Plant growth regulator daminozide is a selective inhibitor of human KDM2/7 histone demethylases

Article abstract of DOI:10.1021/jm300677j

The JmjC oxygenases catalyze the N-demethylation of N^ε- methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.

Full text of DOI:10.1021/jm300677j

Brief Article
pubs.acs.org/jmc
Plant Growth Regulator Daminozide Is a Selective Inhibitor of
Human KDM2/7 Histone Demethylases
Nathan R. Rose,†,‡,# Esther C. Y. Woon,‡,#,∞ Anthony Tumber,§,# Louise J. Walport,‡
Rasheduzzaman Chowdhury,‡ Xuan Shirley Li,† Oliver N. F. King,‡ Clarisse Lejeune,‡,§ Stanley S. Ng,§,∥
Tobias Krojer,§ Mun Chiang Chan,‡ Anna M. Rydzik,‡ Richard J. Hopkinson,‡ Ka Hing Che,§,∥
Michelle Daniel,§ Claire Strain-Damerell,§ Carina Gileadi,§ Grazyna Kochan,§ Ivanhoe K. H. Leung,‡
James Dunford,∥ Kar Kheng Yeoh,⊥ Peter J. Ratcliffe,⊥ Nicola Burgess-Brown,§ Frank von Delft,§
Susanne Muller,§ Brian Marsden,§ Paul E. Brennan,§ Michael A. McDonough,‡ Udo Oppermann,§,∥
Robert J. Klose,† Christopher J. Schofield,*,‡ and Akane Kawamura*,‡
†Epigenetic Regulation of Chromatin Function Group, Department of Biochemistry, University of Oxford, South Parks Road, Oxford
OX1 3QU, U.K.
‡Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, U.K.
§Structural Genomics Consortium, University of Oxford, Headington OX3 7DQ, U.K.
∥Oxford Biomedical Research Unit, Botnar Research Centre, University of Oxford, Oxford OX3 7LD, U.K.
⊥Henry Wellcome Building for Molecular Physiology, Department of Clinical Medicine, University of Oxford, Roosevelt Drive,
Oxford OX3 7BN, U.K.
S
* Supporting Information
ABSTRACT: The JmjC oxygenases catalyze the N-demethylation of Nε-methyl lysine residues in histones and are current
therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases
and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth
regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates
the active site metal via its hydrazide carbonyl and dimethylamino groups.
inhibitors for agricultural and horticultural applications. Here
we report that daminozide, N-(dimethylamino)succinamic acid,
which was once widely used as a plant growth retardant but
later withdrawn because of toxicity concerns, is a highly
selective inhibitor of the KDM2/7 family of human JmjC
histone demethylases.
INTRODUCTION
■
Histone modifications are central to the regulation of
eukaryotic gene expression. The dynamic methylation of lysine
and arginine residues in histones has diverse transcriptional
outcomes, with different methylation sites and states being
associated with promotion or repression of transcription. There
are two classes of histone lysine demethylases (KDMs), the
largest of which uses 2-oxoglutarate (2OG) as a cosubstrate
(the JmjC enzymes) and comprises ∼18 human enzymes
grouped into five subfamilies (Figure 1). Several JmjC
demethylases are targets for the treatment of diseases including
leukemia, breast, and prostate cancers1,2 and inflammation.3
However, only limited inhibition data have been reported for
the JmjC enzymes with few reports of selective inhibitors.4−8
2OG oxygenases have been targeted for therapeutic and
agricultural applications:9 an inhibitor of γ-butyrobetaine
hydroxylase, BBOX1, is used clinically as a “cardioprotectant”,
inhibition of the collagen prolyl hydroxylases has been
investigated for treating fibrotic disease, and inhibitors of the
hypoxia inducible factor hydroxylases are in clinical trials for the
treatment of anemia. These examples provide evidence that
2OG oxygenases are viable targets for therapeutic inhibition by
small molecules in vivo. 2OG oxygenases involved in the
biosynthesis of gibberellins, plant hormones involved in growth
regulation, have also been targeted using small molecule
RESULTS
■
To enable the identification of selective JmjC demethylase
subfamily inhibitors, we developed a unified screening platform
containing representatives of each of the five human
demethylase subfamilies (KDM2A/FBXL11, KDM3A/
JMJD1A, KDM4E/JMJD2E, KDM5C/JARID1C, KDM6B/
JMJD3) (Supporting Information). Active enzymes were
expressed using bacterial or eukaryotic expression systems
and purified to near homogeneity (Supporting Information).
Kinetic parameters for 2OG and histone fragment substrates
were determined (Table 1), and activity AlphaScreens
(amplified luminescence proximity homogeneous assays) were
developed (Figure S1).10 This assay is based on immunode-
tection of the lysine methylation state of biotin-conjugated
histone peptide product, using streptavidin- and protein A-
Received: May 15, 2012
Published: June 25, 2012
© 2012 American Chemical Society
6639
dx.doi.org/10.1021/jm300677j | J. Med. Chem. 2012, 55, 6639−6643

Journal of Medicinal Chemistry
Brief Article
Figure 1. Subfamilies of the human 2-oxoglutarate (JmjC) histone demethylases, showing their domain architecture. Not all proposed family
members are shown. Each demethylation reaction is coupled to 2OG decarboxylation and formaldehyde production. KIAA1718 has recently been
assigned as KDM7A;11 however, its JmjC domain shows high sequence and structural similarity with the KDM2 subfamily members.
a
Table 1. Inhibition Data (IC50) for Daminozide and Its Analogues across KDMs and Other 2OG Oxygenases
a
Data are reported as IC50 in μM. AlphaScreen was used for all IC50 determinations except where values are given in parenthesis where a MALDI
assay was used. All BBOX assays were run using a fluorescence based assay.12 AlphaScreen assays were optimized to run at the linear range of the
reaction. Assays were performed at concentrations of 2OG near experimentally determined 2OG Km values. Where Km values for 2OG for
enzymes were unknown, the kinetic parameter was determined by using the FDH assay: KDM2A (12.5 1.4 μM), PHF8 (16.6 1.8 μM), KDM3A
(5.3 2.2 μM), KDM5C (41.7 6.3 μM), and KDM6B (5.4 0.4 μM).
app
app
conjugated beads for quantification. For counterscreening, we
selected three clinically relevant oxygenases, the hypoxic
response oxygenases PHD2 and FIH (prolyl and asparaginyl
hydroxylases acting on hypoxia inducible factor, respectively),
and BBOX1 (γ-butyrobetaine hydroxylase) for which Al-
phaScreen or fluorescence assays12 were also developed. This
screening platform is a significant improvement on previous
methods for human 2OG oxygenases;9 it enables the
oxygenases to be used at low nanomolar concentrations.
Standardization of the analytical methods for enzymes
employing different types of substrates enabled quantitative
comparisons of inhibitor potencies to be made.
In work aimed at identifying inhibitor scaffolds for the JmjC
histone demethylases, we assembled a set of 2OG analogues
known to inhibit 2OG oxygenases (Figure 2) and screened
these against the 2OG oxygenase panel using the assay
platform. As expected, known “generic” 2OG oxygenase
inhibitors9 including pyridine 2,4-dicarboxylic acid (11) and
N-oxalylglycine (12) showed inhibition across the 2OG
oxygenases tested, validating this assay platform. Interestingly,
the clinically used histone deacetylase (HDAC) inhibitor
Vorinostat/SAHA (suberoylanilide hydroxamic acid, 15)
inhibited several histone demethylases (Figure 2).
Unexpectedly, the plant growth regulator daminozide (19)
stood out as a selective inhibitor of KDM2A (IC50 = 1.5 μM),
which is selective for dimethyl lysine residues. Daminozide was
identified as a plant growth retardant in the 1960s14 and used to
control stem growth, plant size, and fruit ripening for over 20
years.15 In vivo, daminozide is proposed to inhibit 2OG
oxygenases involved in gibberellin, and possibly ethylene,
biosynthesis in plants.16,17 However, daminozide usage in food
crops was curtailed because of the potential carcinogenicity of
its metabolite, 1,1-dimethylhydrazine (although is still used for
ornamental plants).18−20 A formaldehyde dehydrogenase
(FDH) coupled assay monitoring formaldehyde production
confirmed the potency of daminozide against KDM2A (IC50
=
1.5 μM). To test further for subfamily selectivity, daminozide
was screened against two other members of the KDM2/7
subfamily (PHF8 and KDM7A, Table 1) which are structurally
highly related to KDM2A and KDM2B and are also selective
6640
dx.doi.org/10.1021/jm300677j | J. Med. Chem. 2012, 55, 6639−6643

Journal of Medicinal Chemistry
Brief Article
Figure 2. Heat map of JmjC demethylase inhibition by a set of 2OG analogues. Daminozide 19 is selective for KDM2A. Tricarboxylic acid cycle
intermediates succinate 1 and fumarate 2 were generally poor demethylase inhibitors, though KDM4E7 was an exception to this trend. The (R)- and
(S)-2-hydroxyglutarate enantiomers, produced by gain-of-function mutations to isocitrate dehydrogenase, were inhibitors of the KDM2, KDM3, and
KDM4 histone demethylases.13 Catechols 16 and 17, bipyridyl 18, and 8-hydroxyquinoline 21 inhibited all demethylases screened. By contrast, 5
most potently inhibited PHD2 (prolyl hydroxylase domain enzyme isoform 2). Each compound was screened in an AlphaScreen assay and is
represented as % inhibition of the enzyme at 20 μM compound. Details of the assays are in the Supporting Information.
for demethylation of dimethyl lysine residues but have different
sequence specificities.21−23 The results indicate that daminozide
is at least 60-fold selective as an inhibitor of the KDM2/7
subfamily over the other demethylase subfamily members
tested, with IC50 of 2 μM or less against KDM2A, PHF8, and
KIAA1718 and IC50 of 127 μM for KDM3A (a different
subfamily of dimethyl lysine demethylase) or greater
(millimolar range) against other demethylases. No inhibition
was observed (at 1 mM) against other biologically important
2OG oxygenases that catalyze hydroxylation, i.e., PHD2, FIH,
and BBOX1. Given its simple achiral structure and low
molecular weight (160 Da), the degree of selectivity exhibited
by daminozide is remarkable.
Kinetic analyses revealed that daminozide is predominantly a
competitive inhibitor with respect to 2OG (Ki = 1.97 μM) for
KDM2A but shows mixed inhibition with respect to peptide
substrate, binding predominantly to the enzyme-peptide
complex (Ki = 85 μM, α = 0.13) (Figure 3). The latter
observation is notable given that daminozide contains a
dimethylamino group as do the KDM2/7 subfamily dimethyl
lysine substrates and might thus be expected to compete with
the dimethylated lysine substrate. The pKa of the daminozide
hydrazide amine is 2.8 while that of its carboxylate is 4.6,24
suggesting that it may bind the active site iron; NMR analyses
show daminozide complexes to Fe(II) in solution (Figure S2).
We then investigated the structural basis of the selective
inhibition by daminozide on the KDM2/7 subfamily. We
obtained structures of daminozide complexed with two
demethylases, PHF8 and KDM4A, and a hydroxylase, FIH
(Figure 4). The structures support the proposed general mode
of inhibition by daminozide, i.e., by binding in the 2OG binding
pocket and chelating to the active site metal via its
acylhydrazide carbonyl and dimethylamino groups (Figure 4).
While the coordinate position of the daminozide carbonyl was
invariant, i.e., in all cases trans to the Asp/Glu protein-based
ligand, in the KDM4A structure the dimethylamino group was
observed to complex trans to His276 or trans to His188 in the
two different molecules present in the asymmetric unit.
Although in the cases of PHF8 and FIH the daminozide
dimethylamino group was only observed to bind trans to the
histidine equivalent to His276 of KDM4A, previous work25 on
complexes of 2OG oxygenases with 2OG and the cosubstrate
analogue 12 demonstrates that the 1-carboxylate can bind in
either of the two conformational positions, in a manner
analogous to that observed for the dimethylamino group in the
KDM4A structure. We propose that the selectivity of
daminozide for the KDM2/7 subfamily could, at least in part,
arise from a “snug fit” obtained via binding in the position trans
to His247 wherein its two methyl groups are accommodated in
a tight hydrophobic pocket (formed by Val255, Ile313, and
Tyr257), which is conserved in the KDM2/7subfamily (Figure
4). This pocket might bind the daminozide methyl groups less
tightly in other demethylases/oxygenases because it is more
hydrophilic (as demonstrated by crystallographic analysis) or
Figure 3. Mode of inhibition of the KDM2/7 subfamily by
daminozide. Inhibition of KDM2A by daminozide is competitive
with 2OG but not peptide substrate.
6641
dx.doi.org/10.1021/jm300677j | J. Med. Chem. 2012, 55, 6639−6643

Journal of Medicinal Chemistry
Brief Article
subfamilies. In the long term, we aim to help develop screens
for all human 2OG oxygenases and see them applied to the
discovery of medicinally useful inhibitors. By use of a relatively
focused set of 2OG analogues, the screening platform led to the
discovery that daminozide is a selective inhibitor of the KDM2/
7demethylase subfamily. While daminozide itself is unlikely to
be of medicinal use, the work has revealed a new class of 2OG
oxygenase inhibitor, which employs alkylamino iron chelation.
Of particular note is the high degree of selectivity exhibited by
daminozide, especially given its achiral nature and low
molecular weight. The precise mode of action of daminozide
as a plant growth regulator16,17 and its potential human toxicity
(if any) under physiologically relevant conditions are unknown.
We have no evidence that our results are directly relevant to the
toxicity issues relating to daminozide. However, the results do
suggest a potential of daminozide or its derivatives to exert
biological effects via the inhibition of 2OG oxygenases involved
in epigenetics in animals and, potentially, in other organisms
including plants. Given the link between JmjC enzymes and
diseases including cancer,2,3 further work on the biological
effects of daminozide is of interest.
Figure 4. Crystal structures reveal the mode of inhibition of the
KDM2/7 subfamily by daminozide: PHF8 (with Zn(II) substituting
for Fe(II)) and KDM4A (with Ni(II) substituting for Fe(II)) and FIH
(with Zn(II) substituting for Fe(II)) (Table S3). Daminozide binds
the metal through its hydrazide amine lone pair and carbonyl oxygen.
Two distinct orientations of daminozide are observed in the two
KDM4A molecules in the asymmetric unit; both are shown (see Table
S2 for electron density maps). Selectivity of daminozide for the
KDM2/7 subfamily may arise because they possess a hydrophobic
region (Tyr257, Val255, and Ile313) into which the two daminozide
methyl groups bind. In contrast, the equivalent regions of KDM4A,
FIH, and the other tested demethylases/oxygenases are more
hydrophilic.
ASSOCIATED CONTENT
* Supporting Information
Protein expression and purification methods, assay methods,
■
S
synthetic procedures, characterization of all synthesized
1
compounds, H NMR spectra of daminozide and analogues
with iron(II), and crystalllographic data collection and
refinement statistics. This material is available free of charge
via the Internet at http://pubs.acs.org.
Accession Codes
PDB codes: 4AI9, 4AI8, 4DO0 for complex of daminozide with
KDM4A, FIH, PHF8 proteins, respectively.
predicted to be more hydrophilic (by structure based on
sequence alignments) for all the other 2OG oxygenases tested
(Figure 1) (e.g., for KDM4A, they are Ser196, Thr270, and
Asn198; for KDM6B, they are Ser225, Ile291, and Asn227; for
FIH, they are Asn205, Ile273, and Phe257).
AUTHOR INFORMATION
Corresponding Author
*For C.J.S.: phone, (01865) 275625; e-mail, christopher.
schofield@chem.ox.ac.uk. For A.K.: phone, (01865) 275677;
e-mail, akane.kawamura@chem.ox.ac.uk.
■
We also synthesized and tested analogues of daminozide
(Table 1 and Table S3). The trimethylated analogue 22 and 27,
both of which lack the terminal amine lone pair, displayed
little/no KDM inhibition, consistent with the proposed mode
of daminozide inhibition involving chelation by its terminal
hydrazide amine. The monomethylated 23 and unmethylated
analogues 24 were more potent than daminozide against
KDM2A. However, when tested against other 2OG subfamilies,
these compounds were substantially less selective than
daminozide (and were somewhat unstable in aqueous solution).
Succinylhydroxamic acid 25 and dioxoheptanoic acid 28, in
which the acylhydrazinamide of daminozide is replaced by
metal-chelating hydroxamic acid and malonyl groups, respec-
tively, were also relatively potent but nonselective inhibitors
(Table 1). Notably, 26, in which the amide nitrogen of
daminozide is N-methylated, is also selective for the KDM2/
7demethylases, albeit with reduced potency. Like daminozide,
26 has two methyl groups on the acylhydrazide amine,
suggesting that their presence confers selectivity. The other
tested analogues were less active (Table S3).
Present Address
∞Department of Pharmacy, National University of Singapore,
18 Science Drive 4, Singapore 117543.
Author Contributions
#These authors contributed equally to the work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported in part by the The Wellcome
Trust, The Commonwealth Scholarship Commission in the
United Kingdom, the Biotechnology and Biological Research
Council (U.K.), and the European Research Council. The
Structural Genomics Consortium is a registered charity (No.
1097737) that receives funds from the Canadian Institutes for
Health Research, the Canadian Foundation for Innovation,
Genome Canada through the Ontario Genomics Institute,
GlaxoSmithKline, Karolinska Institutet, the Knut and Alice
Wallenberg Foundation, the Ontario Innovation Trust, the
Ontario Ministry for Research and Innovation, Merck & Co.,
Inc., the Novartis Research Foundation, the Swedish Agency
for Innovation Systems, the Swedish Foundation for Strategic
Research, and the Wellcome Trust.
CONCLUSIONS
■
Overall, we have demonstrated how the development of a
screening platform employing multiple human 2OG oxygenases
can enable the discovery of compounds selective for particular
6642
dx.doi.org/10.1021/jm300677j | J. Med. Chem. 2012, 55, 6639−6643

Journal of Medicinal Chemistry
Brief Article
(14) Riddell, J. A.; Hageman, H. A.; J’Anthony, C. M.; Hubbard, W.
L. Retardation of plant growth by a new group of chemicals. Science
1962, 136 (3514), 391.
(15) Rademacher, W. Growth retardants: effects on gibberellin
biosynthesis and other metabolic pathways. Ann. Rev. Plant Physiol.
Plant Mol. Biol. 2000, 51 (1), 501−531.
(16) Brown, R. G. S.; Kawaide, H.; Yang, Y.-Y.; Rademacher, W.;
Kamiya, Y. Daminozide and prohexadione have similar modes of
action as inhibitors of the late stages of gibberellin metabolism. Physiol.
Plant. 1997, 101, 309−313.
(17) Looney, N. E. Inhibition of apple ripening by succinic acid, 2,2-
dimethyl hydrazide and its reversal by ethylene. Plant Physiol 1968, 43,
1133−1137.
(18) Hasegawa, R.; Cabral, R.; Hoshiya, T.; Hakoi, K.; Ogiso, T.;
Boonyaphiphat, P.; Shirai, T.; Ito, N. Carcinogenic potential of some
pesticides in a medium-term multi-organ bioassay in rats. Int. J. Cancer
1993, 54 (3), 489−493.
(19) Mott, L. Alar again: science, the media, and the public’s right to
know. Int. J. Occup. Environ. Health 2000, 6 (1), 68−70.
(20) Toth, B.; Wallcave, L.; Patil, K.; Schmeltz, I.; Hoffmann, D.
Induction of tumors in mice with the herbicide succinic acid 2,2-
dimethylhydrazide. Cancer Res. 1977, 37 (10), 3497−3500.
(21) Horton, J. R.; Upadhyay, A. K.; Qi, H. H.; Zhang, X.; Shi, Y.;
Cheng, X. Enzymatic and structural insights for substrate specificity of
a family of jumonji histone lysine demethylases. Nat. Struct. Mol. Biol.
2010, 17 (1), 38−43.
(22) Liu, W.; Tanasa, B.; Tyurina, O. V.; Zhou, T. Y.; Gassmann, R.;
Liu, W. T.; Ohgi, K. A.; Benner, C.; Garcia-Bassets, I.; Aggarwal, A. K.;
Desai, A.; Dorrestein, P. C.; Glass, C. K.; Rosenfeld, M. G. PHF8
mediates histone H4 lysine 20 demethylation events involved in cell
cycle progression. Nature 2010, 466 (7305), 508−512.
(23) Qi, H. H.; Sarkissian, M.; Hu, G. Q.; Wang, Z.; Bhattacharjee,
A.; Gordon, D. B.; Gonzales, M.; Lan, F.; Ongusaha, P. P.; Huarte, M.;
Yaghi, N. K.; Lim, H.; Garcia, B. A.; Brizuela, L.; Zhao, K.; Roberts, T.
M.; Shi, Y. Histone H4K20/H3K9 demethylase PHF8 regulates
zebrafish brain and craniofacial development. Nature 2010, 466
(7305), 503−507.
ABBREVIATIONS USED
■
2OG, 2-oxoglutarate; BBOX, γ-butyrobetaine hydroxylase;
FBXL, F-box, leucine-rich repeat protein; FIH, factor inhibiting
hypoxia inducible factor; HDAC, histone deacetylase; JARID,
jumonji, AT rich interactive domain containing protein; JmjC,
jumonji-C domain; JMJD, jumonji-C domain containing
protein; PHD2, human prolyl hydroxylase; PHF, plant
homeodomain containing protein
REFERENCES
■
(1) He, J.; Nguyen, A. T.; Zhang, Y. KDM2b/JHDM1b, an
H3K36me2-specific demethylase, is required for initiation and
maintenance of acute myeloid leukemia. Blood 2011, 117 (14),
3869−3880.
(2) Yang, J.; Jubb, A. M.; Pike, L.; Buffa, F. M.; Turley, H.; Baban, D.;
Leek, R.; Gatter, K. C.; Ragoussis, J.; Harris, A. L. The histone
demethylase JMJD2B is regulated by estrogen receptor alpha and
hypoxia, and is a key mediator of estrogen induced growth. Cancer Res.
2010, 70 (16), 6456−6466.
(3) De Santa, F.; Totaro, M. G.; Prosperini, E.; Notarbartolo, S.;
Testa, G.; Natoli, G. The histone H3 lysine-27 demethylase Jmjd3
links inflammation to inhibition of polycomb-mediated gene silencing.
Cell 2007, 130 (6), 1083−1094.
(4) Chang, K. H.; King, O. N.; Tumber, A.; Woon, E. C.; Heightman,
T. D.; McDonough, M. A.; Schofield, C. J.; Rose, N. R. Inhibition of
histone demethylases by 4-carboxy-2,2′-bipyridyl compounds. Chem-
MedChem 2011, 6 (5), 759−764.
(5) Hamada, S.; Suzuki, T.; Mino, K.; Koseki, K.; Oehme, F.;
Flamme, I.; Ozasa, H.; Itoh, Y.; Ogasawara, D.; Komaarashi, H.; Kato,
A.; Tsumoto, H.; Nakagawa, H.; Hasegawa, M.; Sasaki, R.; Mizukami,
T.; Miyata, N. Design, synthesis, enzyme-inhibitory activity, and effect
on human cancer cells of a novel series of jumonji domain-containing
protein 2 histone demethylase inhibitors. J. Med. Chem. 2010, 53 (15),
5629−5638.
(6) Luo, X.; Liu, Y.; Kubicek, S.; Myllyharju, J.; Tumber, A.; Ng, S.;
Che, K. H.; Podoll, J.; Heightman, T. D.; Oppermann, U.; Schreiber, S.
L.; Wang, X. A selective inhibitor and probe of the cellular functions of
Jumonji C domain-containing histone demethylases. J. Am. Chem. Soc.
2011, 133 (24), 9451−9456.
(24) Schoenherr, J.; Bukovac, M. J. Dissociation constants of succinic
acid 2,2-dimethylhydrazide. J. Agric. Food Chem. 1972, 20 (6), 1263−
1265.
(25) Zhang, Z.; Ren, J.; Harlos, K.; McKinnon, C. H.; Clifton, I. J.;
Schofield, C. J. Crystal structure of a clavaminate synthase−Fe(II)−2-
oxoglutarate−substrate−NO complex: evidence for metal centred
rearrangements. FEBS Lett. 2002, 517 (1−3), 7−12.
́
(7) 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.
(8) Sekirnik, R.; Rose, N. R.; Mecinovic, J.; Schofield, C. J. 2-
Oxoglutarate oxygenases are inhibited by a range of transition metals.
Metallomics 2010, 2 (6), 397−399.
(9) Rose, N. R.; McDonough, M. A.; King, O. N.; Kawamura, A.;
Schofield, C. J. Inhibition of 2-oxoglutarate dependent oxygenases.
Chem. Soc. Rev. 2011, 40 (8), 4364−4397.
(10) Kawamura, A.; Tumber, A.; Rose, N. R.; King, O. N.; Daniel,
M.; Oppermann, U.; Heightman, T. D.; Schofield, C. Development of
homogeneous luminescence assays for histone demethylase catalysis
and binding. Anal. Biochem. 2010, 404 (1), 86−93.
(11) Huang, C.; Xiang, Y.; Wang, Y.; Li, X.; Xu, L.; Zhu, Z.; Zhang,
T.; Zhu, Q.; Zhang, K.; Jing, N.; Chen, C. D. Dual-specificity histone
demethylase KIAA1718 (KDM7A) regulates neural differentiation
through FGF4. Cell Res. 2010, 20 (2), 154−165.
(12) Rydzik, A.; Leung, I. K. H.; Kochan, G. T.; Thalhammer, A.;
Opperman, U.; Claridge, T. D. W.; Schofield, C. J. Development and
application of a fluoride detection based fluorescence assay for gamma-
butyrobetaine hydroxylase. ChemBioChem [Online early access]. DOI:
10.1002/cbic.201200256. Published Online: Jun 22, 2012.
(13) Chowdhury, R.; Yeoh, K. K.; Tian, Y. M.; Hillringhaus, L.; Bagg,
E. A.; Rose, N. R.; Leung, I. K. H.; Li, X. S.; Woon, E. C.; Yang, M.;
McDonough, M. A.; King, O. N.; Clifton, I. J.; Klose, R. J.; Claridge, T.
D. W.; Ratcliffe, P. J.; Schofield, C. J.; Kawamura, A. The
oncometabolite 2-hydroxyglutarate inhibits histone lysine demethy-
lases. EMBO Rep. 2011, 12 (5), 463−469.
6643
dx.doi.org/10.1021/jm300677j | J. Med. Chem. 2012, 55, 6639−6643