Analogues of the natural product sinefungin as inhibitors of EHMT1 and EHMT2

Article abstract of DOI:10.1021/ml4002503

A series of analogues of the natural product sinefungin lacking the amino acid moiety was synthesized and probed for their ability to inhibit EHMT1 and EHMT2. This study led to inhibitors 3b and 4d of methyltransferase activity of EHMT1 and EHMT2 and it demonstrates that such analogues constitute an interesting scaffold to develop selective methyltransferase inhibitors. Surprisingly, the inhibition was not competitive toward AdoMet.

Full text of DOI:10.1021/ml4002503

Letter
pubs.acs.org/acsmedchemlett
Analogues of the Natural Product Sinefungin as Inhibitors of EHMT1
and EHMT2
Kanchan Devkota,†,‡ Brian Lohse,‡ Qing Liu,§ Ming-Wei Wang,§ Dan Stærk,‡ Jens Berthelsen,*,†,∥
and Rasmus Prætorius Clausen*,‡
†The NNF Center for Protein Research, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark
‡Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
§The National Center for Drug Screening and the Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai 201203, China
S
* Supporting Information
ABSTRACT: A series of analogues of the natural product sinefungin
lacking the amino acid moiety was synthesized and probed for their
ability to inhibit EHMT1 and EHMT2. This study led to inhibitors 3b
and 4d of methyltransferase activity of EHMT1 and EHMT2 and it
demonstrates that such analogues constitute an interesting scaffold to
develop selective methyltransferase inhibitors. Surprisingly, the
inhibition was not competitive toward AdoMet.
KEYWORDS: Sinefungin, methyltransferase inhibitors, EHMT1, EHMT2
istone modifications have a central role in regulating gene
1
Hexpression and it has been suggested that they constitute
an epigenetic code.2 The enzymes capable of transferring acetyl,
methyl, and other groups are referred to as writers, whereas
enzymes responsible for removing the modifications are
referred to as erasers. Furthermore, a range of domains capable
of recognizing the modified substrates are termed readers.
Protein lysine methyltransferases (PKMTs) are a group of
epigenetic writers, responsible for the transfer of one to three
methyl groups from S-adenosyl-L-methionine(s) (AdoMet, 1,
Figure 1) to the ε-amino group of the target lysine residues in
histone tails3 and some nonhistone targets.4 To date, more than
50 PKMTs have been identified and EHMT1 (GLP, G9a like
proteins) and EHMT2 (G9a) are among the most well-studied
PKMTs. They are capable of mono- and dimethylating lysine 9
residue of histone 3 (H3K9) in euchromatin5 but have also
been found to methylate nonhistone substrates such as tumor
suppressor p53,6 Wiz, and reptin.7
EHMT1/2-mediated methylation has been implicated to be
pivotal for processes such as embryo development,8 germ cell
development and meiosis,9 and lymphocyte functioning,10,11 as
well as cognition and adaptive behaviors.12 Genetic variations of
EHMT1/2 have been associated with human diseases such as
Figure 1. Chemical structures of AdoMet (1), sinefungin (2), and the
cancer, inflammatory diseases, and neurogenerative disor-
analogues 3a−l and 4a−f.
ders.12−17 Removal of EHMT1 causes subtelomeric deletion
syndrome with severe mental retardation,13,14 along with
reduced locomotor activity and exploration.12 Increased
interest to identify potent inhibitors of these enzymes. Since the
discovery of the first PKMT inhibitor, chaetocin,17 in 2005,
expression of EHMT2 has been associated with lung, prostate,
and hepatocellular carcinoma.15,16 Interestingly, studies also
suggest that EHMT2 deficiency impairs immune response6
Received: July 3, 2013
while knock down of EHMT1/2 causes severe growth defects
inducing embryonic lethality.8 There has been a growing
Accepted: January 31, 2014
Published: January 31, 2014
© 2014 American Chemical Society
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Letter
several selective and nonselective inhibitors of PKMTs have
been reported. BIX 01294,18 E72,19 and UNC032120 are some
of the potent substrate (N-terminal peptides of H3)
competitive inhibitors of EHMT1/2. Later, in vivo active
analogues of UNC0321 were also reported.21,22 Some
inhibitors of PKMTs are natural products such as SAH (the
reaction product of methylation) and sinefungin, which
compete with AdoMet instead. In this study, we employed
sinefungin as a lead structure for the synthesis of a series of
methyltransferase inhibitors and tested them for inhibition of
EHMT1/2.
Sinefungin is an amino acid and a natural product from
cultures of Streptomyces incamatus and S. griseolus, which is
structurally related to AdoMet but has an amino methylene
group instead of the methylated sulfonium group. In this study,
two different series of compounds 3a−l and 4a−f were
designed that exchanged the terminal α-amino acid moiety of
sinefungin. One series possessed the additional α-amino group
of sinefungin, and the other series had no amino group (Figure
1).
The two series were synthesized via two different routes
(Figure 2). Route 1 was employed for analogues 3a−l, and
route 2 for analogues 4a−f. Route 1 started with adenosine 5
that was protected to form N6-benzoyl-2′,3′-O-isopropylide-
neadenosine 7 by two steps. Oxidation of 7 was achieved by
treatment with dimethyl sulfoxide and dicyclohexylcarbodii-
mide in the presence of dichloroacetic acid. The addition of
N,N′-diphenylethylenediamine formed 1,3-diphenylimidazoli-
dene derivative of adenosine-5′-aldehyde 8,23 which was treated
with Dowex 50 (H+) resin in aqueous tetrahydrofuran at room
temperature to regenerate adenosine-5′-aldehyde as its stable
monohydrate form 9.
Compound 9 was then dehydrated in benzene and directly
reacted with respective Wittig reagents to yield alkenes 11.
Hydrogenation yielded compound 12 that was deprotected in
one step giving 3a−l. Route 2 started with β-D-ribofuranoside
13, which was subsequently protected and iodinated giving
compound 15. Reaction between diethyl cyanomethylphosph-
onate and 15 in dimethyl sulfoxide gave ethyl phosphonate
16,24 which was then treated with various aldehydes and
ketones under basic conditions to yield conjugated nitriles 17.
Hydrogenation of the alkene gave 18 that was followed by
transformation of the nitrile to amide 19 using alkaline
hydrogen peroxide. Protecting groups were exchanged in two
steps giving acetyl protected 20. Coupling of N6-benzoyl-N6,N9-
bis(trimethylsilyl)adenine with 1′,2′,3′-tri-O-acetyl-β-D-ribofur-
anosides at high temperature (90 °C) in a microwave produced
the more thermodynamically stable 9-isomer 21. Removal of
the benzyl group and reestablishing the acetonide in 22 was
followed by a Hofmann rearrangement yielding Boc-protected
amine 2325 that could be completely deprotected in one step to
give the final products 4a−f as 1:1 mixture of diastereomers.
Initially, the compounds were screened in a FRET-based
LANCE ultra G9a histone H3-lysine N-methyltransferase
assay26 that measures the dimethylation of a biotinylated
histone H3 (1−21) peptide at lysine 9. Previous studies have
suggested that EHMT1 and EHMT2 both can mono-, di-, and
trimethylate histone H3 at lysine 9. This assay is based on the
capture of a modified peptide by the Europium labeled
antibody (Eu−H3K9me2, donor) and ULight-Streptavidin
(SA, acceptor dye), ultimately bringing them in close proximity.
When irradiated at 320 or 340 nm, the energy transfer from the
donor to the acceptor emits light at 665 nm, which is directly
Figure 2. Synthetic routes 1 and 2 to analogues 3a−l and 4a−f,
respectively.
proportional to the amount of modified substrate. (Constructs
for SET domain of EHMT1 and EHMT2 and expression and
purification are described in Supporting Information.)
Initial characterization of the compounds was performed at
100 μM at EHMT1 and EHMT2 (see Supporting Information
for assay procedure), and the results are shown in Figure 3. All
the assay characterization and inhibition studies were done in
triplicates, using the same batch of purified proteins. Most of
the compounds tested showed weak inhibitory activity at this
concentration; however, the inhibition varied and 4 analogues
3b, 3f, 4d, and 4e showed more than 40% inhibition toward
EHMT1 when screened at 100 μM, and the results were
followed up with further inhibition studies. Thus, dose−
response curves were established for the inhibition of these
compounds and sinefungin toward methylation activity of
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Figure 4. Dose−response curves of analogues 3b, 3f, 4d, 4e, and
sinefungin for EHMT1 and EHMT2 determined by LANCE Ultra
assay. All the presented compounds have lower IC50 values compared
to the natural scaffold sinefungin.
Table 1. IC50 Values of Analogues 3b, 3f, 4d, 4e, and
a
Sinefungin on EHMT1 and EHMT2
compds
3b
EHMT1 IC50 (μM)
EHMT2 IC50 (μM)
Figure 3. Inhibition of methyltransferase activity of EHMT1 and
EHMT2 by compounds 3a−l and 4a−4f at 100 μM determined in a
LANCE Ultra assay.
b
b
87 (57−130)
103 (68−156)
b
b
3f
235 (196 −281)
190 (174−206)
4d
1.5 (1.1−1.8)
147 (119−181)
513 (397−662)
1.6 (1.0−2.0)
157 (120−203)
509 (434−595)
b
b
4e
b
b
sinefungin
EHMT1 and EHMT2, and these results are shown in Figure 4.
Furthermore, IC50 values are reported in Table 1.
a
IC50 values are average of experiment done in triplicates. Numbers in
b
brackets indicate 95% confidence interval data. Since a full dose−
response curve was not achieved, these are estimated values. Thus, a
fairly potent analogue 4d was obtained when changing this part. In
addition, the second β-amino group can be removed and still show
weak inhibitory activity as demonstrated with analogue 3b. The
compounds did not discriminate EHMT1 and EHMT2, but the
selectivity changes compared to sinefungin since 4d and 3b did not
show inhibition of DNMT1, PRMT1, and SET7/9 at 200 μM, except
for 4d that is a very weak inhibitor of DNMT1. We cannot exclude
possible activity at other methyltransferases, but the study demon-
strates that selectivity can be achieved among methyltransferases with
this compound series. An inhibition kinetic study of both compounds
showed noncompetitive inhibition kinetics, which is surprising since
they could be expected to compete with SAM.
As seen in Table 1, the four analogues are more potent than
sinefungin with compound 4d as the best inhibitor on EHMT1
and EHMT2 (IC50 values of 1.5 and 1.6 μM, respectively) in
this study.
We then performed a kinetic study of the two most potent
compounds 3b and 4d. Both of the compounds demonstrated
AdoMet noncompetitive inhibition in this study (see Figure S3
and Table S2, Supporting Information), and thus the measured
IC50 values represent Ki values.
Neither of the compounds seems to discriminate significantly
between EHMT1 and EHMT2. However, when probed against
three other methyltransferases (DNMT1, PRMT1, and SET7/
9), 4d and 3b showed no significant inhibition at 200 μM and
below (except for 4d that has an estimated IC50 of 179 μM at
DNMT1) (see Table S3, Supporting Information).
We have presented the synthesis and characterization of a
series of analogues of sinefungin. Recently, a series of N-
alkylated sinefungin analogues were reported27 to suppress
methyltransferase activities leading to a potent N-propyl
substituted SETD2 inhibitor. In this study, we exchanged the
α-amino acid moiety and demonstrated that it is not essential
for inhibitory activity at EHMT1 and EHMT2.
achieving unselective inhibitors, the benefit of this compound
class is that it may be possible to find selective inhibitors toward
different methyltransferases in this compound class. The recent
discovery of a sinefungin analogue as a SETD2 inhibitor
emphasizes this notion. Furthermore, compounds with a
therapeutically favorable profile against several methyltrans-
ferases could be envisioned.
Future studies will focus on improving the synthetic route of
the compounds to extend the series and enable a broader
assessment of the activity of the compound class at various
methyltransferases. Furthermore, it will be important to verify
Thus, these scaffolds appear to be promising for further
modification to increase affinity of these selective non-
competitive inhibitors of EHMTs. While there is a risk for
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed information about compound synthesis, character-
ization, protein purification, assay procedure, and data analysis.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(J.B.) E-mail: jb@sund.ku.dk.
*(R.P.C.) E-mail: rac@sund.ku.dk.
■
Present Address
∥(J.B.) Department of International Health, Immunology and
Microbiology, University of Copenhagen, Blegdamsvej 3, DK-
2200 Copenhagen, Denmark.
Funding
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complex. Neuron 2009, 64 (5), 678−91.
Funding provided by University of Copenhagen for the Ph.D.
fellowship and the Danish Cancer Society, the University of
Copenhagen Programme of Excellence, and the Ministry of
Science and Technology of China (2009ZX09302-001,
2012ZX09304011, and 2013ZX09507002) for grant support.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We are grateful to the Novo Nordisk Foundation Center for
Protein Research and Department of Health Sciences,
University of Copenhagen for the Ph.D. fellowship, Dr.
Thomas Frimurer for valuable inputs in designing the
analogues, Ms. Huili Lu and Ms. Jie Zhang for technical
assistance, the Danish Cancer Society, the University of
Copenhagen Programme of Excellence and the Ministry of
Science and Technology of China (2009ZX09302-001,
2012ZX09304011, and 2013ZX09507002) for grant support,
and the EU COST-action TD0905, for providing a vital
Epigenetic focused network.
ABBREVIATIONS
■
TSA, p-toluenesulfonic acid; BzCl, benzoylchloride; DCA,
dichloroacetic acid; DCC, dicyclohexylcarbodiimide; BSA,
N,O-bis(trimethylsilyl)acetamide; TFA, trifluoroactic acid;
TMS, trimethylsilyl; AC, acetone; TEA, triethylamine
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published ASAP on February 4, 2014 with
errors in the Supporting Information. A corrected Supporting
Information file was reposted with the manuscript on February
18, 2014.
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