcription, maintaining
genomic integrity, and by contributing to epigenetic effects. Here we
describe a variety of inhibitor scaffolds that inhibit the human
2-oxoglutarate-dependent JMJD2 subfamily of histone demethylases.
Combined with structural data, these chemical starting points will be
useful to generate small-molecule probes to analyze the physiological
roles of these enzymes in epigenetic signaling.
A ubiquitous subfamily of Fe(II) and 2-oxoglutarate (2-
OG)a dependent oxygenases (JmjC-domain-containing enzymes)
present in all eukaryotes catalyzes the demethylation of all three
Nε-methylated forms of histone lysyl residues (tri-, di-, and
monomethyl) (for review, see ref 1). Demethylation is proposed
to occur via methyl group hydroxylation followed by fragmen-
tation to produce formaldehyde. Members of the JMJD2 histone
demethylase subfamily are reported to display selectivity for
the demethylation of transcriptionally inactivating tri- and
dimethyl H3K9 (histone H3, Lys-9) and transcriptionally
activating methyl-H3K36 (histone H3, Lys-36). Other JmjC
subfamilies, such as the JARID subfamily, are selective for
demethylation of transcriptionally activating methyl-H3K4
(histone H3, Lys-4).1
Studies on the selective inhibition of 2-OG oxygenases,
including the hypoxia inducible factor (HIF) hydroxylases
(PHD1-3 and FIH), have identified analogues of 2-OG useful
as inhibitors, including N-oxalyl glycine 1a (Table 1). 1a inhibits
the human HIF prolyl hydroxylase PHD2 with Ki ) 8 µM, and
the asparaginyl hydroxylase factor-inhibiting-HIF (FIH) with
Ki ) 1.2 mM.6-8 1a inhibited JMJD2E with an IC50 value of
78 µM; this value decreased to 24 µM with 30 min preincubation
(Table 1). Comparison of the JMJD2A ·Ni(II)·Zn(II)·1a crystal
structure9 complexes with that of FIH ·Fe(II)·1b8 revealed a
largely hydrophobic binding pocket adjacent to the 2-OG
binding site in the JMJD2 enzymes, suggesting that stereospe-
cific modification of 1a at the C-R position may enhance
inhibition or enable selectivity. N-Oxalyl-D-phenylalanine 1b
and N-oxalyl-D-homophenylalanine 1c inhibited JMJD2E with
IC50 ) 320 µM and 100 µM (39 µM with preincubation),
respectively. While these inhibitors are not more potent than
1a, modification of 1a to enable binding in the hydrophobic
pocket enables selective inhibition of 2-OG oxygenases, as
precedented for the HIF hydroxylases;12 1b inhibits FIH with
Ki ) 83 µM but is a much less potent inhibitor of PHD2, with
IC50 > 1 mM.8
PDB ID for crystal structure of JMJD2A complexed with inhibitor
pyridine-2,4-dicarboxylic acid: 2VD7.
* To whom correspondence should be addressed. For C.J.S.: phone,
+441865275625; Fax, +441865275674; E-mail, christopher.schofield@
chem.ox.ac.uk. For U.O.: phone, +441865617585; fax, +441865617575;
E-mail, udo.oppermann@sgc.ox.ac.uk.
†
Department of Chemistry, University of Oxford.
Structural Genomics Consortium and Botnar Research Centre.
These authors contributed equally to the work.
‡
§
a Abbreviations: 2-OG, 2-oxoglutarate; C-P3H, collagen prolyl-3-
hydroxylase; C-P4H, collagen prolyl-4-hydroxylase; DMSO, dimethyl
sulfoxide; ESI-MS, electrospray ionization mass spectrometry; FDH,
formaldehyde dehydrogenase; FH, fumarate hydratase; FIH, factor inhibiting
HIF; H3K4, histone H3 lysine 4; H3K9, histone H3 lysine 9; H3K36, histone
H3 lysine 36; HDAC, histone deacetylase; HEPES, (4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid); HIF, hypoxia-inducible factor; JARID,
Jumonji, AT-rich interactive domain; JmjC, Jumonji-C; JMJD2, JmjC-
domain containing histone demethylase 2; LSD1, lysine-specific demethylase
1; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-
flight mass spectrometry; NAD+, nicotinamide adenine dinucleotide; NADH,
reduced nicotinamide adenine dinucleotide; PHD, prolyl hydroxylase
domain; SAHA, suberoylanilide hydroxamic acid; SDH, succinate dehy-
drogenase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis; SEM, standard error of the mean; TCA, tricarboxylic acid; TSA,
trichostatin A.
Both the histone deacetylases (HDACs) and the JmjC
demethylases employ active site metal cofactors, Zn(II) or Fe(II),
respectively, involved in catalysis. In addition to the active site
Fe(II), the JMJD2 demethylases also contain a Zn binding site
located close to the active site entrance.9 Therefore, we then
tested the known HDAC inhibitors trichostatin A (TSA, 2),2
10.1021/jm800936s CCC: $40.75
2008 American Chemical Society
Published on Web 10/23/2008

7054 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Letters
Table 1. Inhibition Data for 2-OG-Dependent Histone Demethylase JMJD2Ea
a “N.A.” means that formaldehyde dehydrogenase assays were not possible due to partial inhibition of FDH by the inhibitor under pre-incubation conditions.
Note that JMJD2E concentration in all assays was relatively high (2 µM) in order for sufficient NADH to be produced to be detected reliably. The IC50 value
reported for 2 (found to be an FDH inhibitor) was obtained by MALDI-TOF-MS, monitoring turnover of trimethyl peptide, not using the FDH coupled assay
(see Supporting Information for details of methods). Assays were all carried out in triplicate, and SEMs in log(IC50) values were below 10%. The ranking
of inhibitor binding to the JMJD2E.Fe(II).Zn(II) complex was assessed by nondenaturing ESI-MS (see Supporting Information, Methods and Table S1).
suberoylanilide hydroxamic acid (SAHA, 3), and butyric acid
4. 4 did not inhibit JMJD2E, while 2 inhibited FDH, thus making
this assay method unsuitable for 2; assays monitoring demethy-
lation by MALDI-TOF MS gave an IC50 ) 28.4 µM for 2. 3
inhibited JMJD2E with IC50 ) 14 µM with preincubation.
Assays in which the Fe(II) concentration was varied indicated
that the mode of inhibition by 3 was not predominantly due to
Fe(II) chelation by the compound in solution. Further support
for binding of 3 to JMJD2E came from nondenaturing MS
analyses demonstrating that 3 bound to the JMJD2E ·Fe(II)·Zn(II)

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7055
complex in a competitive manner with respect to 2-OG
analogues (Supporting Information, Table S1). In contrast to a
more potent inhibitor (see below), which displayed clear
competitive inhibition in solution with respect to 2-OG, 3
displayed mixed inhibition in solution with respect to both 2-OG
and peptide substrate. Thus we cannot be certain of how 3 binds
to JMJD2E, especially given that JMJD2s contain a Zn(II) as
well as an Fe(II) binding site. However, given that 3 competes
with 2-OG analogues for binding to JMJD2E (Supporting
Information, Table S1) and that in the enzyme-substrate
complexes of both the HDACs and the JMJD2s, the terminus
of the modified lysine side chain binds close to Zn(II) and Fe(II),
respectively, it seems possible that the hydroxamic acid of 3
binds to the Fe(II) of JMJD2E. 3 is reported to inhibit HDAC1
with an IC50 ) 48 nM;10 thus based on inhibition data in vitro,
3 is likely to be selective for HDACs over the JMJD2s; however,
there is at least a possibility of a dual-enzyme family inhibition
mechanism operating in vivo for 3 and, possibly, for other
HDAC inhibitors that are metal chelators.
Following analysis of JMJD2A-substrate-complex crystal
structures9 (JMJD2A and JMJD2E have ∼65% identity over
their catalytic domains), we proposed and synthesized low
molecular weight aromatic hydroxamic acids (5a-5c) intended
to chelate the Fe(II) of JMJD2E in a manner similar to that
predicted for 3. 5a was the most potent of these compounds,
with IC50 ) 4.8 µM, and was significantly more active than 3.
Variation in the Fe(II) concentration indicated that 5a is a 2-OG
competitor and that inhibition was not predominantly due to
Fe(II) chelation in solution. We also investigated several
pyridine carboxylates (6a-6f), some of which are known
inhibitors of collagen and HIF prolyl hydroxylases.11 Pyridine-
2,4-dicarboxylic acid 6a was the most potent JMJD2E inhibitor
tested, with IC50 ) 1.4 µM, and Ki ) 914 ( 85 nM (competitive
inhibition with respect to 2-OG). Inhibition of other Fe(II)/2-
OG dependent oxygenases using 6a has been reported for the
collagen prolyl-4-hydroxylase (C-P4H) (Ki ) 2 µM), the
collagen prolyl-3-hydroxylase (C-P3H) (Ki ) 9 µM)12 and for
the HIF prolyl hydroxylases PHD1-3 (Ki ) 7-40 µM).6 In
contrast, pyridine-2,5-dicarboxylic acid 6b is more potent as
an inhibitor of C-P4H (Ki ) 0.8 µM) but displays no significant
inhibition of C-P3H (Ki > 1000 µM), the PHD enzymes (Ki
> 300 µM) nor of JMJD2E (IC50 ) 184 µM). The bicyclic
analogue 7 [4′-(methoxycarbonyl)-2,2′-bipyridine-4-carboxylic
acid] and the pyrimidine analogue 8 also inhibited JMJD2E
effectively, with IC50 ) 6.6 µM and IC50 ) 27 µM respectively.
Figure 1. View from a crystal structure of JMJD2A ·Ni(II)·Zn(II) (pink
and green) complexed with 6a (yellow) [PDB ID 2VD7]. The
experimental 2Fo-Fc electron density, displayed as blue mesh, is shown
for the metal coordinating residues and ligands (contoured to 1.5σ).
6a interacts with the Ni(II) ion (orange, substituting for Fe(II)) via
bidentate coordination; the pyridinyl nitrogen coordinates to the metal
trans to Oε1 of Glu-190 (2.3 Å), and one of the 6a 2-carboxylate
oxygens, O2, coordinates the metal trans to His-276 (2.2 Å). The
pyridine ring of 6a is positioned to form hydrophobic interactions with
Tyr-177, Phe-185, and Trp-208. The non-metal-ligated 2-carboxylate
oxygen of 6a is positioned to hydrogen bond with Nε of Lys-241 (3.1
Å) and the Tyr-177 OH (3.5 Å) and likely interferes with methyl-lysine
substrate binding (see Supporting Information, Figure S7). The six-
coordinate metal coordination is completed by a water molecule (W1,
O to Fe: 2.3 Å). The 4-carboxylate oxygens of 6a interact with the
2-OG 5-carboxylate binding residues, Lys-206 Nε (2.8 Å) and Tyr-
132 OH (2.6 Å). Asn-198 was observed in two alternative conforma-
tions (green and cyan sticks), one of which is positioned to form a
hydrogen bond with the metal coordinated water.
likely to be mediated via inhibition of the HIF prolyl hydroxy-
lases rather than FIH or the analyzed histone demethylases.
Electrospray ionization (ESI)-MS analyses under nondena-
turing conditions (Supporting Information, Figure S3) verified
binding of the inhibitors to the JMJD2E ·Fe(II)·Zn(II) complex.
Competitive binding experiments were then used to rank the
most potent inhibitors qualitatively in terms of binding affinity
for JMJD2E (Table 1, Supporting Information, Table S1). In
these competitive experiments, the seven inhibitors observed
to bind the best in the initial MS analyses were ranked by
experiments in which pairs of inhibitors were mixed with
JMJD2E and allowed to compete with each other for protein
binding. The results of this ranking of binding affinity correlated
resonably well with the IC50 values obtained in solution, with
6a having the highest affinity for the enzyme (rank order of
binding strength by MS: 6a > 5a > 7 > 3 > 8 > 1c > 1a,
compared to 6a > 5a > 3 > 7 > 8 > 1a > 1c by FDH
inhibition assay IC50s).
To examine the mode of binding of the most potent inhibitor
identified, a crystal structure of the JMJD2A ·Ni(II)·Zn(II)·6a
complex was determined to 2.0 Å resolution (a crystal structure
of JMJD2E has not yet been obtained). The structure (Figure
1) reveals that pyridine-2,4-dicarboxylic acid 6a binds to
JMJD2A between the ꢀ-sheets of its double stranded ꢀ-helix
fold in a different metal coordination mode to that observed
for N-oxalylglycine 1a.9 It binds to the Ni(II) [which replaces
Fe(II)] in a bidentate manner via its N-atom and 2-carboxylate
Variants of the TCA cycle enzymes, succinate dehydrogenase
(SDH) and fumarate hydratase (FH), lead to elevated intracel-
lular levels of succinate 9 and fumarate 10, which are proposed
to stimulate tumor growth by activation of the hypoxic response
via HIF hydroxylase inhibition.13,14 Cellular levels of 9 and 10
in SDH- and FH-deficient tumor cells are estimated at 30-100
µmol/g protein and 400-600 µmol/g protein respectively.13
and 10 were relatively poor inhibitors of JMJD2E, with IC50
9
)
0.32 mM (9, preincubation) and 2.3 mM (10). Smith et al. have
reported apparent inhibition of JMJD2D activity by high
concentrations of 9 (1-10 mM) in cells.15 However, the weak
inhibition of JMJD2E by 9 and 10 contrasts with the more potent
inhibition of a HIF prolyl hydroxylase (PHD2) by 10 (IC50
)
3 µM, preincubation) and by 9 (IC50 ) 19 µM, preincubation).16
However, FIH, which is much more closely related to the histone
demethylases in sequence and structure than are the PHDs, was
only weakly inhibited by 9 and 10 (IC50 > 1 mM). Thus, the
available data, albeit limited, suggest that cellular effects
involving 2-OG oxygenase inhibition by 9 and 10 are more

7056 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Letters
(3) Lee, M. G.; Wynder, C.; Schmidt, D. M.; McCafferty, D. G.;
Shiekhattar, R. Histone H3 Lysine 4 Demethylation Is a Target of
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and occupies the 2-OG binding site in the enzyme. This binding
mode is consistent with our kinetic analyses, showing that 6a
is a competitive inhibitor with respect to 2-OG. There was no
evidence for any binding of 6a at the Zn(II) site, which is present
in both JMJD2A and JMJD2E. 6a was also shown to inhibit
JMJD2A in an FDH coupled assay with IC50 ) 0.7 µM
(although the data quality for JMJD2A was inferior to that for
JMJD2E, possibly due to lower levels of enzyme activity).
Overall we have identified inhibitors of the JMJD2 histone
demethylases, of which the most potent is 6a. Other identified
inhibitors that are also 2-OG analogues include the hydroxamic
acid 5a and the oxalylamino acids 1a and 1c, all of which should
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inhibit the JMJD2 histone demethylases, albeit much less
potently than they inhibit the HDAC enzymes. Succinate 9 and
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than the histone demethylases. Our work marks a starting point
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demethylases for use as therapeutic agents and as chemical tools
for investigating the complex interplay between the many
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Acknowledgment. Help with synchrotron data collection by
F. von Delft is gratefully acknowledged. We thank the Com-
monwealth Scholarship Commission, The Wellcome Trust, and
the BBSRC for funding. The Structural Genomics Consortium
is a registered charity (no. 1097737) that receives funds from a
number of sources, details of which are found in the Supporting
Information.
Supporting Information Available: Sequence alignments of
JMJD2 histone demethylases, reactions catalyzed by histone
demethylases, cloning and expression of JMJD2E, peptide synthesis,
inhibitor synthesis, 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.
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