Identification of cell-active lysine specific demethylase 1-selective inhibitors

Article abstract of DOI:10.1021/ja907055q

(Chemical Equation Presented) Lysine specific demethylase 1 (LSD1) plays a key role in the regulation of gene expression by removing the methyl groups from methylated Lys4 of histone H3 (H3K4). Here we report the identification of the first small-molecule LSD1-selective inhibitors. These inhibitors show in vivo H3K4-methylating activity and antiproliferative activity and should be useful as lead structures for anticancer drugs and as tools for studying the biological roles of LSD1.

Full text of DOI:10.1021/ja907055q

Published on Web 11/16/2009
Identification of Cell-Active Lysine Specific Demethylase 1-Selective
Inhibitors
Rie Ueda,† Takayoshi Suzuki,*,† Koshiki Mino,‡ Hiroki Tsumoto,† Hidehiko Nakagawa,†
Makoto Hasegawa,‡ Ryuzo Sasaki,§ Tamio Mizukami,*,‡ and Naoki Miyata*,†
Graduate School of Pharmaceutical Sciences, Nagoya City UniVersity, 3-1 Tanabe-dori, Mizuho-ku,
Nagoya, Aichi 467-8603, Japan, Graduate School of Bio-Science, Nagahama Institute of Bio-Science Technology,
1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan, and Frontier Pharma Inc., 1281-8 Tamura-cho,
Nagahama, Shiga 526-0829, Japan
Received August 20, 2009; E-mail: suzuki@phar.nagoya-cu.ac.jp; mizukami@nagahama-i-bio.ac.jp;
miyata-n@phar.nagoya-cu.ac.jp
The methylation status of histone lysine residues, which is tightly
controlled by two counteracting enzyme families, the histone methyl
transferases and the histone demethylases, plays a pivotal role in
the regulation of gene expression.1 Lysine specific demethylase 1
(LSD1), the first histone demethylase to have been discovered,
removes the methyl groups from mono- and dimethylated Lys4 of
histone H3 (H3K4me1/2) through flavin adenine dinucleotide
(FAD)-dependent enzymatic oxidation (Figure 1a).2
LSD1-selective inhibitors are useful as tools for elucidating in
detail the biological functions of the enzyme.3 To date, only a few
types of LSD1 inhibitors have been identified. Monoamine oxidase
(MAO) inhibitors such as trans-2-phenylcyclopropylamine (PCPA)
(Figure 1b) and pargyline have been reported to inhibit LSD1,
Figure 1. (a) Proposed catalytic mechanism for the demethylation of
although their inhibitory activity and selectivity for LSD1 are very
methylated lysine by LSD1. (b) Proposed mechanism of inactivation of
low.4 Among MAO inhibitors, PCPA is the most potent LSD1
LSD1 by PCPA. (c) Proposed mechanism of inactivation of LSD1 by
N-propargyl lysine peptides.
inhibitor. LSD1 inhibition by PCPA occurs via formation of a
covalent adduct at the N5 and C4a positions of the flavin ring
following one-electron oxidation and cyclopropyl ring opening
(Figure 1b). N-Propargyl lysine-containing H3 peptides (Figure 1c)
have been reported to be LSD1-selective inhibitors.5 The mecha-
nism of LSD1 inhibition by the peptides involves conjugate addition
of the flavin N5 to the γ carbon of the electrophile following two-
electron oxidation of the iminium ion (Figure 1c). The propargyl
lysine peptides are selective for LSD1 over MAO-B and can be
used as biochemical tools for in vitro study of LSD1. However, it
is difficult to use peptide inhibitors for cellular studies because of
their poor membrane permeability. In addition to the inhibitors
mentioned above, polyamine analogues have been reported,6
although their selectivity for LSD1 over other flavin-containing
oxidases was not examined. Therefore, LSD1-selective inhibitors
that show activity in in vivo assays are still required for elucidation
of the cellular functions of LSD1. Herein we report the LSD1-
inhibitory activity, selectivity, inhibitory mechanism, and cellular
activity of small molecules designed on the basis of the reported
X-ray crystal structures of LSD1.
In designing small-molecule LSD1-selective inhibitors, we
focused on the X-ray crystal structures of the FAD-PCPA adduct4a
and the FAD-N-propargyl lysine peptide adduct5b in the active
site of LSD1. Figure 2a shows the superimposition of the two
structures. The FAD parts of the two adducts are well-superimposed,
and the benzene ring of the FAD-PCPA adduct overlaps with the
ε-N and δ-C of the FAD-N-propargyl lysine peptide adduct. On
the basis of these superimposed structures, we designed PCPA-lysine
Figure 2. (a) Superimposition of the FAD-PCPA adduct (PDB entry
2UXX) (green tube) and the reduced FAD-N-propargyl lysine peptide
adduct (PDB entry 2UXN) (magenta wire) in the active site of LSD1. Amino
acid residues in the active site are not shown for the sake of clarity. (b)
Structures of compounds 1-4.
hybrid compounds 1 and 2 (Figure 2b) in which the side chain of
the amino acid is linked with the phenyl ring of PCPA through an
ether bond at the meta and para positions, respectively. We chose
benzylamino and benzoyl groups as substituents of the carbonyl
and amino groups of the amino acid, respectively, since they are
expected to be recognized by hydrophobic amino acid residues (Val
333, Ile 356, Phe 382, Leu 386, Leu 536, Ala 539, Thr 566, and
Leu 677) at the entrance to the N-methylated lysine binding channel
of LSD1 (Figure S1a in the Supporting Information). In addition,
the attachment of these small, hydrophobic groups should enhance
the membrane permeability. Furthermore, compounds 1 and 2 were
expected to selectively inhibit LSD1 over MAO-A and MAO-B,
as the X-ray crystal structures of MAO-A7 and MAO-B8 indicate
that their active-site cavities are not capacious enough to accom-
modate the large group attached to the phenyl ring of PCPA in
compounds 1 and 2 (Figure S1b,c). Compounds 3 and 4 (Figure
† Nagoya City University.
‡ Nagahama Institute of Bio-Science Technology.
§ Frontier Pharma Inc.
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17536 J. AM. CHEM. SOC. 2009, 131, 17536–17537
10.1021/ja907055q  2009 American Chemical Society

C O M M U N I C A T I O N S
Table 1. In Vitro LSD1-, MAO-A-, and MAO-B-Inhibitory Activities
of Compounds 1 and 2 and PCPA
compound FAD-1 adduct was generated as a result of LSD1-
catalyzed reaction of compound 1 and FAD. The data from the
kinetics and mass spectroscopic analyses support the idea that
compound 1 inactivates LSD1 by mechanism-based enzyme inhibi-
tion in a manner similar to PCPA.
IC50 (µM)
selectivitya
cmpd
LSD1
MAO-A
MAO-B
MAO-A/LSD1
MAO-B/LSD1
PCPA
32
7.3
4.3
0.23 (1)
0.13 (1)
Unlike peptide inhibitors, compounds 1 and 2 are small molecules
that might be active in cellular assays. We performed a cellular
assay using Western blot analysis. Since LSD1 is known to catalyze
the demethylation of H3K4me2, the methylation level of H3K4 in
HEK293 cells was analyzed. As Figure S5 shows, the level of
H3K4me2 was dose-dependently elevated in the presence of 1 or
2. These results suggest that compounds 1 and 2 inactivate LSD1
in cells and can be used as tools for probing the biological role of
LSD1.
1
2
2.5
1.9
230
290
500
>1000
92 (400)
150 (650)
200 (1500)
>520 (>11000)
a Numbers in parentheses are the selectivity values divided by the
(MAO IC50)/(LSD1 IC50) value for tranylcypromine.
Since it has been reported that RNAi-mediated knockdown of
LSD1 suppresses the growth of tumor cells,9 we carried out cell-
growth inhibition assays of compounds 1 and 2, the most selective
and active compounds in this study, using HEK293 cells. Cell-
growth suppression by the inhibitors was observed over the
concentration range in which distinct H3K4 methylation was
detected in the Western blot analysis (Figure S6). Thus, the
demethylase function of LSD1 appears to be deeply involved in
cell growth. Next, we evaluated growth inhibition by inhibitors 1
and 2 against various other human cancer cell lines (Table S3 and
Figure S7). Compounds 1 and 2 exhibited growth inhibition with
GI50 values ranging from 6.0 to 67 µM, suggesting that LSD1-
selective inhibitors are anticancer-agent candidates.
Thus, we have identified the first cell-active LSD1-selective
inhibitors 1 and 2, which should be useful as lead structures in the
development of more potent and selective LSD1 inhibitors through
modification of the benzoyl and benzylamino groups. Such inhibi-
tors are anticancer-agents candidate as well as tools for studying
the biological roles of LSD1 in cells.
Figure 3. Mass spectrometric detection of the FAD-1 adduct.
2b), which lack the R-amino moiety of compounds 1 and 2, were
designed as reference compounds.
Compounds 1-4 were synthesized, and their inhibitory activities
toward human LSD1 and MAO-A and -B were evaluated. The
results are summarized in Table 1 and Table S1 as IC50 values (also
see Figure S2). The LSD1-inhibitory activities of compounds 1-4
were more potent than that of PCPA (IC50 values: PCPA, 32 µM;
1, 2.5 µM; 2, 1.9 µM; 3, 22 µM; 4, 9.7 µM). Furthermore, while
PCPA inhibited MAO-A and MAO-B more potently than LSD1
[(MAO-A IC50)/(LSD1 IC50) ) 0.23; (MAO-B IC50)/(LSD1 IC50)
) 0.13], compounds 1-4 inhibited LSD1 more potently than
MAO-A and MAO-B [(MAO-A IC50)/(LSD1 IC50) ) 2.8 to 150;
(MAO-B IC50)/(LSD1 IC50) ) 1.0 to >520]. In particular, the LSD1
selectivity of compounds 1 and 2 was 400 to >11000 times higher
than that of PCPA while compounds 3 and 4 showed less selectivity,
indicating the importance of the amino acid structure of the
inhibitors for potency and selectivity toward LSD1.
Acknowledgment. We thank Mie Tsuchida, Sayaka Nakagawa,
and Haruka Komaarashi for their technical support. This work was
supported in part by a Grant-in-Aid for Scientific Research from
the Japan Society for the Promotion of Science (N.M.) and a Grant-
in Aid for Research at Nagoya City University (T.S.).
Supporting Information Available: Experimental procedures,
including spectral data for compounds 1-4 and biological methods,
and supporting figures and tables. This material is available free of
charge via the Internet at http://pubs.acs.org.
To investigate the mechanism of LSD1 inhibition, we initially
examined whether inhibition by compound 1 or 2 is time-dependent.
The time course of product formation was monitored in the absence
or presence of compound 1 or 2. As shown in Figure S3, compounds
1 and 2 were found to be time-dependent inhibitors of LSD1,
showing nonlinear progress curves and reaching a plateau value.
These data suggest that compounds 1 and 2 are irreversible
inhibitors. The values of kinact, KI, and kinact/KI for compounds 1, 2,
and PCPA were obtained from kinetic assays using LSD1, MAO-
A, and MAO-B (Table S2). The kinact/KI values toward LSD1 for
compounds 1 and 2 are much larger than those toward MAO-A
and MAO-B, while the kinact/KI value toward LSD1 for PCPA is
smaller than those toward MAO-A and MAO-B, confirming that
compounds 1 and 2 are highly selective for LSD1.
To gain further mechanistic insight, a mass spectroscopic analysis
of an incubation mixture of LSD1 with compound 1, the most potent
inhibitor in this study, was performed. If compound 1 reacts with
FAD as expected, an FAD-1 conjugate should be generated. As
depicted in Figure 3, while the peak for FAD was observed at m/z
783.9, significant peaks at m/z 1228.1 and 1210.1 were also
observed. These peaks correspond to the predicted molecular
weights of the FAD-1 adduct. Furthermore, they were not detected
in the absence of LSD1 (Figure S4). These results indicate that
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