Inhibition of histone demethylases by 4-Carboxy-2,2'-Bipyridyl compounds

Article abstract of DOI:10.1002/cmdc.201100026

Exploiting epigenetics: 2-Oxoglutarate (2OG)-dependent histone lysine demethylases, such as JMJD2E, are potential therapeutic targets in a range of diseases. Through structure-activity relationship studies and analyses, we identified a potent 4-carboxy-2,2'-bipyridyl compound, which inhibits JMJD2E with an IC₅₀ value of 110nM, representing a 66-fold improvement over the lead compound. These bipyridyl derivatives bind in the 2-oxoglutarate binding site.

Full text of DOI:10.1002/cmdc.201100026

DOI: 10.1002/cmdc.201100026
Inhibition of Histone Demethylases by 4-Carboxy-2,2’-Bipyridyl Compounds
Kai-Hsuan Chang,[a] Oliver N. F. King,[a] Anthony Tumber,[b] Esther C. Y. Woon,[a] Tom D. Heightman,[b]
Michael A. McDonough,[a] Christopher J. Schofield,*[a] and Nathan R. Rose*[a]
In eukaryotes, nuclear DNA is packaged into chromatin by
binding to histones and associated factors. Covalent modifica-
tions to histone tails are associated with specific transcriptional
states of the associated DNA. Acetylation of lysine side chains
normally correlates with transcriptional activation, while deace-
tylation leads to transcriptional silencing. The regulatory roles
of lysine and arginine methylation appear to be more complex.
Methylation of certain lysine residues is associated with active
transcription, while methylation of others is associated with si-
lencing and heterochromatin formation. Each methylation
marker is placed, removed and interpreted in a site-specific
manner by histone methyltransferases, demethylases and
methyl binding domains, respectively. The biological functions
of the individual enzymes are
prolyl hydroxylases are in clinical trials for the treatment of
anaemia.[6] Inhibitors of the collagen prolyl hydroxylases have
also been evaluated as potential therapeutics for the treatment
of liver fibrosis.[7,8] The discovery of the JmjC domain histone
demethylases, and the suggestion that some of them are po-
tential therapeutic targets for cancer treatment,[9] has stimulat-
ed interest in their inhibition, but relatively few studies have
been described. Reported inhibitors of the JmjC demethylases
include N-oxalyl amino acids, 8-hydroxyquinolines, pyridine di-
carboxylates, hydroxamic acids and catechol-type flavonoids
(Figure 1).[10–13] Compounds that catalyse the ejection of a
structural ZnII ion from the JMJD2 demethylases have also
been reported (Figure 1).[14]
largely undefined and are the
focus of current investigations
(for Reviews see References
[1,2])
The JmjC histone demethylas-
es are 2-oxoglutarate (2OG)-de-
pendent oxygenases that cata-
lyse Ne-lysyl demethylation via
hydroxylation of the methyl
group in a 2OG- and FeII-depen-
dent manner (Scheme 1). Human
2OG oxygenases catalyse
a
range of reactions, including hy-
droxylation of amino acids, DNA,
and small molecules, and deme-
thylation of proteins and DNA.[3]
2OG oxygenases show promise
as therapeutic targets; an inhibi-
tor of g-butyrobetaine hydroxy-
lase (BBOX) is used for the treat-
ment of cardiovascular dis-
ease,[4,5] and inhibitors of the hy-
poxia inducible factor (HIF)
Figure 1. Structures of some previously reported histone demethylase inhibitors. Compounds 5 and 6 inhibit by
ejecting ZnII from the enzyme.
[a] K.-H. Chang,+ Dr. O. N. F. King,+ Dr. E. C. Y. Woon, Dr. M. A. McDonough,
Prof. Dr. C. J. Schofield, Dr. N. R. Rose
Chemistry Research Laboratory
In a study describing various template inhibitors of the JmjC
demethylases, we found that 2,2’-bipyridyl compounds with at
least one 4-carboxylate group inhibit the histone demethylase
JMJD2E.[11] A related series of compounds, 5,5’-dicarboxylate-
2,2’-bipyridyl derivatives, is reported to inhibit the collagen
prolyl-4-hydroxylases.[15] 2,2’-Bipyridine and bipyridyl com-
pounds have also been used as inhibitors of the HIF hydroxy-
lases.[16] Although it is likely that in some cases the enzyme in-
hibition effects of bipyridyl compounds result from metal che-
lation in solution, they also have the potential to inhibit via
active site binding, as is the case for some 2OG oxygenases.
However, to date there is no structural information on their
University of Oxford
Oxford, OX1 3TA (UK)
Fax: (+44)1865-275-674
E-mail: christopher.schofield@chem.ox.ac.uk
nathan.rose@chem.ox.ac.uk
[b] Dr. A. Tumber,+ Dr. T. D. Heightman
Structural Genomics Consortium, University of Oxford
Old Road Campus Research Building, Oxford, OX3 7DQ (UK)
[+] These authors contributed equally.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100026.
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Scheme 1. The JmjC-domain histone demethylases catalyse N-demethylation. For each demethylation reaction, 2OG is oxidised by molecular oxygen to gen-
erate succinate, CO2 and a reactive ferryl-oxo species which hydroxylates lysyl Ne-methyl groups; the unstable hemiaminal intermediate then fragments to
release formaldehyde and the demethylated lysine residue.
mechanism of action. Here, we report structure–activity rela-
tionship studies and analyses on bipyridyl inhibitors of
JMJD2E.
The bipyridyl compounds tested were synthesised according
to Scheme 2. Thus, 4,4’-dicarboxy-2,2’-bipyridine 9 was esteri-
fied to give the dimethyl or diethyl esters, which were then hy-
drolysed to yield the monoesters 8a or 8b, respectively. 8a
was coupled to a set of suitably protected primary amines to
yield compounds 11 a–b, 14a–e, 16, 18a–b, 20, 22, 24 and 27,
which were then hydrolysed and deprotected to yield the free
carboxylic acids 12a–b, 13a–b, 15a–e, 17, 19a–b, 21, 23, 25
and 28, respectively (Table 1). A derivative of 4-carboxy-2,2’-bi-
pyridine (30) was synthesised to evaluate the importance of a
4-carboxyl group (Scheme 2).
Inhibition assays for the histone demethylase JMJD2E were
carried out using two complementary assay methods. Inhibi-
tion of histone demethylation was measured using a coupled
enzyme assay, employing formaldehyde dehydrogenase (FDH)
to convert formaldehyde to formate, with concomitant reduc-
tion of NAD+ to NADH, which was monitored spectrophoto-
metrically.[11,17,18] Compounds were screened in eight-point
serial dilutions against JMJD2E (100 nm), generating dose–re-
sponse curves and IC50 data (Table 1). Each compound was also
screened in a similar dose–response manner against FDH only,
using 1 mm formaldehyde as a substrate. This was done to
ensure that inhibitory effects were due to inhibition of JMJD2E
and not FDH. To evaluate whether compounds were inhibiting
Figure 2. Nondenaturing ESI MS binding assay of inhibitors to JMJD2E.
by binding to the active site—as opposed to, or in addition to,
a) JMJD2E·FeII (labelled E) in the presence of compounds b) 40, c) 25 and
d) 13a, showing examples of relatively weak, medium and strong binding
chelation of iron in solution—nondenaturing electrospray ioni-
sation protein mass spectrometry (ESI MS) was used to detect
binding of compounds to the intact protein complex
(Figure 2).[11] Although there are caveats on the use of mass
spectrometry for screening binding strength,[17] the results of
both assay methods correlated well (Table 1), with more
potent compounds showing a stronger binding interaction
with the protein complex by ESI MS.
compounds, respectively.
ate, while the 4-carboxylate is positioned to interact with the
2OG binding residues Lys206 and Tyr132, effectively anchoring
the compound in the active site (see Figure 4).[11] The bipyridyl
compounds were, by analogy, predicted to bind in a similar
manner, with the pyridinyl nitrogens chelating the active site
metal, and the 4-carboxylate positioned to interact with
Lys206 and Tyr132. In support of this proposal, all the bipyridyl
derivatives tested were relatively potent inhibitors of JMJD2E
Previous work on inhibitors of the JMJD2 demethylases
demonstrated that the parent compound 2,4-pyridinedicarbox-
ylate is a 2OG-competitive inhibitor. This compound was
shown by crystallographic analyses (PDB: 2VD7) to bind the
active site metal via the pyridine nitrogen and the 2-carboxyl-
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Scheme 2. Reagents and conditions: a) SOCl2, MeOH, reflux, overnight, 90%; b) KOH, MeOH/THF (1:1), reflux, overnight, 70%; c) 1-Ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDCI), 1-hydroxybenzotriazole (HOBT), Et3N, DMF, overnight, RT; d) KOH, MeOH/THF (1:1), reflux, 4 h; e) CF3COOH, 2% H2O, 4 h, RT.
(IC50 <30 mm), with the exception of compound 30, which
lacks the 4-carboxylate group, which exhibited an IC50 value of
86 mm. This compound was 300-fold less potent than the
equivalent compound with the 4-acid moiety (15d).
The bipyridyl derivative resulting from coupling with ethyle-
nediamine (13a) inhibits JMJD2E with an IC50 value of 180 nm,
showing a 36-fold improvement of inhibition compared to 4’-
tency than 13a. Similarly, compounds 25 and 26 were signifi-
cantly less potent than 13a, suggesting that the amine of 13a
is important in binding.
The optimal position of the amine was then investigated.
Compound 13b (IC50 =470 nm) exhibited a twofold potency
decrease over 13a, which could reflect a suboptimal length
between the bipyridine core and Asp135. Consistent with a
preference for the positive charge at this position, the IC50
values of 12a and 12b, where the amine is neutralized by a
Boc group, were elevated and found to be 3.2 mm and 5.3 mm
for 12a and 12b, respectively. Introduction of unfunctionalised
aromatic amides (15a: IC50 =1.1 mm; 15b: IC50 1.0 mm) resulted
in a decrease in potency relative to 13a. The naphthalene de-
rivative (21) showed increased inhibition relative to its phenyl
analogue (15a), suggesting that hydrophobic or p–p interac-
tions in the active site might play a role in binding.
(methoxycarbonyl)-2,2’-bipyridine-4-carboxylic
acid
(IC50 =
6.6 mm). Analysis of a model of JMJD2A in complex with 13a
suggests that this improvement might in part be due to for-
mation of a salt bridge between the amine of bipyridyl deriva-
tive 13a and Asp135 at the active site. To investigate the im-
portance of the amine, the inhibition of JMJD2E activity by
compound 23, with a hydrogen atom substituting for an
amino group, was evaluated. Compound 23 was found to
have an IC50 value of 13 mm, representing a 72-fold lower po-
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Table 1. Inhibition of the histone demethylase JMJD2E (100 nm) by bipyridyl derivatives.[a]
Compd
IC50[b] [mM] Binding rank[c] Compd
IC50[b] [mM] Binding rank[c]
12a
3.2
5.3
medium
medium
17
1.1
strong
strong
12b
19a
0.69
13a
0.18
strong
19b
17
strong
13b
15a
15b
15c
15d
0.47
strong
medium
strong
strong
strong
21
23
25
8b
28
0.43
strong
1.1
13
medium
medium
medium
N.D.
1.0
25
27
0.11
0.27
2.3
15e
0.32
strong
30
86
N.D.
[a] All compounds were counter-screened against FDH at the same concentrations used for IC50 determinations, and inhibition of FDH was not observed
under these conditions, implying that the compounds only inhibited JMJD2E. [b] IC50 values represent the result of three independent experiments, where
standard errors in the log(IC50) are less than 10%. [c] Ranking of binding strength determined by ESI MS is as shown in Figure 2. N.D. not determined.
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Based on the possibility that a
combination of aromatic ring
and amine might give improved
potency, anilines 19a and 19b
were synthesized and evaluated.
The IC50 values of 19a and 19b
(690 nm and 17 mm, respectively)
showed no improvements rela-
tive to 13a, but demonstrated
the importance of the regio-
chemistry of the aromatic amine.
Compounds 15c, 15d and 15e
were synthesized with the aim
of tuning the length between
the bipyridine and phenyl
group. Compounds 15c, 15d
and 15e (IC50 =110 nm, 270 nm
and 320 nm, respectively) had
similar activities to 13a, demon-
strating that, at least for the
compounds analysed, chain
lengths longer than one methyl-
Figure 3. Views from the crystal structure of JMJD2A in complex with compound 13a (yellow sticks). The double-
stranded b-helix (conserved in 2OG oxygenases) is in red. Residues that bind FeII and 2OG are shown as green
sticks; NiII, which replaces FeII for crystallography, is shown as a green sphere. Other residues likely interacting
with 13a are shown as blue sticks.
ene all conferred similar potencies. These compounds might
form p–p interactions between their phenyl ring and Tyr175,
which is close to Asp135 in the substrate binding site. Howev-
er, the addition of a second benzene ring in compound 28
caused a 20-fold loss of potency relative to its analogue 15c.
Although bipyridyl compounds are expected to chelate FeII
in solution, it is clear from the ESI MS binding data that these
compounds inhibit by binding to the active site. Further, the
IC50 values for most compounds are 10- to 100-fold less than
the concentration of FeII used in the assays (10 mm), also indi-
cating that their inhibitory effects are not predominantly due
to iron chelation in solution. Finally, the substantial differences
(>100-fold) in IC50 values between compounds anticipated to
chelate iron similarly in solution (e.g., between 4-carboxylate
derivatives 12a–28 and compound 30 lacking the 4-carboxyl-
ate group) implies that these compounds inhibit, at least in
part, as a result of specific interactions in the active site. To
confirm this proposal, we also determined IC50 values for com-
pounds 13a, 15c and 15d at 50 mm FeII. The results showed
that IC50 values did not shift with the increase in FeII concentra-
tion (data not shown), indicating that these compounds inhibit
JMJD2E predominantly by mechanisms other than FeII chela-
tion in solution (Figure 2).
ence in potency resulting from addition of a 4-carboxylate to
one of the pyridinyl rings. The amide nitrogen of 13a is posi-
tioned to form two hydrogen bonds with water molecules that
in turn form hydrogen bonds to the phenol oxygen atom of
Tyr177 and to the backbone carbonyl oxygen atom of Glu169,
and an electrostatic interaction with Asp135 (2.7 ꢁ). For other
potent inhibitors such as 15c–e, it is likely that the lack of a
salt bridge with Asp135, and the flexibility of the aliphatic
chain, would allow the inhibitors to adopt conformations plac-
ing the aliphatic/aromatic side chains in the substrate binding
region of the active site. This general mode of binding would
also explain the relatively similar potencies of compounds
15c–e, if the side chains are projecting into the solvent-acces-
sible region of the substrate binding groove.
Comparison of the crystal structure of the JMJD2A–13a
complex with structures of JMJD2A in complex with fragments
of its histone H3K9 and K36 substrates (PDB: 2OQ6 and 2OS2)
reveals that, although 13a does not occupy the Ne-methyl-
lysine binding site, it is likely to block binding of the polypep-
tide substrate by occupying part of the binding site for the
polypeptide backbone.
Comparison of the binding mode of compound 13a with
crystal structures of other inhibitors in complex with JMJD2A
We then worked to obtain a crystal structure for JMJD2A in
complex with one of the more potent bipyridyl inhibitors, 13a.
A structure was solved for JMJD2A in complex with zinc, nickel
(substituting for iron) and 13a to 2.0 ꢁ resolution using a re-
ported structure (PDB: 2OX0) as a search model (Figure 3). The
structure reveals that 13a binds the active site metal by biden-
tate chelation through both pyridinyl nitrogens, verifying the
proposed overall mode of iron binding of bipyridyl com-
pounds to JMJD2A and likely other 2OG oxygenases including
the HIF hydroxylases. Notably, the carboxylate group of 13a is
positioned to interact with Lys206 and Tyr132 in a manner
analogous to that observed for 2OG, so rationalising the differ-
(2,4-PDCA,
5-carboxy-8-hydroxyquinoline,
N-oxalyl-d-(O-
benzyl)tyrosine and N-oxalylglycine) reveals that the bipyridyl
compound binds in the same plane as the other two aromatic
inhibitors, 2,4-PDCA and 5-carboxy-8-hydroxyquinoline, occu-
pying the two coordination sites opposite His276 and Glu190
(Figure 4).[11,12,17,19,20] In contrast, 2OG, N-oxalylglycine and N-
oxalyl-d-(O-benzyl)tyrosine coordinate opposite Glu190 and
His188. In the case of 2OG this presumably leaves the site op-
posite His276 available for the binding of molecular oxygen.
Although the situation with iron may be different to that for
nickel (used for crystallography), these observations suggest
that compounds such as 13a, 2,4-PDCA and 5-carboxy-8-hy-
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Acknowledgements
The authors thank the Wellcome
Trust (UK), the Biotechnology and
Biological
Sciences
Research
Council (UK) and the European
Union for funding.
Keywords: bipyridyl derivatives ·
epigenetics
·
histone
inhibitors ·
demethylases
2-oxoglutarate
·
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Experimental Section
Experimental details, including compound syntheses, inhibition
assays, protein purification and crystallography, are available as
Supporting Information on the WWW under http://dx.doi.org/
10.1002/cmdc.201100026.
The crystallographic data have been deposited in the RCSB Protein
Data Bank (PDB), PDB ID: 3PDQ.
Received: January 27, 2011
Published online on March 15, 2011
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