Cytosine-Based TET Enzyme Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.8b00474

DNA methylation is known as the prima donna epigenetic mark for its critical role in regulating local gene transcription. Changes in the landscape of DNA methylation across the genome occur during cellular transition, such as differentiation and altered neuronal plasticity, and become dysregulated in disease states such as cancer. The TET family of enzymes is known to be responsible for catalyzing the reverse process that is DNA demethylation by recognizing 5-methylcytosine and oxidizing the methyl group via an Fe(II)/alpha-ketoglutarate-dependent mechanism. Here, we describe the design, synthesis, and evaluation of novel cytosine-based TET enzyme inhibitors, a class of small molecule probes previously underdeveloped but broadly desired in the field of epigenetics. We identify a promising cytosine-based lead compound, Bobcat339, that has mid-μM inhibitor activity against TET1 and TET2, but does not inhibit the DNA methyltransferase, DNMT3a. In silico modeling of the TET enzyme active site is used to rationalize the activity of Bobcat339 and other cytosine-based inhibitors. These new molecular tools will be useful to the field of epigenetics and serve as a starting point for new therapeutics that target DNA methylation and gene transcription.

Full text of DOI:10.1021/acsmedchemlett.8b00474

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Letter
Cytosine-based TET Enzyme Inhibitors
Gabriella N.L. Chua, Kelly L. Wassarman, Haoyu Sun, Joseph A. Alp, Emma I. Jarczyk, Nathanael
J. Kuzio, Michael J Bennett, Beth G. Malachowsky, Martin Kruse, and Andrew John Kennedy
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00474 • Publication Date (Web): 31 Jan 2019
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Cytosine-based TET Enzyme Inhibitors
Gabriella N. L. Chua1, Kelly L. Wassarman1, Haoyu Sun1, Joseph A. Alp1, Emma I. Jarczyk1,
Nathanael J. Kuzio1, Michael J. Bennett1, Beth G. Malachowsky1, Martin Kruse2, Andrew J.
Kennedy1,*
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1Department of Chemistry and Biochemistry, Bates College, 2 Andrews Road, Lewiston, ME 04240
2Department of Biology and Program in Neuroscience, Bates College, 44 Campus Avenue, Lewiston, ME 04240
KEYWORDS: TET Inhibitors, TET Enzymes, DNA Methylation, Cytosine Methylation, Epigenetics,
Neuroepigenetics
ABSTRACT: DNA methylation is known as the prima donna epigenetic mark for its critical role in regulating local gene
transcription. Changes in the landscape of DNA methylation across the genome occur during cellular transition, such as
differentiation and altered neuronal plasticity, and become dysregulated in disease states such as cancer. The TET family
of enzymes is known to be responsible for catalyzing the reverse process that is DNA demethylation by recognizing 5-
methylcytosine and oxidizing the methyl group via an Fe(II)/alpha-ketoglutarate-dependent mechanism. Here we
describe the design, synthesis, and evaluation of novel cytosine-based TET enzyme inhibitors, a class of small molecule
probes previously underdeveloped but broadly desired in the field of epigenetics. We identify a promising cytosine-based
lead compound, Bobcat339, that has mid-µM inhibitor activity against TET1 and TET2, but does not inhibit the DNA
methyltransferase, DNMT3a. In silico modeling of the TET enzyme active site is used to rationalize the activity of
Bobcat339 and other cytosine-based inhibitors. These new molecular tools will be useful to the field of epigenetics and
serve as a starting point for new therapeutics that target DNA methylation and gene transcription.
DNA methylation of cytosine is a long-lived and self-
perpetuating epigenetic mark necessary for maintaining
cell-type transcriptional programs.1 Methylation of
cytosine at cytidine-guanosine (CpG) sites in promoters is
an inhibitory epigenetic mechanism capable of
completely silencing a gene in perpetuity. In addition,
patterns of DNA methylation contribute to the creation of
precise epigenetic landscapes associated with active gene
transcription.2 Such durability is owed to a rigorous
system wherein de novo DNA methyltransferases
(DNMTs) catalyze the methyl transfer from S-adenosyl
methionine to cytosines on previously unmethylated
DNA strands, while maintenance DNMTs methylate the
cytosine on the complimentary strand of the CpG site,
producing a dimethyl epigenetic mark.3-6
reversal back to unmodified cytosine.14 The result is a
cyclical epigenetic mechanism that is enduring, yet
dynamically regulated (Scheme 1).
Disruption of DNA methylation patterns is known to be
a hallmark of cancer with Tet2 being one of the most
frequently
mutated
genes
in
hematopoietic
malignancies.15 Likewise, mutations in Tet1, Tet2, and
Tet3, eliciting reduced expression, impaired enzymatic
activity, and concomitant decrease in levels of 5hmC,
appear to impart a diverse range of mutational landscapes
in a wide variety of different cancer types including liver,
lung, gastric, prostate, and breast cancer as well as
melanoma and glioblastoma.15-20 However, the precise
impact of altered TET activity on the progression and
maintenance of these cancers is largely unknown,
contributing to the need for further molecular tools for
DNA methylation patterns tend to be maintained in
differentiated cells; however, the methylation of DNA
exists as a dynamic process, reversible by the Ten-eleven
translocation methylcytosine dioxygenase (TET) family of
dioxygenases, coded by three separate genes (Tet1, Tet2,
and Tet3). These isoenzymes recognize and oxidize 5-
methylcytosine (5mC) to 5-hydroxymethylcytosine
(5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine
(5caC) via an Fe(II)/alpha-ketoglutarate-dependent
intensifying
investigative
efforts
surrounding
premalignant and malignant transformation of TET-
mutated cells.15
mechanism.7-10 These oxidized cytosine derivatives,
8, 11-13
themselves persistent epigenetic marks,7,
can then
function as intermediates subject to deamination and
glycosylase-dependent excision and repair, leading to the
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Scheme 1. Active DNA methylation and demethylation.
help explain the long-lasting changes in gene expression
DNA methylation is dynamically regulated by methyl writing
enzymes (DNMTs) and methyl erasing enzymes (TETs), allowing
the mechanism to govern local gene expression, a process
important for cell function and identity.
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associated with long-term memory in the central nervous
system. Active DNA methylation has been found to occur
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at memory-associated genes within memory circuits21,
23-25
and is required for long-term memory formation.6,
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Consequently,
studies
using
With growing consensus, it is hypothesized that
epigenetic mechanisms, namely DNA methylation, can
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Figure 1. Crystal Structure of TET2-DNA complex.26 a. TET2 binds dsDNA, breaks the double helix, and inserts 5mC into its active site.
b. View of the TET2 active site binding 5mC by forming hydrogen bonds with Asn1387, His1904, and Arg1261, all of which are critical
residues for TET2 catalytic activity and methylated DNA binding. The oxidative iron center is shown in proximity to the methyl group
on 5mC. c. A 2D rendering of the 5mC-bound TET2 active site and critical residue interactions; blue = basic residue, red = acidic residue,
and black = neutral residue.
DNMT inhibitors have shown DNMT activity to be crucial
for memory formation and stabilization.6, 21, 27-29 Moreover,
novo design of competitive inhibitors based on cytosine.
The deoxyribose also makes positive contacts with the
active site in the form of a water-mediated hydrogen
bond between the hydrofuran oxygen and Arg1261, a
critical residue that also binds alpha-ketoglutarate. The
methyl substituent on cytosine increases binding affinity
to TET2;26 however, installing a methyl group into the
design would likely produce a competitive substrate
rather than an inhibitor. Thus, a search was undertaken
to identify a suitable bioisostere for the methyl at the 5
position.
support for active DNA methylation as
regulator is in accordance with evidence situating TET-
mediated active DNA demethylation in negative
a positive
a
regulatory role within memory systems. Viral
overexpression of Tet1 has been shown to generate deficits
in long-term contextual fear memory,29 and Tet1 knockout
mice exhibit impaired memory extinction and enhanced
31
long-term memory.30,
With implications for other
cognitive disorders such as Alzheimer’s disease,
understood to be associated with altered DNA patterns at
Initial candidates included halides, particularly
chlorine, which is able to approximate the size of a
33
a number of disease-specific genes,32,
as well as for
mechanisms of drug addiction,23, 34 a deeper interrogation
into TET-mediated demethylation of DNA has become
increasingly imperative.
methyl group. Additionally,
a
CF3 group was
contemplated for its ability to mimic the tetrahedral
shape of the methyl as well as remain protected against
oxidation via fluorination. Finally, to aid in the ease of
analog synthesis and refrain from designing compounds
that may be incorporated into DNA, such as the cytosine-
based DNMT inhibitors azacitidine and decitabine, the
replacement of the deoxyribose for a phenyl group was
considered as a starting point. Therefore, a two-step
synthesis was undertaken (Scheme 2): First, the 5 position
of cytosine was halogenated (or alkylated) by taking
advantage of the preference for cytosine to undergo
electrophilic aromatic substitution at this position. For
example, 5-chlorosytosine was synthesized using N-
chlorosuccinimide (NCS) in acetic acid at reflux.35 Then,
the N1 position of the cytosine derivative was coupled
Unfortunately, there is currently no class of selective
TET inhibitors to pharmacologically probe these
biochemical processes sufficiently. Thus, the design and
development of potent inhibitors of the TET enzymes
were undertaken, starting with an assessment of the
solved crystal structure of human TET2 (Figure 1a).26 The
structure, which shows the TET2 enzyme bound to
methylated double stranded DNA (dsDNA), reveals how
the enzyme isolates and recognizes 5mC by orientating its
methyl substituent proximal to the oxidative Fe center.
Several critical hydrogen bonds are formed for enzymatic
recognition of 5mC. Asn1387 accepts a hydrogen bond
from N7 and His 1904 donates a hydrogen bond to N3 of
the 5mC ring (Figure 1b and c). Mutating either residue
leads to the loss of enzymatic function and reduced
binding affinities to methylated DNA.26 Therefore, it was
considered desirable to maintain these contacts in the de
using
copper-mediated
Ullman
conditions36
to
phenylboronic acid.
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NH2
100
75
50
25
0
1
2
3
4
5
Cl
N
TET1
TET2
NH2
NH2
NCS,
AcOH, ∆
Cu(OAc)2,
phenylboronic acid
NH2
Cl
N
N
O
N
R1
N
H2O, MeOH
***
O
N
H
O
N
H
O
N
***
Scheme 2. Cytosine-based TET enzyme inhibitor synthesis.
The 5 position of cytosine was chlorinated using NCS in acetic
acid and then coupled to phenylboronic acid using copper-
mediated Ullman conditions. All other N1 and 5-substituted
cytosine derivatives were synthesized using similar conditions.
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H
Cl
Br
CF3
R1 Group
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Figure 2. Examining possible methyl bioisosteres at the R1
position. Chloro, bromo, and trifluoromethyl-substituted
derivatives were tested for inhibition of TET1 or TET2-mediated
oxidation of methylated dsDNA. Each compound was tested at
100 µM in an ELISA. All data presented are N=3, error bars
indicate +/- sem. Two-way ANOVA, *P < 0.05, **P < 0.01, ***P <
0.001.
Figure 3. Inhibitor optimization. a. Several aryl groups were tested at the R2 position for inhibition of TET1 and TET2 at 100 µM. 3-
biphenyl substitution significantly increased TET1 inhibition (P = 0.002) over simple phenyl substitution, while 2-biphenyl (P = 0.0001)
and 4-biphenyl (P < 0.0001) substitution significantly reduced TET1 inhibition as compared to a phenyl substitution. b. 5-chloro
substitution at the R1 position is necessary to maintain the activity of 3-biphenyl substitution at the R2 position for both TET1 (P <
0.0001) and TET2 (P = 0.0003). c. Bobcat339 inhibits TET1 (IC50 = 33 µM) and TET2 (IC50 = 73 µM), but not DNMT3a. d. Bobcat339
docked into a homology model of TET1. e. The predicted binding conformations of Bobcat339 (teal), its 2-biphenyl isomer (blue), and its
4-biphenyl isomer (gold). Molecular surface; white = hydrophobic, blue = electropositive, and red = electronegative. All data presented
are N=3, error bars indicate +/- sem.
Next, these simple 5mC derivatives were tested for
inhibition of recombinant human TET1 or TET2 enzyme-
mediated oxidation of methylated dsDNA in an ELISA
with an inhibitor concentration of 100 µM (Figure 2). As
expected, hydrogen substitution at the R1 position was
insufficient to elicit significant inhibition. Three
bioisosteres of a methyl group, chlorine, bromine, and
trifluoromethyl, were then tested as substituents. Our
original hypothesis was that the trifluoromethyl group,
with its similar size and tetrahedral geometry, would be
the most effective at mimicking the binding conformation
of the endogenous methyl group, while being protected
from oxidation by fluorine substitution. However, this
derivative failed to perform with any greater potency than
no substitution at all. The 5-chloro substitution
(Bobcat212) proved the most effective with 57% and 43%
inhibition of TET1 and TET2 respectively.
Our attention then turned to the optimization of the
aryl substituent R2 position on the N1 of the 5-
chlorocytosine. Using the same synthetic strategy as the
phenyl series, 5-chlorocytosine was coupled to a variety of
arylboronic acids under Ullman conditions. These R2
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derivatives, which included tolyl, chlorophenyl, naphthyl, TET2 (calc. ∆GBinding = -10.08 kcal/mol). The 4-biphenyl
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quinolyl, and biphenyl substitution, were then tested for
inhibitory activity against TET1 and TET2 (Figure 3a).
Most performed with similar or worse potency to the
original phenyl derivative; however, results demonstrate
R2 substitution to be vital for activity, as most aryl
derivatives displayed significantly stronger inhibition
than unsubstituted 5-chlorocytosine. Notably, compound
Bobcat339, substituted at the R2 position with 3-biphenyl,
showed significantly enhanced activity at TET1 (P = 0.002,
two-way ANOVA) with comparable activity to the original
phenyl derivative at TET2. This observed activity was also
5-chlorocytosine-dependent (Figure 3b), and removal of
the chlorine substituent significantly reduced activity at
both TET1 (P < 0.0001) and TET2 (P = 0.0003).
derivative fails to take advantage of these interactions,
while the 2-biphenyl derivative is forced into a different
binding orientation entirely due to what would be steric
clashing with Tyr 1902 (Figure 3e), leading to a decreased
association with the active site for both the 4-biphenyl
(calc. ∆∆GBinding = +0.99 kcal/mol) and the 2-biphenyl
(calc. ∆∆GBinding = +0.52 kcal/mol) derivatives compared to
Bobcat339 when docked into the TET1 model. Similar
weaker binding affinities were also observed for the 4-
biphenyl (calc. ∆∆GBinding = +0.37 kcal/mol) and the 2-
biphenyl (calc. ∆∆GBinding = +0.72 kcal/mol) derivatives
when docked into the TET2 model. These calculated
differences in binding affinity align with the observed
differences in enzyme inhibition elicited by these isomers.
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Since any impediment to active DNA methylation may
counteract the potential therapeutic effects of inhibiting
the TET enzymes, Bobcat339 was evaluated for its desired
role as a selective inhibitor of TET1 and TET2 using an
inhibitory assay for DNMT3a. Bobcat339 elicited a dose-
response relationship for both TET1 (IC50 = 33 µM) and
TET2 (IC50 = 73 µM), while failing to show substantial
inhibition of DNMT3a at a concentration of 500 µM
(Figure 3c). These results underscore the potential of
these compounds to inhibit the removal, but not the
placement of methyl marks on the genome.
The activity of Bobcat339 was not shared by its
constitutional isomers, the 2-biphenyl and 4-biphenyl
derivatives, suggesting that the three-dimensional
structure of the 3-biphenyl isomer is preferentially
accommodated by the TET active site. To interrogate this
hypothesis computationally, we docked these cytosine
derivatives into the active site of the TET2 crystal
structure and a homology model of TET1 using the
Molecular Operating Environment (MOE) software
package. To generate the TET1 homology model, its
primary protein sequence was aligned to that of the
residues present in the published crystal structure of
TET2 (supporting information). Based on the binding
orientation of 5mC, our cytosine derivatives were placed
into the active site of the TET1 homology model. Then,
allowing all bonds to rotate, a series of potential binding
conformations was created. These conformations were
next docked into both the TET1 and TET2 models,
allowed to relax to a local energy minimum using the
Amber10:EHT force field, and scored using the London
∆G algorithm. The proposed binding mode of Bobcat339
for both isoenzymes situates the 5-chlorocytosine head
group directly into the active site in a fashion that mimics
the arrangement of 5mC, forming two base-pairing-like
hydrogen bonds with the enzyme and placing the
chlorine into the small pocket that typically
accommodates the methyl substrate (Figure 3d).
Furthermore, the 3-biphenyl group is oriented at an angle
to make hydrophobic contacts with the side wall of the
binding pocket, which is disrupted when the biphenyl
substitution pattern is altered. There is a high degree of
homology between the active sites of both isoenzymes,
and the calculated binding affinities of Bobcat339 are
similar for TET1 (calc. ∆GBinding = -10.23 kcal/mol) and
Figure 4. Bobcat339 reduces DNA 5hmC levels in hippocampal
neurons. Lead compounds Bobcat212 and Bobcat339 were dosed
at 10 µM in 1% DMSO onto HT-22 mouse hippocampal neurons
for 24 hrs. Cells were then lysed, DNA extracted, and TET
enzyme inhibition determined by 5hmC-specfic antibody
binding. Data presented are N=6 biological replicates per group,
error bars indicate +/- sem. One-way ANOVA, ns = not
significant, *P < 0.05, **P < 0.01.
Lastly, to determine whether these TET inhibitors
could act as molecular probes for reducing 5hmC levels in
live cells, Bobcat 339 and Bobcat212 were dosed onto HT-
22 cells, an immortalized mouse hippocampal neuronal
cell line. HT-22 cells were treated with 10μM Bobcat339 or
Bobcat212 in 1% DMSO for 24 hrs and lysed. DNA was
extracted, and 5hmC levels were detected using a 5hmC-
specific antibody, allowing for percent 5hmC of all
cytosine species to be determined by colorimetric
detection (Figure 4). Cells treated with Bobcat339 had
significantly reduced global 5hmC levels as compared to
the vehicle control, both demonstrating its ability to
reduce DNA 5hmC abundance by inhibiting TET enzyme
function in living cells and providing support for its utility
as a viable pharmacological probe.
Here, we have identified and evaluated a new class of
cytosine-based TET enzyme inhibitors. The most potent
in this series, Bobcat339, generated mid-μM IC50s for
TET1 and TET2 without inhibiting DNMT3a, a cytosine-
recognizing DNA methyltransferase, and reduced 5hmC
abundance in the DNA of cultured neurons. The TET
enzymes are critical epigenetic modifiers that regulate
gene imprinting to consequently govern cell
differentiation and development. Namely, TET enzymes
have been implicated in the epigenetic dysregulation
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driving several cancer states as well as in the modulation
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of neuronal plasticity and memory. As there does not
currently exist a selective pharmacological tool for
probing TET enzyme function, Bobcat339 and the other 5-
chlorocytosine derivatives described here present as
important contributions toward such a reagent.
ASSOCIATED CONTENT
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The Supporting Information is available free of charge on the
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to
5-
Hydroxymethylcytosine in Mammalian DNA by MLL Partner
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Heguy, A.; Viale, A.; Morris, L. G. T.; Huse, J. T.; Mellinghoff, I.
K.; Chan, T. A., IDH1 mutation is sufficient to establish the
glioma hypermethylator phenotype. Nature 2012, 483 (7390),
479-483.
AUTHOR INFORMATION
Corresponding Author
Tel: 207-755-5920.
Email: akennedy@bates.edu
Author Contributions
GNLC and AJK wrote the manuscript. Docking studies were
performed by GNLC. Chemical synthesis was performed by
KLW, HS, JAA, EIJ, and NJK. Chemical characterization was
performed by NJK. In vitro biological analysis was performed
by KLW and HS. Cell culture was performed by MJB and MK.
BGM provided research support for the project. All authors
have given approval to the final version of the manuscript.
Funding Sources
Research reported in this publication was supported by an
Institutional Development Award (IDeA) from the National
Institute of General Medical Sciences of the National
Institutes of Health under grant number P20GM103423, the
Pitt-Hopkins Research Foundation, the Orphan Disease
Center’s 2018 MDBR Pilot Grant Program, the Sherman
Fairchild Foundation, and Bates College.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work is dedicated to the memory of Kindal Kivisto.
ABBREVIATIONS
5caC, 5-carboxylcytosine; 5fC, 5-formylcytosine; 5hmC, 5-
hydroxymethylcytosine; 5mC, 5-methylcytosine; CpG,
cytidine-guanosine; DNMT, DNA methyltransferase;
dsDNA, double stranded DNA; MOE, Molecular
Operating Environment; TET, Ten-eleven translocation
methylcytosine dioxygenase.
19. Liu, C.; Liu, L.; Chen, X.; Shen, J.; Shan, J.; Xu, Y.; Yang, Z.;
Wu, L.; Xia, F.; Bie, P.; Cui, Y.; Bian, X.-w.; Qian, C., Decrease
of 5-hydroxymethylcytosine is associated with progression of
hepatocellular carcinoma through downregulation of TET1. PloS
one 2013, 8 (5), e62828.
20. Yang, H.; Liu, Y.; Bai, F.; Zhang, J.-Y.; Ma, S.-H.; Liu, J.; Xu, Z.-
D.; Zhu, H.-G.; Ling, Z.-Q.; Ye, D.; Guan, K.-L.; Xiong, Y.,
Tumor development is associated with decrease of TET gene
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NH2 OH
NH2
NH2
CH3
DNMT
SAM
TET
N
N
N
1
2
KG, O2
O
N
H
O
N
H
O
N
H
3
4
5
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8
C
5mC
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Asn
1387
a.
b.
c.
Asp
1384
Asn
1387
Fe
O
1
2
3
4
5
6
7
8
-O
O-
Tet2
H
O
H
N
His
1904
N
N
KG
Fe
O
O
H3C
Val
1900
His
1904
Arg
1261
Tyr
1902
O
5mC
Tyr
1902
Arg
1261
O H
H
9
O
10
11
12
13
14
O
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NH2
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Cl
N
NH2
NH2
NCS,
AcOH, ∆
Cu(OAc)2,
phenylboronic acid
Cl
1
2
3
4
5
N
N
O
N
H O, MeOH
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N
H
O
N
H

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100
TET1
TET2
NH2
1
2
3
75
R1
N
***
4
5
6
7
8
9
10
11
12
13
14
O
N
50
25
0
***
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Cl
Br
CF
3
R1 Group

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a.
b.
100
75
50
25
0
100
75
50
25
0
TET1
TET1
TET2
1
2
3
4
5
6
7
8
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NH2
N
TET2
NH2
R1
N
Cl
N
O
O
N
R2
H
H
Cl
4-tolyl
R1 Group
phenyl
3-quinolyl
4-bipheny3l-bipheny2l-biphenyl
1-naphthy2l-naphthyl
4-chloroph3e-cnhyllorophenyl
R2 Group
c.
d.
e.
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23
24
25
Asn
1387
Fe
TET1
TET2
100
75
50
25
0
NH2
Cl
N
DNMT3a
26
KG
Tyr
1902
O
N
27
28
29
30
31
32
33
His
1386
Arg
1261
Bobcat339
34
Bobcat339
1
10
100
1000
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0.20
NH2
NH2
Cl
Cl
1
2
0.15
0.10
0.05
0.00
N
N
ns
3
N
O
N
O
4
5
6
7
**
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12
13
14
Bobcat212
Bobcat339
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Asn
1387
Fe
1
2
3
4
5
KG
Tyr
1902
6
7
His
1386
8
9
10
11
12
13
14
15
Arg
1261
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Bobcat339