A selective inhibitor and probe of the cellular functions of jumonji C domain-containing histone demethylases

Article abstract of DOI:10.1021/ja201597b

Histone methylations are important chromatin marks that regulate gene expression, genomic stability, DNA repair, and genomic imprinting. Histone demethylases are the most recent family of histone-modifying enzymes discovered. Here, we report the character

Full text of DOI:10.1021/ja201597b

ARTICLE
pubs.acs.org/JACS
A Selective Inhibitor and Probe of the Cellular Functions of Jumonji C
Domain-Containing Histone Demethylases
Xuelai Luo,†,O,z Yongxiang Liu,†,z Stefan Kubicek,‡,§,|| Johanna Myllyharju,^ Anthony Tumber,#
Stanley Ng,#,r Ka Hing Che,#,r Jessica Podoll,† Tom D. Heightman,#,[ Udo Oppermann,#,r
Stuart L. Schreiber,‡,§ and Xiang Wang*,†
†Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, United States
‡The Howard Hughes Medical Institute at the Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts
02142, United States
§Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United
States
CeMM ꢀ Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, A-1090 Vienna, Austria
^The Oulu Center for Cell-Matrix Research, Biocenter Oulu, Department of Medical Biochemistry and Molecular Biology, University of
Oulu, FIN-90014, Oulu, Finland
#Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Headington OX3 7DQ, United Kingdom
rThe Botnar Research Center, NIHR, Oxford Biomedical Research Unit, Oxford OX3 7LD, United Kingdom
S Supporting Information
b
ABSTRACT: Histone methylations are important chromatin marks that
regulate gene expression, genomic stability, DNA repair, and genomic
imprinting. Histone demethylases are the most recent family of histone-
modifying enzymes discovered. Here, we report the characterization of a
small-molecule inhibitor of Jumonji C domain-containing histone demethy-
lases. The inhibitor derives from a structure-based design and preferentially
inhibits the subfamily of trimethyl lysine demethylases. Its methyl ester
prodrug, methylstat, selectively inhibits Jumonji C domain-containing his-
tone demethylases in cells and may be a useful small-molecule probe of
chromatin and its role in epigenetics.
’ INTRODUCTION
(H3K4), H3K9, H3K27, H3K36, H3R2, or H4R3 methylations.5
However, HDMs that specifically modify H3K79me3 and
H4K20me3 have not yet been identified. Recent studies have
shown that HDMs often display tissue-specific expression and
play critical roles in gene expression, meiosis, and embryonic
stem cell self-renewal.6
HDMs can be categorized into two classes based on their
enzymatic mechanisms: flavin adenine dinucleotide (FAD)-
dependent HDMs and Jumonji C domain-containing HDMs
(JHDMs).5,7 There are two FAD-dependent HDMs, both of
which are monoamine oxidases and can demethylate mono- and
dimethylated H3K4 and H3K9.4,8 As compared to FAD-depen-
dent HDMs, JHDMs appear to be more versatile in terms of their
substrate specificities. These proteins are Fe2þ- and R-ketoglu-
tarate-dependent hydroxylases, and their reported substrate
residues include H3K4, H3K9, H3K27, and H3K36 at all
methylation states.5 As the DNA repair protein AlkB,9 JHDMs
The covalent attachment of functional groups to chromatin,
including DNA methylation and histone modifications, is asso-
ciated with heritable changes that regulate cellular transcrip-
tomes without altering DNA sequence. Histone methylation is
one of the most important chromatin marks; these play impor-
tant roles in transcriptional regulation, DNA-damage response,
heterochromatin formation and maintenance, and X-chromo-
some inactivation.1 A recent discovery also revealed that histone
methylation affects the splicing outcome of pre-mRNA by
influencing the recruitment of splicing regulators.2 Histone
methylation includes mono-, di-, and trimethylation of lysines,
and mono-, symmetric di-, and asymmetric dimethylation of
arginines. These modifications can be activating or repressing,
depending on the site and degree of methylation. Two classes of
enzymes regulate the maintenance of histone methylation:
histone methyltransferases (HMTs) and histone demethylases
(HDMs). HDMs are the most recent family of histone-modify-
ing enzymes discovered. Since the human HDM, LSD1, was first
detected in 19983 and characterized in 2004,4 over a dozen
HDMs have been discovered that modify histone H3 lysine 4
Received: February 25, 2011
Published: May 17, 2011
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hydroxylate the CꢀH bond of methyl group, and the resulting
hemiaminal collapses to form the demethylated product.
Small-molecule modulators of histone-modifying enzymes not
only play important roles in understanding the structures and
functions of these enzymes, but possibly also provide unique
opportunities for treating diseases such as cancer and mental
retardation.10 Small molecules specifically inhibiting FAD-
dependent HDMs have been discovered recently.11 As with
other Fe2þ- and R-ketoglutarate-dependent hydroxylases,
JHDMs are inhibited by general inhibitors such as desferox-
amine (DFO, a metal-chelating agent),12 and R-ketoglutarate
mimics N-oxalylglycine13 and pyridine-2,4-dicarboxylic acid.14 In
addition, small-molecule inhibitors that show in vitro selectivity
for JHDMs have been discovered.15 However, their cellular
specificities have not been reported yet.
A number of JHDMs crystal structures have been solved,
several of which are complexed with methyllysine-containing
histone peptides and cofactor mimics.16 On the basis of these
crystal structures and the enzymatic mechanism of JHDMs,
we designed and synthesized potential JHDM-selective small-
molecule inhibitors, each of which contains a methyllysine
mimic (substrate mimic), an R-ketoglutarate mimic (cofactor
mimic), and a linker combining these two (Figure 1). Herein,
we characterize compound 1 (Figure 1) as a selective JHDM
inhibitor in vitro, and its corresponding methyl ester prodrug
2 as a selective JHDM inhibitor in vivo.
’ RESULTS AND DISCUSSION
Design and Synthesis of JHDM Inhibitor 1. The lysine-
mimicking fragment of compound 1 was derived from a well-
known histone deacetylase (HDAC) inhibitor MS-275.17 The
4-carbon linker was selected on the basis of the relative distance
and geometry of R-ketoglutarate and methyl lysine in crystal
structures. The synthesis of 1 began with oxidation of a commer-
cially available amine, 3 (Scheme 1), using benzoyl peroxide to
afford compound 4.18 Acylation of 4 with acyl chloride 5 gave
amide 6, which was sequentially deprotected to afford amine 7
using potassium carbonate in anhydrous methanol and trifluor-
oacetic acid (TFA). Synthesis of the lysine-mimicking fragment 8
started with monocarbamate formation of diol 9 with 2-naphthy-
lene isocyanate 10. Oxidation of the remaining alcohol using
pyridinium dichromate (PDC) provided an aldehyde 8, which
underwent a reductive amination with amine 7 to afford methyl-
stat (2). The corresponding acid 1 was prepared by hydrolysis of
2 using sodium hydroxide. To examine if under physiological
conditions, the positively charged ammonium ion is an important
functional group in the substrate mimicking fragment of 1, we
also synthesized its analogue 12 from amine 7 by a carbamate
formation reaction followed by hydrolysis of the ester (Scheme 1).
In Vitro Profiling of Compound 1. To evaluate the inhibitory
activity of 1 in vitro, we cloned and purified a GST-fusion protein
of the catalytic N-terminus of human JMJD2C(1ꢀ420).19 The
JHDM, JMJD2C, predominantly uses H3K9me3 substrates, but
has also been shown to demethylate H3K36me3. By using
electrospray time-of-flight (ESI-TOF) mass spectrometry, we
found this purified protein can remove the methyl group of a
synthetic histone H3 fragment peptide substrate (H3K9me3,
Supporting Information, Figure S1). Compound 1 shows a half
maximal inhibitory concentration (IC50) for JMJD2C of approxi-
mately 4.3 μM determined by a dissociation-enhanced lantha-
nide fluorescent immunoassay (DELFIA, Figure 2a). It is
noteworthy that compound 12 showed no inhibition of JMJD2C
activity at up to 100 μM, the highest concentration tested. This
result suggests that a positively charged substrate-mimicking
Figure 1. Structure-based design of a JHDM-selective inhibitor 1 and
the structure of its methyl ester methylstat (2).
Scheme 1. Synthesis of the Designed Bivalent JHDM Inhibitor 1 and Related Moleculesa
a Reagents and conditions: (a) dibenzoyl peroxide (1.2 equiv), buffer (pH = 10.5), CH2Cl2, 25 ꢀC, 2.5 h, 75%; (b) 5 (1.3 equiv), oxalyl chloride (1.2
equiv), DMF (0.05 equiv), CH2Cl2, 0ꢀ25 ꢀC, 1 h; then Et3N (2.0 equiv), DMAP (0.01 equiv), CH2Cl2, 0ꢀ25 ꢀC, 2 h, 72%; (c) K2CO3 (1.2 equiv),
MeOH, 30 min; (d) TFA (20 equiv), CH2Cl2, 0ꢀ25 ꢀC, 2 h; (e) 10 (0.3 equiv), DMAP (0.016 equiv), DMF, 25 ꢀC, 12 h, 56%; (f) PDC (1.0 equiv), 4 Å
molecular sieves, CH2Cl2, 25 ꢀC, 5 h, 85%; (g) NaBH3CN (5.0 equiv), HOAc (2.0 equiv), MeOH, 25 ꢀC, 12 h, 38% over three steps; (h) NaOH (2.0
equiv), THF, H2O, 25 ꢀC, 3 h, 85%; (i) CbzCl (1.0 equiv), Et3N (2.0 equiv), CH2Cl2, 0 ꢀC, 5 h, 82%; (j) LiOH H2O (3.2 equiv), THF, H2O, 2 h, 87%.
3
Abbreviations: Cbz = carboxybenzyl, DMF = N,N-dimethylformamide, DMAP = 4-dimethylaminopyridine, PDC = pyridinium dichromate, TFA =
trifluoroacetic acid.
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Figure 2. In vitro profiling of compound 1. (a) Enzyme inhibition data of compound 1 against H3K9me3 demethylases JMJD2A, JMJD2C, and
JMJD2E, H3K9me2 demethylase PHF8, and H3K27me3 demethylase JMJD3. (b) Enzyme inhibition data of compound 1 against prolyl hydroxylases
PHD1ꢀ3, factor inhibiting hypoxia-inducible factor FIH, an asparaginyl hydroxylase, FAD-dependent histone demethylase LSD1, and histone
deacetylases HDACs.
Figure 3. Methylstat as a selective JHDM inhibitor in vivo. (a) Methylstat, but not 1, inhibits KYSE150 cell growth after treatment for 48 h.
(b) Methylstat recovers H3K9me3 level in HeLa cells ectopically expressing JMJD2C(1ꢀ420)-GFP fusion protein. HeLa cells were transfected with
JMJD2C(1ꢀ420)-GFP and then treated with DMSO or 10 μM methylstat. Immunostaining experiments using anti-H3K9me3 antibody (red) and
DAPI nuclear stain (blue) were performed after 48 h of incubation. Yellow arrowheads indicate transfected cells. (c) Methylstat induces
hypermethylation of histone proteins in a concentration-dependent manner. KYSE150 cells were treated with DMSO, DFO at 150 μM, DMOG at
1 mM, and methylstat at 5, 10, and 15 μM for 48 h, respectively. (d) EC50 values of methylstat for H3K4me3 and H3K9me3 in KYSE150 cells were
determined by quantifying images from immunostaining assays as 10.3 and 8.6 μM, respectively. (e) Methylstat does not inhibit prolyl hydroxylases in
cells. MCF7 cells were treated with DMSO, DFO at 150 μM, DMOG at 1 and 2 mM, and methylstat at 5, 10, and 15 μM for 24 h, respectively. Histone
levels are based on coomassie stain.
fragment of our designed inhibitor 1 is important for its inhibi-
tory activity against JHDMs.
against other subfamily members such as the dimethyl-
specific H3K9 demethylase PHF8 (Figure 2a).22 Although
comparison of IC50 values for different enzymes should be
approached with caution, these data indicate that compound
1 might be in vitro a better inhibitor for the trimethyl
demethylase families JMJD2 and JMJD3 as compared to
dimethyl demethylases such as FBXL11/PHF8 subfamily.
To further evaluate the specificity of compound 1, we tested it
against a distinct Jumonji C domain-containing enzyme, the
To evaluate the enzyme selectivity of 1, we next examined
its activity against several other JHDM isoforms in vitro.
Compound 1 inhibits JMJD2A (H3K9me3 and H3K36me3
demethylase), JMJD2E (H3K9me3 demethylase),20 and
JMJD3 (H3K27me3 demethylase)21 with IC50 values on the
same order of magnitude (Figure 2a) as seen for JMJD2C;
however, compound 1 shows considerably higher IC50 values
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Figure 4. Methylstat prevents myogenesis through inhibition of H3K27me3-demethylase UTX. (a) Methylstat prevented myotube formation in a
concentration-dependent manner. C2C12 cells were treated with DMSO and methylstat at 1, 2, or 4 μM in differentiation media, respectively.
Immunostaining experiments using antimyosin heavy chain (MHC) antibody (red) and DAPI nuclear stain (blue) were performed after 6 days of
incubation. (b) Methylstat inhibits the expressions of UTX-targeted genes, Myog and Ckm, during myogenesis. C2C12 cells were treated with DMSO or
methylstat at 1, 2, and 4 μM in differentiation media, respectively. mRNAs were collected after 6 days of incubation, and RT-PCR experiments were
performed. (c) Methylstat induced hypermethylation of H3K27me3 in C2C12 cells. C2C12 cells were treated with DMSO or methylstat at 2 and 4 μM
in differentiation media for 48 h, respectively. Histone levels are based on coomassie stain.
asparaginyl hydroxylase FIH,23 as well as other, even more
distantly related R-ketoglutarate-dependent hydroxylases such
as prolyl hydroxylases PHD1ꢀ3,24 which also show higher IC50
values against compound 1 (Figure 2b). Although compound 1
contains a methyl lysine mimicking fragment, its IC50 against the
FAD-dependent demethylase, LSD1, was determined as 620 μM
(Figure 2b), which is over 100-fold higher than its IC50 against
the JMJD2 histone demethylases. In addition, compound 1 does
not inhibit class I or class II HDACs at up to 800 μM (Figure 2b),
which are also transition metal-dependent histone lysine mod-
ifying enzymes.
treatment of the cells with methylstat. Next, we further evaluated
the cellular effects of methylstat on a variety of histone methyla-
tion marks. DMOG and DFO were used as positive controls,
because they are known cell-active inhibitors of Fe2þ- and
R-ketoglutarate-dependent hydroxylases. Western blotting ex-
periments showed that methylstat induces hypermethylation of
lysine residues including H3K4, H3K9, H3K27, and H3K36 at
almost all methylation states investigated in a concentration-
dependent manner (Figure 3c). Similar results were also ob-
served in human breast adenocarcinoma MCF7 cells (Suppor-
ting Information, Figure S3). Of substantial interest is the
observation that methylstat also induces increased cellular levels
of H3K79me3 and H4K20me3. These results suggest that these
histone methylation marks can also be removed by some as yet
unknown Jumonji C domain-containing proteins.
To quantify the cellular activity of methylstat, we used a quan-
titative image-based assay. Immunostaining of KYSE150 cells
treated with DMSO or methylstat showed that the cellular levels
of both H3K4me3 and H3K9me3 increase in a concentration-
dependent fashion (Figure 3d). Quantification of the images
provided the half maximal effective concentrations (EC50) for
H3K4me3 and H3K9me3 as 10.3 and 8.6 μM, respectively. The
cellular effects of methylstat are also cell type-specific. In MCF7
cells, the EC50 values of methylstat for H3K4me3 and H3K9me3
are 6.7 and 6.3 μM, respectively (Supporting Information, Figure
S4). It is noteworthy that methylstat induced a much higher
increase of the H3K9me3 mark in KYSE150 cells as compared to
that in MCF7 cells (10.8 fold vs 4.5 fold), probably due to higher
expression level of JMJD2C in KYSE150 cells.19
Cellular Activity and Specificity of Methylstat. To examine
the cellular activity of the small molecules we synthesized, we
chose to use JMJD2C-sensitive esophageal carcinoma cell line
KYSE150 in a growth inhibition assay.19 However, compound 1
did not show significant growth inhibition at up to 100 μM,
possibly due to the poor cellular uptake arising from the high
polarity of the zwitterions form in which it typically exists under
physiological conditions (c log P ꢀ0.99). Therefore, we tested its
methyl ester prodrug 2 (Figure 1, c log P 1.02),13,14 which we
have named methylstat. Methylstat inhibited KYSE150 cell
growth with a half maximal growth inhibitory concentration
(GI50) at approximately 5.1 μM (Figure 3a). It should be noted
that methylstat does not inhibit the enzymatic activities of
JMJD2C, JMJD2A, LSD1, or HDACs at up to 50 μM, the
highest concentration tested (Supporting Information, Figure
S2). To test the stability of methylstat, it was incubated in
KYSE150 cell growth media at 37 ꢀC for 48 h, then extracted
using organic solvent (3:1 chloroform:isopropanol). We found
that 88% of methylstat was recovered unchanged, the structure of
To assess the selectivity of methylstat in vivo, we examined
another class of Fe2þ- and R-ketoglutarate-dependent hydro-
xylases, prolyl hydroxylases (PHDs). PHDs have been reported
to regulate normoxic inactivation of hypoxia-inducible factors
(HIFs) by hydroxylating specific prolyl residues of HIFs.12,25
Inhibition of PHDs results in higher stability of HIFs and
accumulation of HIFs in cells. DMOG and DFO have been used
to mimic hypoxia conditions by inhibiting PHDs in vivo. MCF7
cells treated with DMOG or DFO showed significantly increased
1
which was confirmed by H NMR; 89% of compound 1 was
recovered when this procedure was performed with 1.
To prove methylstat inhibits JMJD2C in cells, we ectopically
expressed a GFP-fusion protein of the catalytic N-terminus of
JMJD2C(1ꢀ420) in human epithelial carcinoma HeLa cells.
Successfully transfected cells (green cells in Figure 3b) showed a
significant reduction of H3K9me3 level as indicated by an
immunostaining assay, and this effect was reversed by the
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cellular levels of HIF-1R, whereas methylstat did not affect the
cellular HIF-1R levels at up to 15 μM, the highest concentration
tested (Figure 3e). Similar results were also observed in HeLa
cells (Supporting Information, Figure S5).
Author Contributions
zThese authors contributed equally.
’ ACKNOWLEDGMENT
Inhibition of H3K27me3 Demethylase UTX-Dependent
Myogenesis Using Methylstat. To demonstrate methylstat as
a cellular probe for the studies of JHDMs further, we investigated its
action during myogenesis. Recent studies have shown that UTX, an
H3K27me3-specific JHDM, is recruited to the regulatory regions of
muscle-specific genes Myog and CKm to remove the repressive
chromatin mark H3K27me3 during myogenesis.26 Knockdown of
UTX in C2C12 cells using shRNA prevents the formation of
multinucleated myotubes. We found that treatment of C2C12 cells
with 2 μM of methylstat is sufficient to prevent the formation myosin
heavy chain (MHC)-positive myotubes (Figure 4a). Reverse tran-
scription-polymerase chain reaction (RT-PCR) experiments also
showed loss of expression of UTX-targeted genes Myog and CKm in
differentiating myoblast upon treatment with methylstat (Figure 4b).
It is also noteworthy that no obvious global hypermethylation of
H3K27me3 was observed by Western blot in differentiating myo-
blast treated with 2 μM methylstat, but only at higher concentration
(Figure 4c).
We thank S. Kato (University of Colorado) for his help with
mass spectrometry and S. Fisch (Broad Institute of Harvard and
MIT) and Ferran Fece de la Cruz (CeMM) for help with
biochemical assays. This work was supported by the University
of Colorado, Colorado Initiative of Molecular Biotechnology, the
National Institute of General Medical Sciences (GM38627),
European Union FP7Marie Curie grant PIOF-GA-2008-
221135 (to S.K.), and Oxford NIHR Biomedical Research Unit.
The Structural Genomics Consortium is a registered charity (No.
1097737) funded by the Canadian Institutes for Health Re-
search, the Canadian Foundation for Innovation, Genome
Canada through the Ontario Genomics Institute, GlaxoSmithK-
line, Karolinska Institutet, the Knut and Alice Wallenberg
Foundation, the Ontario Innovation Trust, the Ontario Ministry
for Research and Innovation, Merck and Co., Inc., the Novartis
Research Foundation, the Swedish Agency for Innovation Sys-
tems, the Swedish Foundation for Strategic Research, and the
Wellcome Trust.
’ CONCLUSIONS
’ REFERENCES
We have described a cell-active selective small-molecule
inhibitor of Jumonji C domain-containing histone demethylases.
Compound 1 showed selective inhibitory activity against tri-
methyl-specific JHDMs in vitro, and its methyl ester methylstat
(2) showed selective activity against a large set of JHDMs in cells.
In addition, induction of hypermethylation of H3K79me3 and
H4K20me3 in cells by methylstat also indicates the existence of
novel JHDMs specifically targeting these important chromatin
marks. This discovery provides a useful small-molecule probe
that selectively targets histone demethylases. Together with other
small-molecule regulators of histone-modifying enzymes,10,11,13ꢀ15,27
methylstat should find applications for studies of histone methy-
lation dynamics in a wide range of biological processes, such as
embryonic development and differentiation, germline mainte-
nance and meiosis, and regulation of gene expression, and of
disease processes, such as those leading to the development of
cancer. Further studies on the structureꢀactivity relationship
and the inhibitory mechanism of methylstat are underway and
will be reported in due course.
(1) Klose, R. J.; Zhang, Y. Nat. Rev. Mol. Cell Biol. 2007, 8, 307–318.
(2) Luco, R. F.; Pan, Q.; Tominaga, K.; Blencowe, B. J.; Pereira-Smith,
O. M.; Misteli, T. Science 2010, 327, 996–1000.
(3) Tong, J. K.; Hassig, C. A.; Schnitzler, G. R.; Kingston, R. E.;
Schreiber, S. L. Nature 1998, 395, 917–921.
(4) Shi, Y.; Lan, F.; Matson, C.; Mulligan, P.; Whetstine, J. R.; Cole,
P. A.; Casero, R. A.; Shi, Y. Cell 2004, 119, 941–953.
(5) For recent reviews on histone demethylases, see: (a) Shi, Y. Nat.
Rev. Genet. 2007, 8, 829–833. (b) Loenarz, C.; Schofield, C. J. Nat. Chem.
Biol. 2008, 4, 152–156. (c) Cloos, P. A.; Christensen, J.; Agger, K.; Helin,
K. Genes Dev. 2008, 22, 1115–1140. (d) Marmorstein, R.; Trievel, R. C.
Biochim. Biophys. Acta 2009, 1789, 58–68.
(6) Lan, F.; Nottke, A. C.; Shi, Y. Curr. Opin. Cell. Biol. 2008,
20, 316–325.
(7) Tsukada, Y.; Fang, J.; Erdjument-Bromage, H.; Warren, M. E.;
Borchers, C. H.; Tempst, P.; Zhang, Y. Nature 2005, 439, 811–816.
(8) (a) Wissmann, M.; Yin, N.; M€uller, J. M.; Greschik, H.; Fodor,
B. D.; Jenuwein, T.; Vogler, C.; Schneider, R.; G€unther, T.; Buettner, R.;
Metzger, E.; Sch€ule, R. Nat. Cell Biol. 2007, 9, 347–353. (b) Ciccone,
D. N.; Su, H.; Hevi, S.; Gay, F.; Lei, H.; Bajko, J.; Xu, G.; Li, E.; Chen, T.
Nature 2009, 461, 415–418.
(9) (a) Yi, C.; Yang, C. G.; He, C. Acc. Chem. Res. 2009, 42, 519.
(b) Yi, C.; Jia, G.; Hou, G.; Dai, Q.; Zhang, W.; Zheng, G.; Jian, X.; Yang,
C. G.; Cui, Q.; He, C. Nature 2010, 468, 330–333.
(10) (a) Cole, P. A. Nat. Chem. Biol. 2008, 4, 590–597. (b) Spannhoff,
A.; Hauser, A. T.; Heinke, R.; Sippl, W.; Jung, M. ChemMedChem 2009,
4, 1568–1582. (c) Natoli, G.; Testa, G.; De Santa, F. Curr. Opin. Drug
Discovery Dev. 2009, 12, 607–615.
’ ASSOCIATED CONTENT
S
Supporting Information. Complete refs 15b, 15c, 16b,
b
22a, supporting figures, materials, experimental procedures, and
characterization data for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(11) (a) Culhane, J. C.; Szewczuk, L. M.; Liu, X.; Da, G.; Marmorstein,
R.; Cole, P. A. J. Am. Chem. Soc. 2006, 128, 4536–4537. (b) Ueda, R.;
Suzuki, T.; Mino, K.; Tsumoto, H.; Nakagawa, H.; Hasegawa, M.; Sasaki,
R.; Mizukami, T.; Miyata, N. J. Am. Chem. Soc. 2009, 131, 17536–17537.
(12) Ivan, M.;Kondo, K.;Yang, H.;Kim, W.;Valiando, J.;Ohh, M.;Salic,
A.; Asara, J. M.; Lane, W. S.; Kaelin, W. G., Jr. Science 2001, 292, 464–468.
(13) Hamada, S.; Kim, T. D.; Suzuki, T.; Itoh, Y.; Tsumoto, H.;
Nakagawa, H.; Janknecht, R.; Miyata, N. Bioorg. Med. Chem. Lett. 2009,
19, 2852–2855.
’ AUTHOR INFORMATION
Corresponding Author
xiang.wang@colorado.edu
Present Addresses
ODepartment of Surgery, Tongji Hospital, Huazhong University
of Science and Technology, 1095 Jiefang Road, Wuhan, Hubei,
430030, People’s Republic of China.
(14) Mackeen, M. M.; Kramer, H. B.; Chang, K. H.; Coleman, M. L.;
Hopkinson, R. J.; Schofield, C. J.; Kessler, B. M. J. Proteome. Res. 2010,
9, 4082–4092.
[Astex Therapeutics, 436 Cambridge Science Park, Cambridge
CB4 0QA, United Kingdom.
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(15) (a) Rose, N. R.; Woon, E. C.; Kingham, G. L.; King, O. N.;
Mecinovic, J.; Clifton, I. J.; Ng, S. S.; Talib-Hardy, J.; Oppermann, U.;
McDonough, M. A.; Schofield, C. J. J. Med. Chem. 2010, 53, 1810–1818.
(b) Hamada, S.; et al. J. Med. Chem. 2010, 53, 5629–5638. (c) King,
O. N.; et al. PLoS One 2010, 5, e15535.
(16) (a) Chen, Z.; Zang, J.; Whetstine, J.; Hong, X.; Davrazou, F.;
Kutateladze, T. G.; Simpson, M.; Mao, Q.; Pan, C. H.; Dai, S.; Hagman,
J.; Hansen, K.; Shi, Y.; Zhang, G. Cell 2006, 125, 691–702. (b) Ng, S. S.;
et al. Nature 2007, 448, 87–91. (c) Couture, J. F.; Collazo, E.;
Ortiz-Tello, P. A.; Brunzelle, J. S.; Trievel, R. C. Nat. Struct. Mol. Biol.
2007, 14, 689–695. (d) Horton, J. R.; Upadhyay, A. K.; Qi, H. H.; Zhang,
X.; Shi, Y.; Cheng, X. Nat. Struct. Mol. Biol. 2010, 17, 38–43.
(17) Simonini, M. V.; Camargo, L. M.; Dong, E.; Maloku, E.; Veldic,
M.; Costa, E.; Guidotti, A. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 1587–1592.
(18) Wang, Q. X.; King, J.; Phanstiel, I. O. J. Org. Chem. 1997, 62,
8104–8108.
(19) Cloos, P. A.; Christensen, J.; Agger, K.; Maiolica, A.; Rappsilber,
J.; Antal, T.; Hansen, K. H.; Helin, K. Nature 2006, 442, 307–311.
(20) Kawamura, A.; Tumber, A.; Rose, N. R.; King, O. N.; Daniel,
M.; Oppermann, U.; Heightman, T. D.; Schofield, C. Anal. Biochem.
2010, 404, 86–93.
(21) (a) Lan, F.; Bayliss, P. E.; Rinn, J. L.; Whetstine, J. R.; Wang,
J. K.; Chen, S.; Iwase, S.; Alpatov, R.; Issaeva, I.; Canaani, E.; Roberts,
T. M.; Chang, H. Y.; Shi, Y. Nature 2007, 449, 689–694. (b) De Santa, F.;
Totaro, M. G.; Prosperini, E.; Notarbartolo, S.; Testa, G.; Natoli, G. Cell
2007, 130, 1083–1094. (c) Agger, K.; Cloos, P. A.; Christensen, J.;
Pasini, D.; Rose, S.; Rappsilber, J.; Issaeva, I.; Canaani, E.; Salcini, A. E.;
Helin, K. Nature 2007, 449, 731–734.
(22) (a) Qi, H. H.; et al. Nature 2010, 466, 503–507. (b) 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. Nature 2010, 466, 508–
512.
(23) Koivunen, P.; Hirsil€a, M.; Gunzler, V.; Kivirikko, K. I.; Myllyharju, J.
J. Biol. Chem. 2004, 279, 9899–9904.
(24) (a) Hirsil€a, M.; Koivunen, P.; Gunzler, V.; Kivirikko, K. I.;
Myllyharju, J. J. Biol. Chem. 2003, 278, 30772–30780. (b) Hirsil€a, M.;
Koivunen, P.; Xu, L.; Seeley, T.; Kivirikko, K. I.; Myllyharju, J. FASEB J.
2005, 19, 1308–10.
(25) Asikainen, T. M.; Schneider, B. K.; Waleh, N. S.; Clyman, R. I.;
Ho, W. B.; Flippin, L. A.; G€unzler, V.; White, C. W. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 10212–10217.
(26) Seenundun, S.; Rampalli, S.; Liu, Q. C.; Aziz, A.; Palii, C.; Hong,
S.; Blais, A.; Brand, M.; Ge, K.; Dilworth, F. J. EMBO J. 2010,
29, 1401–1411.
(27) Bradner, J. E.; West, N.; Grachan, M. L.; Greenberg, E. F.;
Haggarty, S. J.; Warnow, T.; Mazitschek, R. Nat. Chem. Biol. 2010,
6, 238–243.
9456
dx.doi.org/10.1021/ja201597b |J. Am. Chem. Soc. 2011, 133, 9451–9456