Discovery of a Highly Selective Cell-Active Inhibitor of the Histone Lysine Demethylases KDM2/7

Article abstract of DOI:10.1002/anie.201706788

Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure–activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.

Full text of DOI:10.1002/anie.201706788

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Discovery of a highly selective cell-active inhibitor of KDM2/7
Authors: Philip Gerken, Jamie Wolstenhulme, Anthony Tumber,
Stephanie Hatch, Yijia Zhang, Susanne Müller, Shane
Chandler, Barbara Mair, Fengling Li, Sebastian Nijman,
Rebecca Konietzny, Tamas Szommer, Clarence Yapp, Oleg
Fedorov, Justin Benesch, Masoud Vedadi, Benedikt Kessler,
Akane Kawamura, Paul Brennan, and Martin Derwyn Smith
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201706788
Angew. Chem. 10.1002/ange.201706788
Link to VoR: http://dx.doi.org/10.1002/anie.201706788
http://dx.doi.org/10.1002/ange.201706788

10.1002/anie.201706788
Angewandte Chemie International Edition
COMMUNICATION
Discovery of a highly selective cell-active inhibitor of KDM2/7
Philip A. Gerken,a Jamie R. Wolstenhulme,a Anthony Tumber,b Stephanie B. Hatch,b Yijia Zhang,a
Susanne Müller,b Shane A. Chandler,a Barbara Mair,b Fengling Li,c Sebastian M. B. Nijman,b Rebecca
Konietzny,b Tamas Szommer,b Clarence Yapp,b Oleg Fedorov,b Justin L. P. Benesch,a Masoud
Vedadi,c Benedikt M. Kessler,b Akane Kawamura,a Paul E. Brennanb* and Martin D. Smitha*
Previously discovered KDM2/7 inhibitors:
Abstract: Histone lysine demethylases (KDMs) are of critical
HO
O
HO
O
N
HO
O
importance in the epigenetic regulation of gene expression, yet there
are few selective, cell-permeable inhibitors or suitable tool
compounds for these enzymes. Here we describe the discovery of a
new class of inhibitor that is highly potent towards the histone lysine
demethylases KDM2A/7A. A modular synthetic approach was used
to explore chemical space and accelerate investigation of key
structure-activity relationships, leading to the development of a small
molecule with ≥75-fold selectivity towards KDM2A/7A vs other KDMs,
as well as cellular activity at low micromolar concentrations.
H
O
N
N
HO
7
N
O
OH
Hopkinson et al. [8]
IOX-1 (pan-KDM)
Rose et al. [9]
Daminozide (KDM2/7)
Suzuki et al. [12]
(KDM2/7)
CO2H
N
Ph
O
MeO
H2N
N
N
N
O
4
N
N
N
N
N
H
N
England et al. [14b]
(KDM2A)
Upadhyay et al. [22]
E67-2 (KDM7A)
Chemical modifications of DNA and its associated histones
regulate gene expression across the entire genome, and
therefore have a profound impact on a number of fundamental
biological processes.[1] As a result, targeting the epigenetic
pathways responsible for these chemical modifications may
represent a pivotal approach to addressing disease at the
transcription level.[2] In order to realize the potential of
epigenetics in drug discovery, a toolkit of chemical probes that
selectively target individual epigenetic proteins and allow
researchers to clearly identify their downstream effects is
invaluable.[3] Significant progress has been made towards the
development of a library of chemical probes that target the
proteins involved in histone acetylation, in particular the
bromodomain family of epigenetic readers.[4] In contrast, proteins
involved in the dynamic methylation of histone lysine residues
have proven to be more challenging targets, especially the
histone lysine demethylases (KDMs).[5]
This work: small molecule cell-active selective inhibitors of KDM2A/7A
R1
X
in vitro IC50 up to 0.12 µM
cellular IC50 = 2.5 µM
75-fold selectivity for KDM2A/7A vs
other lysine demethylase enzymes
N
R2
r
ke
n
i
l
N
O
R3
Y
Figure 1: Previously-discovered KDM2/7 inhibitors, and this work.
may be divided into six sub-families (KDM2-KDM7) based on
substrate specificity, with KDM2 and KDM7 proteins being
closely related.[7] A major challenge in generating a toolkit of
chemical probes for KDMs is achieving selectivity between these
structurally similar sub-families. Currently, most inhibitors of the
JmjC KDMs are iron-chelating 2-OG competitors (Figure 1).[8-14]
Although many of these molecules achieve high levels of in vitro
potency, they are frequently limited by a lack of selectivity and
activity in cells. Peptide inhibitors that either mimic the histone
substrate or bind KDMs allosterically have also been reported,[15]
however peptides are often limited by their cellular permeability.
The majority of KDMs belong to the Jumonji C (JmjC)
family of enzymes, containing a catalytically-active FeII ion in the
active site and requiring a 2-oxoglutarate (2-OG) cofactor for
demethylation in the catalytic JmjC-domain.[6] The JmjC KDMs
[a]
[b]
[c]
Dr P. A. Gerken, Dr J. R. Wolstenhulme, S. A. Chandler, Y. Zhang
Prof. Dr J. L. P. Benesch, Dr A. Kawamura, Prof. Dr M. D. Smith
Chemistry Research Laboratory
University of Oxford
12 Mansfield Road, Oxford, OX1 3TA (UK)
E-mail: martin.smith@chem.ox.ac.uk
Here, we describe the discovery of
permeable KDM2A/7A inhibitor that exhibits ≥75-fold selectivity
relative to other JmjC KDM sub-families.
a first-in-class, cell-
KDM2A catalyzes the demethylation of mono- and
dimethylated lysine 36 on histone H3 (H3K36).[16] The enzyme
has been reported to be involved in the regulation of NF-κB[17]
and the control of stem cell differentiation and proliferation.[18] Its
overexpression in gastric and small-cell lung cancer cells
suggests that inhibiting KDM2A may represent a strategy for
targeting certain cancers at the transcription level.[19,20] All
KDM2A inhibitors described to date are 2-OG competitors, and
none are truly selective. In addition, many 2-OG competitors
have reduced activity in cells, mainly due to poor cellular
permeability and high intracellular concentration of 2-OG.[21] We
therefore envisioned an inhibitor that would mimic the structure
of the enzyme’s histone substrate rather than the 2-OG co-
substrate, as postulated for the
Homepage: http://msmith.chem.ox.ac.uk
Dr A. Tumber, Dr S. Hatch, Dr S. Müller, T. Szommer, Dr B. Mair,
Prof. Dr S. Nijman, Dr O. Fedorov, Dr R. Konietzny,
Prof. Dr B. M. Kessler, Prof. Dr P. E. Brennan
Structural Genomics Consortium and Target Discovery Institute,
Nuffield Department of Medicine, University of Oxford
Roosevelt Drive, Oxford, OX3 7DQ (UK)
E-mail: paul.brennan@sgc.ox.ac.uk
Prof. Dr M. Vedadi, F. Li
Structural Genomics Consortium, University of Toronto,
Toronto, Ontario, M5G 1L7, Canada
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
This article is protected by copyright. All rights reserved.

10.1002/anie.201706788
Angewandte Chemie International Edition
COMMUNICATION
R1
R1
R3
N
R1
R1
R1
N
A
R2
R2
r
R2
R2
ke
n
i
l
(i), (ii)
R2
(iii)
(iv)
(v)
Br
Br
Br
N
N
N
N
H
NO2
O
X
O
R3
X
X
X
CN
CN
MeO
O
N
Ph
Ph
N
B
N
O
N
N
H
N
N
N
N
N
N
N
N
H
N
Me
Me
Me
O
N
Me
Me
Me
O
N
1. IC50 5.0 µM
2. IC50 0.82 µM
3. IC50 0.62 µM
CN
CN
CN
Ph
Ph
Ph
N
N
N
N
N
N
Me
Me
O
N
O
N
O
N
4. IC50 0.67 µM
5. IC50 0.22 µM
6. IC50 0.25 µM
CN
CN
CN
Ph
Ph
Ph
O
O
N
N
N
O
O
O
7. IC50 0.24 µM
8. IC50 0.33 µM
9. IC50 1.0 µM
N
N
S
CO2Me
Ph
CN
CN
Ph
N
N
N
N
H2N
Me
O
N
Me
O
N
O
N
12. IC50 0.12 µM
10. IC50 1.6 µM
11. IC50 2.4 µM
CN
Ph
Ph
O
Ph
C
CN
CN
CN
O
(iv), (v)
(i)
(ii),(iii)
Ph
OBn
OH
N
3
OBn
N
H
N
N
N
N
Me
N
Me
O
N
N
H
13
15
17
(R,R)-3. IC50 0.84 µM
(S,S)-3. IC50 0.39 µM
Ph
OMe
CN
Br-
OMe
(vi)-(viii)
CF3
N+
OH
Br-
N
N
Ar =
N+
Ar
Me
O
N
OH
14
CF3
H
16
Ar
N
(R,R)-6. IC50 0.39 µM
(S,S)-6. IC50 0.16 µM
N
Scheme 1: [A] General synthetic strategy for racemic synthesis of KDM2A inhibitors. Reagents and conditions: (i) Zn powder (10 eq.), NH4Cl (15 eq.), 5:1
acetone/H2O or H2, Pd/C (10% w/w), EtOAc; (ii) ArCHO (1.3 eq.), MgSO4 (5 eq.), PhMe or ArCHO (1.3 eq.), pyrrolidine (0.1 eq.), 3 Å sieves, CH2Cl2; (iii) KOtBu
(1.1 eq.), PhMe, 0 °C; (iv) R3COCl (2-5 equiv), pyridine (2-5 equiv), CH2Cl2; (v) metal-catalysed cross-coupling. [B] Key structure-activity relationships. IC50 values
were determined by RapidFire MS and confirmed by AlphaScreen. All compounds are racemic. [C] Catalytic enantioselective synthesis. Reagents and conditions:
(i) CsOH·H2O (2.0 eq.), catalyst 14/16 (10 mol %), PhMe, -30 °C. Catalyst 14: 89%, d.r. 10:1, e.r. 88:12; catalyst 16: 84%, d.r. 7:1, e.r. 17:83; (ii) CH3COCl (2.0
eq.), pyridine (2.0 eq.), CH2Cl2; (iii) H2, Pd/C (10% w/w), 88% (2 steps); (iv) K2CO3 (5 eq.), Br(CH2)7Br (4 eq.), acetone, reflux, 38%; (v) K2CO3 (5 eq.) pyrrolidine
(4 eq.), CH3CN, 65%; (vi) PhN(SO2CF3)2 (1.1 eq.), DIPEA (2.0 eq.), CH2Cl2, 0 °C, 85%; (vii) 7-pyrrolidine-hept-1-yne (1.5 eq.), PdCl2(PPh3)2 (5 mol %), CuI (5
mol %), HN(iPr)2, 75 °C, 79%; (viii) H2, Pd/C (10% w/w), MeOH, 74%.
KDM7A inhibitor E67-2.[22] To identify a starting point, a library of
known binders to methyl-lysine reader domains and histone
methyl transferases was screened for inhibitory activity against a
panel of KDMs, as we reasoned that such a specialized library
would be more likely to contain molecules that also interact with
demethylases. Compound 1, which was prepared as a putative
methyl lysine binding domain inhibitor,[23] was identified as a
promising candidate for further optimization against KDM2A.
However, attempts to significantly improve potency by
functionalizing at the indole NH position and varying the
aromatic substituent on the indole C-2 position were
unsuccessful. The pyrrolidine moiety in compound 1 was shown
to be critical for potency, that we postulated was due to its role
as a H3K36me2 mimic. Preliminary docking studies of 1 with
occupancy of the enzyme’s peptide binding site. Based on this
initial model, we subsequently hypothesized that inhibitory
activity towards KDM2A might be improved by replacing the
original indole scaffold with a saturated indoline ring system. We
envisioned an exploration of three-dimensional chemical space
around this core, with the aim of augmenting selectivity through
increasing complexity.[24] To achieve this, a modular synthetic
approach was employed to generate a library of indoline-
containing compounds and identify key structure-activity
relationships (Scheme 1A).
A
base-mediated 5-endo-trig cyclization of
a
C-2
substituted aromatic imine afforded the racemic indoline core
with two adjacent stereocenters. Subsequent acylation of the
indoline ring system conferred stability towards oxidation and
KDM2A (PDB ID 2YU1) (SI-8) indicated
a
potential for
provided a handle for modulating polarity. Finally, metal-
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10.1002/anie.201706788
Angewandte Chemie International Edition
COMMUNICATION
catalysed cross coupling of the aryl bromide provided access to
a variety of linkers between the indoline core and pyrrolidine
capping group. In total, 45 racemic compounds were
synthesized, and IC50 values for inhibition of KDM2A were
determined using two orthogonal enzyme activity assays:
AlphaScreen[25] and RapidFire MS[26] (see SI-3.1 for complete
inhibition data). Key structure-activity relationships are
summarized above (Scheme 1B, compounds 2-12). We
examined different linkers and found that triazole 2, ether 3 and
alkyne 4 linkers were well tolerated, with significantly lower IC50
values than the original hit. Reduction of the alkyne functional
group in 4 to an alkene 5 or an alkane 6 also improved potency.
Molecules containing a pyridine ring at the indoline C-2 position
were marginally more active than analogues bearing other
aromatic groups such as furan (7 or 8) and significantly more
active than the substituted benzene 9. In addition, pyridine-
containing compounds displayed the highest selectivity towards
KDM2A (SI-3.1). Exploration of substituents at the all-carbon
quaternary stereocenter as in 10 and 11 demonstrated that the
Ph,CN combination gave rise to the most potent series of
compounds. Unfortunately, 12, the most potent inhibitor
identified, was found to be reactive in aqueous solution due to
the susceptibility of the a-aminoacetyl group to hydrolysis.
However, the N-acetyl group present in compounds 2-10 proved
inert to hydrolytic cleavage. The optimal length of the linker
connecting the indoline core to the pyrrolidine capping group
was found to be 7-8 atoms, and replacing pyrrolidine with other
secondary amines or a cyclopentyl ring led to a significant drop
in potency (SI-3.1).
effects of KDM2A inhibition within cells. To profile its selectivity,
(S,S)-6 was tested for inhibitory activity against a panel of KDMs,
methyl-lysine binding motifs, and epigenetic enzymes. It was
found to be remarkably selective towards KDM2A (≥100-fold)
relative to representatives of the other KDM sub-families, except
closely related KDM7A (H3K9/K27me2/me1 demethylase),[29]
where it was similarly potent (Figure 2B).[30] (S,S)-6 was inactive
towards a representative panel of methyl-lysine binding domains,
methyl transferases and histone acetyl transferases (SI-3.2). To
our knowledge, this is the first time a KDM2A/7A selective small
molecule has been shown to inhibit demethylation in cells, with a
significant reduction of demethylation achievable at low µM
concentrations. To explore its cellular activity further, the effect
of (S,S)-6 on gene expression in HAP1 cells was monitored
using a highly multiplexed 3’ mRNA sequencing method.[31]
Within a diverse panel of in-house compounds, our series of
indoline-containing inhibitors was represented by (S,S)-6 and a
less active close analogue 18 (IC50 17 µM, Figure 2C).
A
200
150
100
Mutant
WT
WT + 0.4 µM (S,S)-6
WT + 3.1 µM (S,S)-6
WT + 6.2 µM (S,S)-6
50
0
B (S,S)-6 Selectivity vs other KDMs (IC50 µM)
KDM3A KDM4A KDM4C KDM4D KDM5B KDM5C KDM6B KDM7A KDM7B
39
639
430
321
17
16
20
0.19
34
Having succeeded in augmenting the potency of our initial
hit compound, we focused on the development of
enantioselective syntheses of 3 and 6 using a counterion-
mediated strategy (Scheme 1C).[27] Cyclization of imine 13 with
CsOH·H2O in the presence of quinine-derived salt 14 afforded
(R,R)-15 as the major product (10:1 d.r.) and 88:12 e.r. The OBn
substituent on the pyridine ring was found to be crucial for
attaining good levels of stereoselectivity. Pseudoenantiomeric
ammonium salt 16 afforded (S,S)-15 as the major product (7:1
d.r.) and with acceptable e.r. (17:83). Enantiopurity was
subsequently augmented (to >99:1 e.r.) by preparative HPLC.
N-Acetylation and hydrogenolysis of the benzyl group afforded
common intermediate 17, which could be converted to (S,S)-
and (R,R)-3 by alkylation with 1,7-dibromoheptane and
subsequent treatment with pyrrolidine. Alternatively, (S,S)- and
(R,R)-6 could be synthesized via O-triflation, Sonogashira
coupling (with 1-(6-heptyn)-pyrrolidine), and reduction of the
resulting alkyne.
The (S,S)-enantiomers of 3 and 6 were found to be slightly
more potent than their respective (R,R)-analogues, and (S,S)-6
(IC50 – 0.16 µM) was assessed further in a variety of biological
assays. In immunofluorescence assays using HeLa cells
ectopically expressing catalytically active KDM2A, a dose-
dependent increase in H3K36me2 staining was observed upon
incubation with (S,S)-6, reflecting augmented cellular H3K36me2
levels (Figure 2A, SI-3.3). No significant change in H3K36me2
fluorescence was observed for cells containing constitutively
inactive KDM2A (SI-3.3).[28] Cytotoxicity towards HeLa and
HAP1 cells was observed at higher concentrations (EC50 22 µM
and 7.1 µM respectively), and (S,S)-6 showed a similar effect on
the viability of human fibroblasts (HDFa) (EC50 10 µM) to GSK-
J4, a well-characterized chemical probe for KDM5/6[9] (SI-3.4).
This suggests a potential activity window for investigating the
C
CN
CN
Ph
Ph
Et
N
O
Et
N
N
3
N
Me
O
N
Me
O
(S,S)-6. KDM2A IC50 0.16 µM
18. KDM2A IC50 17 µM
D
E
300
80
(S,S)-6
(1 µM)
increased
expression
32
86
0
60
40
200
reduced
expression
18
(10 µM)
21
0
100
0
20
0
18
(1 µM)
0
18
18
(S,S)-6 (S,S)-6
(S,S)-6 (S,S)-6
18
(10 µM) (1 µM) (10 µM) (1 µM)
(10 µM) (1 µM) (10 µM)
Figure 2: [A] (S,S)-6 inhibits KDM2A catalysed demethylation of H3K36me2 in
HeLa cells at µM concentrations. Mutant - cells contain constitutively-inactive
KDM2A. WT - cells contain active KDM2A. [B] AlphaScreen IC50 values (μM)
of (S,S)-6 against other KDMs. [C] 18 is a less active close analogue of (S,S)-
6. [D] Both (S,S)-6 and 18 affect gene expression in HAP1 cells at high
concentrations, but only (S,S)-6 has an effect at low concentrations.
[E] Overlap of gene expression changes for (S,S)-6 and 18.
When dosed at a concentration of 10 µM, both molecules
influenced expression levels of more than 200 genes. However,
at a concentration of 1 µM, only the active analogue (S,S)-6 had
a significant effect on expression levels (Figure 2D and SI-4).
We postulate that this concentration dependence is
a
consequence of predominantly off-target effects at high
concentrations, as opposed to a more specific effect resulting
from KDM2A/7A inhibition at low concentrations. The overlap of
gene expression signatures of (S,S)-6 and 18 is depicted in
Figure 2E.
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10.1002/anie.201706788
Angewandte Chemie International Edition
COMMUNICATION
Obtaining a co-crystal structure of (S,S)-6 bound to
KDM2A proved challenging, and hence, non-denaturing mass
spectrometry (MS) experiments were performed to determine
the binding stoichiometry of (S,S)-6 to KDM2A. KDM2A was
incubated with (S,S)-6 and subsequently introduced into a mass
spectrometer under conditions optimized for the preservation of
noncovalent interactions.[32] The native mass spectrum (Figure
3B) shows 1:1 binding of (S,S)-6 to KDM2A. To verify the
identity of bound (S,S)-6 we performed tandem-MS on the 14+
charge state, resulting in the removal of (S,S)-6 as a singly
charged species (Figure 3C, see inset).
how the generation of three-dimensional scaffolds bearing
significant saturation and multiple chiral centers can lead to the
discovery of selective compounds that may be useful in the
study of a challenging epigenetic target.
Acknowledgements
We are indebted to Stephen V. Frye (UNC Chapel Hill), Tomasz
Konopka and Erica De Zan (Oxford), and Guillermo Senisterra,
(SGC Toronto), for assistance. We thank Prof. Xiang Wang (CU
Boulder) for the gift of fluorescent methylstat. We are grateful to
the ERC (grant no. 259056), the EPSRC and MRC
(EP/L016044/1), Waters Corporation/BBSRC (BB/L017067/1),
the National Institutes of General Medical Sciences
(GM100919), Innovative Medicines Initiative (EU/EFPIA
[ULTRA-DD grant no. 115766]), the Wellcome Trust
[092809/Z/10/Z], Cancer Research UK (C8717/A18245) and the
Royal Society Dorothy Hodgkin Fellowship (A.K.). The SGC is a
charity (number 109773 7).
Keywords: epigenetics, lysine demethylase, enantioselective
catalysis, inhibitor, selective
[1]
[2]
a) M. A. Dawson, T. Kouzarides, Cell 2012, 150, 12. b) J. R. Tollervey,
V. V. Lunyak, Epigenetics 2012, 7, 823. c) P. Sen, P. P. Shah, R.
Nativio, S. L. Berger, Cell 2016, 166, 822.
a) H. Kantarjian, et al., Cancer 2006, 106, 1794. b) P. K. Mazur et al.,
Nat. Med. 2015, 21, 1163.
Figure 3: Non-denaturing MS indicates 1:1 binding of (S,S)-6 to KDM2A. [A]
Non-denaturing mass spectrum of apo KDM2A. [B] Non-denaturing mass
spectrum of KDM2A (2.5 µM) and the 1:1 complex with (S,S)-6 (12.5 µM). [C]
The 14+ charge state of the complex was selected (lower) and subjected to
collisional activation (upper) to release bound (S,S)-6 (inset). The spectrum
intensity has been magnified 1.5-fold above 3500 m/z (CID = collision-induced
dissociation).
[3]
[4]
[5]
S. Ackloo, P. J. Brown, S. Müller, Epigenetics 2017, 12, 378.
M. Moustakim et al., Med. Chem. Commun. 2016, 7, 2246.
For reviews of histone demethylase inhibitors see: a) T. E. McAllister, K.
S. England, R. J. Hopkinson, P. E. Brennan, A. Kawamura, C. J.
Schofield, J. Med. Chem. 2016, 59, 1308. b) J. W. Højfeldt, K. Agger, K.
Helin, Nat. Rev. Drug Discov. 2013, 12, 917. c) H. Ümit Kaniskan, J. Jin,
Chem. Rev. 2017, DOI: 10.1021/acs.chemrev.6b00801.
I. J. Clifton, M. A. McDonough, D. Ehrismann, N. J. Kershaw, N.
Granatino, C. J. Schofield, J. Inorg. Biochem. 2006, 100, 644.
R. J. Klose, E. M. Kallin, Y. Zhang, Nat. Rev. Genet. 2006, 7, 715.
R. J. Hopkinson et al., Chem. Sci. 2013, 4, 3110.
[6]
[7]
[8]
[9]
Kinetic analyses subsequently revealed that (S,S)-6 does not
display competitive inhibition kinetics with respect to either 2-OG
or the peptide substrate (SI-6), suggesting a different mode of
inhibition to the majority of previously-discovered KDM
inhibitors.[33] Consistent with this observation, (S,S)-6 did not
displace fluorescent methylstat (a ‘bivalent’ substrate-cofactor
tracer for KDM2A) in fluorescence polarisation assays. To probe
the (S,S)-6 binding site further, KDM2A was subjected to
photoaffinity labelling profile by a diazirine-containing analogue
of (S,S)-6, and LC-MS/MS experiments were conducted (SI-7).
The majority of covalently-modified residues were found to be
either aspartic or glutamic acids, suggesting the formation of a
relatively long-lived electrophilic intermediate following photo-
induced isomerization of the diazirine to a diazo compound.[34]
While this precludes the unambiguous determination of the
inhibitor binding site, the observed lack of labelling within the
JmjC domain active site (SI-7) is consistent with the observed
lack of competitive inhibition with respect to either 2-OG or the
peptide substrate. This may indicate the presence of an
alternative (allosteric) binding site specific to KDM2A/7A, though
further investigation is necessary to demonstrate this clearly.
In conclusion, we have developed a potent and highly
selective first-in-class inhibitor of the histone lysine
demethylases KDM2A/7A. Compound (S,S)-6 displays >75 fold
selectivity towards KDM2A/7A versus other JmjC lysine
demethylases and is, to our knowledge, the first reported
selective KDM2A/7A inhibitor demonstrated to reduce
H3K36me2 demethylation within cells. This study demonstrates
N. R. Rose et al., J. Med. Chem. 2012, 55, 6639.
[10] L. Kruidenier, et.al., Nature 2012, 488, 404.
[11] J. Liang et al., Bioorg. Med. Chem. Lett. 2016, 26, 4036.
[12] T. Suzuki et al., J. Med. Chem. 2013, 56, 7222.
[13] J. R. Horton et al., Cell Chem. Biol. 2016, 23, 769.
[14] a) X. Luo et al., J. Am. Chem. Soc. 2011, 133, 9451. b) K. S. England
et al., MedChemComm 2014, 5, 1879.
[15] a) U. Leurs, B. Lohse, K. D. Rand, S. Ming, E. S. Rise, P. A. Cole, J. L.
Kristensen, R. P. Clausen, ACS Chem. Biol. 2014, 9, 2131. b) A.
Kawamura et al., Nat. Commun. 2017, 8, 14773.
[16] Y. Tsukada, J. Fang, H. Erdjument-Bromage, M. E. Warren, C. H.
Borchers, P. Tempst, Y. Zhang, Nature 2006, 439, 811.
[17] T. Lu, M. W. Jackson, A. D. Singhi, E. S. Kandel, M. Yang, Y. Zhang, A.
V. Gudkov, G. R. Stark, Proc. Natl. Acad. Sci. U. S. A. 2009, 106,
16339.
[18] J. Du, Y. Ma, P. Ma, S. Wang, Z. Fan, Stem Cells 2013, 31, 126.
[19] Y. Huang, Y. Liu, L. Yu, J. Chen, J. Hou, L. Cui, D. Ma, W. Lu, Tumor
Biol. 2015, 36, 271.
[20] S. S. Dhar, H. Alam, N. Li, K. W. Wagner, J. Chung, Y. W. Ahn, M. G.
Lee, J. Biol. Chem. 2014, 289, 7483.
[21] a) V. Bavetsias et al., J. Med. Chem. 2016, 59, 1388. b) S. B. Hatch et
al., Epigenetics Chromatin, 2017, 10:9.
[22] A. K. Upadhyay et al., J. Mol. Biol. 2012, 416, 319.
[23] J. M. Herold et al., J. Med. Chem. 2011, 54, 2504.
[24] a) A. R. Leach, M. M. Hann, Curr. Opin. Chem. Biol. 2011, 15, 489. b)
F. Lovering, Med. Chem. Commun. 2013, 4, 515. c) P. A. Clemons et
al. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 18787.
[25] A. Kawamura, A. Tumber, N. R. Rose, O. N. F. King, M. Daniel, U.
Oppermann, T. D. Heightman, C. J. Schofield, Anal. Biochem. 2010,
404, 86.
[26] S. E. Hutchinson et al., J. Biomol. Screening 2012, 17, 39.
[27] a) E. E. Maciver, S. Thompson, M. D. Smith, Angew. Chem. Int. Ed.
2009, 48, 9979. b) K. Sharma, J. R. Wolstenhulme, P. P. Painter, D.
Yeo, F. Grande-Carmona, C. P. Johnston, D. J. Tantillo, M. D. Smith, J.
Am. Chem. Soc. 2015, 137, 13414.
[28] S. B. Hatch et al., Epigenetics Chromatin 2017, 10, 9.
[29] J. R. Horton, A. K. Upadhyay, H. H. Qi, X. Zhang, Y. Shi, X. Cheng,
Nat. Struct. Mol. Biol. 2010, 17, 38.
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[30] We are unable to investigate the activity of (S,S)-6 towards KDM2B.
[31] B. V. Gapp, T. Konopeka, T. Penz, V. Dalal, T. Buerckstuemmer, C.
Bock, S. M. B. Nijman, Mol. Syst. Biol. 2016, 12, 879.
[32] F. D. Kondrat, W. B. Struwe, J. L. P. Benesch, Methods Mol. Biol. 2014,
1261, 349.
[33] For a review on allosteric regulation of epigenetic modifying enzymes
see: B. E. Zucconi, P. A. Cole, Curr. Opin. Chem. Biol. 2017, 39, 109.
[34] a) J. M. Perez, J. Chem. Soc., Faraday Trans. 1 1982, 78, 3509. b) K.
A. Mix, R. T. Raines, Org. Lett. 2015, 17, 2358.
This article is protected by copyright. All rights reserved.