Identification of Jumonji AT-Rich Interactive Domain 1A Inhibitors and Their Effect on Cancer Cells

Article abstract of DOI:10.1021/acsmedchemlett.5b00083

Jumonji AT-rich interactive domain 1A (JARID1A), one of the jumonji C domain-containing histone demethylase (JHDM) family members, plays key roles in cancer cell proliferation and development of drug tolerance. Therefore, selective JARID1A inhibitors are

Full text of DOI:10.1021/acsmedchemlett.5b00083

Letter
pubs.acs.org/acsmedchemlett
Identification of Jumonji AT-Rich Interactive Domain 1A Inhibitors
and Their Effect on Cancer Cells
†
†
†
†
‡
‡
Yukihiro Itoh, Hideyuki Sawada, Miki Suzuki, Toshifumi Tojo, Ryuzo Sasaki, Makoto Hasegawa,
,
‡
,†,§
Tamio Mizukami,* and Takayoshi Suzuki*
†Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamohangi-Cho, Sakyo-Ku, Kyoto
06-0823, Japan
6
‡
Graduate School of Bio-Science, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829,
Japan
§
CREST, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
S Supporting Information
*
ABSTRACT: Jumonji AT-rich interactive domain 1A (JAR-
ID1A), one of the jumonji C domain-containing histone
demethylase (JHDM) family members, plays key roles in
cancer cell proliferation and development of drug tolerance.
Therefore, selective JARID1A inhibitors are potential anti-
cancer agents. In this study, we searched for cell-active
JARID1A inhibitors by screening hydroxamate compounds in
our in-house library and the structural optimization based on
docking study of the hit-compound to a homology model of
JARID1A. As a result, we identified compound 6j, which
selectively inhibits JARID1A over three other JHDM family members. Compound 7j, a prodrug form of compound 6j, induced a
selective increase in the level of trimethylation of histone H3 lysine 4, a substrate of JARID1A. Furthermore, compound 7j
synergistically enhanced A549 human lung cancer cell growth inhibition induced by vorinostat, a histone deacetylase inhibitor.
These findings support the idea that JARID1A inhibitors have potential as anticancer agents.
KEYWORDS: Epigenetics, histone demethylase, drug design
eversible methylation of the ε-amino groups of histone
with histone deacetylases (HDACs) as polycomb proteins to
8
−10
R
lysine residues is involved in epigenetic gene activation or
regulate gene transcription.
cancer cell growth and tumorigenesis.
JARID1A (also known as KDM5A and RBP2) has been
In addition, they are involved in
1
−3
11,12
inactivation independently of DNA sequence.
It occurs at
In particular,
several histone lysine residues, and each methylation site is
thought to have a distinct role. Therefore, compounds that can
regulate site-specific methylation are of great interest as
chemical tools for biological studies. Such compounds are
also candidate therapeutic agents for diseases associated with
aberrant histone methylation.
1
3,14
suggested to be associated with cancer cell proliferation
and
15
the development of drug tolerance. Therefore, selective
inhibitors of JARID1A are of interest not only as biological
tools to elucidate the biology of JARID1A and H3K4me2/3 but
also as candidate anticancer agents.
Jumonji C domain-containing demethylases (JHDMs) are
To date, several groups, including ours, have reported JHDM
7
,16−18
19
Fe(II)/α-ketoglutarate-dependent oxygenases that catalyze
inhibitors,
such as N-oxalylglycine (NOG, 1), 4-
4,5
20
demethylation of methylated lysines of histones. JHDM
family members identified so far have been categorized into five
subfamilies, namely, JHDM1 (also known as KDM2/7),
jumonji domain-containing protein 1 (JMJD1, also known as
KDM3), JMJD2 (also known as KDM4), jumonji AT-rich
interactive domain 1 protein (JARID1, also known as KDM5),
carboxy-4′-carbomethoxy-2,2′-bipyridine 2, NCDM-32a
21 22
(3), NCDM-64 (4), and 2-(4-methylphenyl)-1,2-benziso-
23
thiazol-3(2H)-one (PBIT, 5) (Figure 1). Though some of
these inhibitors, such as NCDM-64 (4), selectively inhibit a
subfamily of JHDMs, most of them lack selectivity toward
subfamily proteins or individual isozymes. It has been reported
that compounds 2, 5, and an inhibitor from EpiTher-
apeutics inhibit JARID1, but few details have been disclosed,
and their intracellular activity is unknown. Therefore, there is
24
23
6
and UTX/JMJD3 (also known as KDM6). Because each
12
subfamily can distinguish the number of methyl groups and can
demethylate lysine in a site-specific manner, JHDMs function-
ally regulate the state of histone lysine methylation and
7
subsequent gene expression. Among the JHDM subfamilies,
Received: February 17, 2015
Accepted: April 23, 2015
JARID1s are unique in that their substrates are limited to di-
and trimethylated H3K4 (H3K4me2/3), and JARID1s interact
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00083
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Letter
2
1,22
among the enzymes.
For example, there is a unique
hydrophobic pocket around the active site of JHDM1F, where
22
the alkyl chain of compound 4 is thought to be located. We
assumed that the pocket around the active site might be unique
in each JHDM and could contribute to the substrate specificity,
8
25
i.e., H3K4 for JARID1A, H3K9 and H3K36 for JMJD2C,
26
and H3K9 and H3K27 for JHDM1F. We also hypothesized
that derivatives of compounds 3 and 4 whose alkyl amino
group fits the unique pocket of JARID1A could be selective
inhibitors of JARID1A. On the basis of this hypothesis, we
screened our JHDM inhibitor candidate library, including
various alkyl amino group-hydroxamate hybrid compounds. As
a result of the screening using previously reported JARID1A,
Figure 1. Structures of reported small-molecular JHDM inhibitors.
21,22
JMJD2C, and JHDM1F assays,
we identified compound 6a,
which has an N-methylbutan-1-amino group. Compound 6a
still a need to develop selective JARID1 inhibitors and to
elucidate the structural requirements for JARID1 inhibition.
Thus, we set out to identify cell-active JARID1-selective
inhibitors. Herein, we describe the discovery, structural
optimization, and biological evaluation of JARID1A inhibitors.
To find selective inhibitors of JARID1A, we screened
analogues of hydroxamates 3 and 4, which we had identified
efficiently and selectively inhibited JARID1A (JARID1A IC =
50
4
.3 μM; JMJD2C IC = 55 μM; JMJD1A > 100 μM; JHDM1F
50
IC50 > 100 μM) (Table 1, entry 4), showing much higher
selectivity than the hydroxamates 3 and 4. Encouraged by this
finding, we next attempted to optimize the structure of
compound 6a.
2
1,22
To guide optimization of the inhibitor structure, we initially
performed a binding simulation of compound 6a with
JARID1A and JMJD2C. The binding model of compound 6a
to JARID1A (Figure 2A,B) showed three characteristic features:
(i) the NH group forms a hydrogen bond with Asp 412, which
is positioned near Ser 464 and Tyr 472; (ii) the n-butyl group is
positioned near a small hydrophobic pocket formed by Pro 449,
Val 468, and Trp 470; (iii) the methyl group is located in a
large space at the entrance to the catalytic site of JARID1A.
However, , in the binding model of JMJD2C (Figure 2C,D), (i)
the NH group forms a hydrogen bond with Asn 292, (ii) the n-
butyl group is located in a spacious tunnel-like pocket of the
as JMJD2 and JHDM1 inhibitors, respectively.
previous docking studies of these inhibitors with JMJD2C
also known as KDM4C and GASC1) and JHDM1F (also
Our
(
known as KDM7B and PHF8) suggested that the hydroxamate
group could bidentately coordinate to a catalytic Fe(II) ion in
the catalytic site, and the carboxylate group could form
hydrogen bonds with amino acid residues such as tyrosine,
lysine, or asparagine in the active site of the two enzymes. The
positions of the catalytic Fe(II) ion and the above-mentioned
amino acid residues appeared to be similar among JARID1A,
JMJD2C, and JHDM1F (Figure S1). On the other hand, it was
suggested that the pockets around the active site are different
a
Table 1. In Vitro JARID1A, JMJD2C, JMJD1A, and JHDM1F Inhibitory Activities of Compounds 1−4 and 6
structure
R2
IC50 (μM)
JMJD2C
entry
compd
R1
n
JARID1A
JMJD1A
569
JHDM1F
1
2
3
4
5
6
7
8
9
1
250
430
640
19
b
c
3
b
13
2.2
ND
c
4
55
83
ND
1.2
6a
6b
6c
6d
6e
6f
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
4
2
3
5
6
4
4
4
4
4
4
4
4
4
4.3 ± 0.9
53 ± 8.5
6.3 ± 1.8
4.0 ± 0.6
1.8 ± 0.1
18 ± 2.0
7.8 ± 1.0
8.9 ± 3.0
2.3 ± 0.9
3.3 ± 1.3
14 ± 5.0
19 ± 6.2
4.5 ± 0.9
3.1 ± 0.3
63 ± 16
21 ± 3.7
55 ± 11
>100
>100
>100
c
c
ND
ND
c
c
63 ± 6.4
61 ± 1.6
6.5 ± 0.3
51 ± 6.3
62 ± 11
76 ± 4.7
37 ± 26
43 ± 11
65 ± 13
>100
ND
ND
c
ND
58 ± 16
c
c
ND
ND
c
c
ND
ND
c
c
10
11
12
13
14
15
16
17
18
19
6g
6h
6i
Et
ND
ND
c
c
n-Pr
ND
ND
n-pentyl
n-hexyl
n-Bu
n-Bu
n-Bu
n-Bu
>100
>100
>100
6j
73 ± 9
c
c
6k
6l
ND
ND
c
c
n-Bu
ND
ND
6m
6n
7i
n-hexyl
n-octyl
>100
>100
>100
29 ± 13
24 ± 8
83 ± 17
c
c
c
ND
ND
ND
c
c
c
7j
ND
ND
ND
a
b
c
Value are means of at least three experiments. Taken from the literature (ref 22). ND = no data available.
B
DOI: 10.1021/acsmedchemlett.5b00083
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Figure 2. (A) View of the conformation of compound 6a (ball and stick) docked into the JARID1A active site. (B) Schematic diagram of binding of
compound 6a to JARID1A (a homology model based on the crystal structure of JMJD2A). (C) View of the conformation of compound 6a (ball and
stick) docked into the JMJD2C active site (PDB code 2MXL). (D) Schematic diagram of binding of compound 6a to JMJD2C.
catalytic site where no interaction is observed between the n-
butyl group and the amino acid residues of JMJD2C, and (iii)
the methyl group is located near the backbone amide between
Ser 290 and Thr 291. On the basis of these simulation results,
we designed compounds 6b−n and evaluated their JARID1A-
and JMJD2C inhibitory activity.
6f−h with a shorter alkyl group (Table 1, entries 9−11) were
inferior. As we had expected, compounds 6f−j showed
moderate activity against JMJD2C (IC50 = 37−76 μM)
(Table 1, entries 9−13), similar to compound 6a (IC50 = 55
μM) (Table 1, entry 4). Compound 6i showed the highest
selectivity among compounds 6f−j.
Compounds 6b−e with various linker lengths were expected
to form a hydrogen bond with Ser 464 or Tyr 472 of JARID1A
Next, we focused on changing the methyl group of
compound 6a to various alkyl groups (6k−n). We expected
(
Figure 2A,B), but should not form a hydrogen bond with
that the R2 group of compounds 6k−n would be
JMJD2C because of loss of the interaction between the NH
group and Asn 292 of JMJD2C. As shown in Table 1, JARID1A
inhibitory activity and selectivity were distinctly dependent on
linker length (Table 1, entries 5−8). Among compounds 6b−e,
compound 6e (n = 6) showed the most potent JARID1A
inhibitory activity with an IC of 1.8 μM. However, compound
accommodated in the large space at the entrance of JARID1A,
whereas there could be steric repulsion between the alkyl group
and the backbone amides of Ser 290 and Thr 291 of JMJD2C
(
Figure 2). As we had expected, the longer alkyl group of
compounds 6m and 6n was effective for JARID1A inhibition
Table 1, entries 16 and 17), but the shorter alkyl group of
5
0
(
6e also potently inhibited JMJD2C (IC50 = 6.5 μM), which
compounds 6k and 6l was not (Table 1, entries 14 and 15). In
addition, compounds 6m and 6n with a longer alkyl group
showed only weak activity against JMJD2C (Table 1, entries 16
and 17).
We next investigated the JMJD1A (also known as KDM3A)
and/or JHDM1F inhibitory activity of compounds 6d, 6i, 6j,
resulted in low JARID1A selectivity as compared with
compound 6a. With respect to the selectivity for JARID1A
over JMJD2C, compounds 6a (n = 4) and 6d (n = 5) were
superior to the other compounds (Table 1, entries 4 and 7).
Next, we examined the activity of compounds 6f−j, in which
the n-butyl group of compound 6a is replaced with various alkyl
groups (Table 1, entries 9−13). The longer alkyl chains of
compounds 6i and 6j were expected to be located at the
hydrophobic pocket formed by Pro 449, Val 468, and Trp 470
of JARID1A (Figure 2A,B) without interacting with any amino
acid residues of JMJD2C (Figure 2C and 2D). Compounds 6i
and 6j with a longer alkyl group (Table 1, entries 12 and 13)
were slightly superior JARID1A inhibitors to compound 6a
with the n-butyl group (Table 1, entry 4), although compounds
6m, and 6n (Table 1, entries 7, 12, 13, 16, and 17). These
compounds showed moderate JHDM1F inhibitory activities
(
IC50 > 24 μM) and no JMJD1A inhibitory activities (IC50
>
1
00 μM). As a result, they showed high selectivity for JARID1A
over JMJD1A and JHDM1F as compared with compounds 3
and 4. Among them, compound 6i and 6j displayed relatively
high JARID1A inhibitory activity and selectivity (Table 1, entry
12; Table S1).
C
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The structure−activity and structure−selectivity relationships
may be summarized as follows. With regard to the length
between the amino group and the hydroxamate, n > 4 is better
for JARID1A inhibition, and n = 4 or n = 5 is suitable for
1
2
JARID1A-selective inhibition. As for the R and R groups, the
combination of a methyl group and a long alkyl group, such as
n-butyl, n-pentyl, and n-hexyl, is preferred for both JARID1A
inhibition and JARID1A selectivity. These structure−activity
and structure−selectivity relationships are consistent with the
calculation results of the binding mode of compounds 6a, 6e,
6
i, and 6j (Figures 2 and S2−S4 and Table S2).
Next, we investigated whether the identified inhibitors
behave as JARID1 inhibitors in cell-based assays by using
Western blot analysis to evaluate accumulation of H3K4me3, a
8
physiological substrate of JARID1A. We applied JARID1A
inhibitors 6i and 6j to human lung cancer A549 cells, which
27
overexpress JARID1A, and analyzed the cell extracts after 24
h treatment. However, compounds 6i and 6j had little effect on
the level of H3K4me3 (Figure 3A). On the basis of the idea
that this might be due to poor membrane permeability, we next
examined the methyl ester prodrugs 7i and 7j (Figure 3A),
which were expected to permeate the cell membrane more
efficiently than the parent compound and to be converted to 6i
20
and 6j, respectively, by enzymatic hydrolysis within the cell.
Though compounds 7i and 7j did not inhibit JARID1A
strongly in enzyme assays (Table 1, entries 18 and 19), they
increased the accumulated amount of H3K4me3 in a dose-
dependent matter in cell-based assays (Figure 3B). The activity
of compound 7j was a little higher than that of compound 7i.
Then, to examine the selectivity in cells, we evaluated the effect
of compound 7j on H3K9me3 and H3K27me2, substrates of
2
5,26
JMJD2s and JHDM1s,
respectively. Treatment with 100
μM compound 7j had little effect on the level of H3K9me3 and
H3K27me2 (Figure 3C). The Western blot analysis data
suggest that compound 7j inhibits JARID1 selectively over
JMJD2 and JHDM1 in the cells.
Figure 3. (A) Structures of compounds 7i and 7j. (B) Western blot
detection of methylated histone levels in A549 cells treated with
JARID1 inhibitors. H3K4me3 levels after 24 h incubation with 6i, 6j,
Finally, we evaluated the effects of compound 7j on
proliferation of A549 cells. Contrary to our expectation,
compound 7j was found to be inactive at concentrations up
to 300 μM (Figure S5). We next tested whether compound 7j
could act synergistically with vorinostat, a clinically used HDAC
inhibitor, in growth inhibition assays using A549 cells. We
expected that the combination of compound 7j and vorinostat
would cause synergistic inhibition of A549 cell growth because
it has been reported that HDACs are associated with A549 cell
7
i, and 7j. Values of H3K4me3/H3 ratio determined by optical density
measurement of the blots are shown. (C) Western blot detection of
methylated histone levels in A549 cells treated with 7j. H3K4me3,
H3K9me3, and H3K27me2 levels after 24 h incubation with 7j. Values
of H3K4me3/H3, H3K9me3/H3, and H3K27me2/H3 ratio deter-
mined by optical density measurement of the blots are shown. (D)
Cell growth inhibition of A549 cells after 72 h incubation with
combination of 7j and vorinostat, an HDAC inhibitor. Error bars
represent the mean standard deviation (SD) of at least three samples.
Combination with 100 μM compound 7j. *P < 0.05, **P < 0.01
(Student t test).
2
8,29
proliferation,
and JARID1A is a participant in polycomb
repressive complexes that down-regulate gene transcription
8
cooperatively with HDACs. Combined treatment of A549 cells
with vorinostat and 100 μM compound 7j induced more potent
cell growth inhibition than that obtained with vorinostat alone
induced selective accumulation of H3K4me3. Furthermore, the
combination of compound 7j with vorinostat, a clinically used
HDAC inhibitor, caused synergistic inhibition of A549 lung
cancer cell growth. We believe that the compounds described
here will be useful tools for probing the biology of JARID1A
and are also candidate agents, or at least lead compounds, for
cancer treatment.
(
Figure 3D). However, JMJD2 inhibitor NCDM-32b (8), a
prodrug form of NCDM-32a (3), did not enhance the
antiproliferative activity of vorinostat (Figure S6). In addition,
JARID1A inhibitors 6i and 6j and prodrug 7j did not inhibit
HDAC1 (Figure S7). These results suggest that JARID1
inhibition, but not JMJD2 inhibition or HDAC inhibition, by
compound 7j is involved in the synergistic inhibition of A549
cell growth with vorinostat. These results of cell-based assays
suggest that JARID1A inhibitors could have potential as
anticancer agents in combination with vorinostat.
ASSOCIATED CONTENT
Supporting Information
■
*
S
Figures S1−S7, Table S1 and S2, and experimental procedures.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00083.
In conclusion, we identified compound 6j, which selectively
inhibits JARID1A over three other JHDM family members. In
cell-based assays, compound 7j, a prodrug of compound 6j,
D
DOI: 10.1021/acsmedchemlett.5b00083
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ACS Medicinal Chemistry Letters
Letter
(
11) Teng, Y. C.; Lee, C. F.; Li, Y. S.; Chen, Y. R.; Hsiao, P. W.;
AUTHOR INFORMATION
■
Chan, M. Y.; Lin, F. M.; Huang, H. D.; Chen, Y. T.; Jeng, Y. M.; Hsu,
C. H.; Yan, Q.; Tsai, M. D.; Juan, L. J. Histone demethylase RBP2
promotes lung tumorigenesis and cancer metastasis. Cancer Res. 2013,
Corresponding Authors
(T.M.) E-mail: mizukami@nagahama-i-bio.ac.jp.
(T.S.) E-mail: suzukit@koto.kpu-m.ac.jp.
*
*
7
(
3, 4711−4721.
12) Rasmussen, P. B.; Staller, P. The KDM5 family of histone
demethylases as targets in oncology drug discovery. Epigenomics 2014,
, 277−286.
13) Liang, X.; Zeng, J.; Wang, L.; Fang, M.; Wang, Q.; Zhao, M.; Xu,
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
6
(
X.; Liu, Z.; Li, W.; Liu, S.; Yu, H.; Jia, J.; Chen, C. Histone
demethylase retinoblastoma binding protein 2 is overexpressed in
hepatocellular carcinoma and negatively regulated by hsa-miR-212.
PLoS One 2013, 8, e69784.
Funding
This work was supported in part by CREST, JST (to T.S.),
Takeda Science Foundation (to T.S.), Mochida Memorial
Foundation for Medical and Pharmaceutical Research (to T.S.,
and Y.I.), and Kyoto Prefectural Public University Corporation
for Young Researcher Grant (to Y.I.).
(
̈
14) Zeng, J.; Ge, Z.; Wang, L.; Li, Q.; Wang, N.; Bjorkholm, M.; Jia,
J.; Xu, D. The histone demethylase RBP2 is overexpressed in gastric
cancer and its inhibition triggers senescence of cancer cells.
Gastroenterology 2010, 138, 981−992.
Notes
(15) Hou, J.; Wu, J.; Dombkowski, A.; Zhang, K.; Holowatyj, A.;
The authors declare no competing financial interest.
Boerner, J. L.; Yang, Z. Q. Genomic amplification and a role in drug-
resistance for the KDM5A histone demethylase in breast cancer. Am. J.
Transl. Res. 2012, 4, 247−256.
ACKNOWLEDGMENTS
■
(
16) Itoh, Y.; Suzuki, T.; Miyata, N. Small-molecular modulators of
We thank Ms. Mie Tsuchida, Dr. Yasunao Hattori, and Prof.
Kenichi Akaji for their technical support.
cancer-associated epigenetic mechanisms. Mol. Biosyst. 2013, 9, 873−
96.
17) Pachaiyappan, B.; Woster, P. M. Design of small molecule
epigenetic modulators. Bioorg. Med. Chem. Lett. 2014, 24, 21−32.
18) Luo, X.; Liu, Y.; Kubicek, S.; Myllyharju, J.; Tumber, A.; Ng, S.;
8
(
ABBREVIATIONS
■
(
JARID, jumonji AT-rich interactive domain; JHDM, jumonji C
domain-containing demethylases; JMJD, jumonji domain-
containing protein; HDAC, histone deacetylase; NOG, N-
oxalyl glycine
Che, K. H.; Podoll, J.; Heightman, T. D.; Oppermann, U.; Schreiber, S.
L.; Wang, X. A selective inhibitor and probe of the cellular functions of
Jumonji C domain-containing histone demethylases. J. Am. Chem. Soc.
2
(
011, 133, 9451−9456.
19) Cloos, P. A.; Christensen, J.; Agger, K.; Maiolica, A.; Rappsilber,
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