3,5-Diamino-1,2,4-triazoles as a novel scaffold for potent, reversible LSD1 (KDM1A) inhibitors

Article abstract of DOI:10.1039/c4md00283k

The chromatin remodeling amine oxidase lysine-specific demethylase 1 (LSD1) has become an attractive target for the design of specific inhibitors with therapeutic potential. We, and others, have described LSD1 inhibitors that have potential as antitumor agents. Many of the currently known LSD1 inhibitors are poor drug candidates, or are structurally based on the tranylcypromine backbone, thus increasing the potential for off-target effects mediated by other amine oxidases. We now describe a series of potent LSD1 inhibitors based on a novel 1,2,4-triazole scaffold; these inhibitors show a high degree of specificity for LSD1 in vitro, and cause increases in cellular histone 3 dimethyllysine 4 (H3K4me2), a gene transcription activating mark. Importantly, these inhibitors exhibit low toxicity towards mammalian cells in vitro, and thus they may show utility in the treatment of epigenetically-based diseases where cell death is not a desired endpoint. This journal is

Full text of DOI:10.1039/c4md00283k

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3
,5-Diamino-1,2,4-triazoles as a novel scaffold for
potent, reversible LSD1 (KDM1A) inhibitors
Cite this: DOI: 10.1039/x0xx00000x
a§
a§
a
Craig J. Kutz, Steven L. Holshouser, Ethan A. Marrow and Patrick M.
Woster
a*
Received 00th January 2012,
Accepted 00th January 2012
The chromatin remodeling amine oxidase lysine-specific demethylase 1 (LSD1) has become an
attractive target for the design of specific inhibitors with therapeutic potential. We, and others,
have described LSD1 inhibitors that have potential as antitumor agents. Many of the
currently known LSD1 inhibitors are poor drug candidates, or are structurally based on the
tranylcypromine backbone, thus increasing the potential for off-target effects mediated by
other amine oxidases. We now describe a series of potent LSD1 inhibitors based on a novel
DOI: 10.1039/x0xx00000x
www.rsc.org/
1
,2,4-triazole scaffold; these inhibitors show a high degree of specificity for LSD1 in vitro,
and cause increases in cellular histone 3 dimethyllysine 4 (H3K4me2), a gene transcription
activating mark. Importantly, these inhibitors are not toxic to mammalian cells in vitro, and
thus they may show utility in the treatment of epigenetically-based diseases where cell death
is not a desired endpoint
INTRODUCTION
The dynamic interplay between histones and chromatin remodeling
is critical for selective control of gene expression, and chromatin
remodeling enzymes have now become attractive therapeutic targets
for multiple diseases with an epigenetic basis. A number of post-
translational histone modifications are known to control gene
expression, including methylation, ubiquitination, sumoylation,
ADP-ribosylation and acetylation of histone lysine or arginine
residues,
ADP-ribosylation
of
glutamate
residues
and
1
phosphorylation of histone serine residues. The FAD-dependent
amine oxidase lysine-specific demethylase 1 (LSD1, also known as
KDM1A or BHC110) bound to the co-repressor CoREST,
preferentially demethylates the mono- and dimethylated forms of the
activating mark histone 3 lysine 4 (H3K4) to repress gene
2, 3
transcription.
Under some conditions (e.g. when bound to the
androgen receptor), LSD1 also demethylates the mono- and
4
dimethylated forms of the deactivating chromatin mark H3K9.
Because LSD1 is overexpressed in a number of human cancers
(
acute myeloid leukemia, neuroblastoma, retinoblastoma, prostate
5
-8
cancer, breast cancer, lung cancer and bladder cancer), the protein
has emerged as an important target for the development of specific
inhibitors as a new class of antitumor agents. Importantly, LSD1 is
9
now regarded as an emerging drug target for diseases other than
1
0, 11
12, 13
cancer, such as neurological disease,
blood disorders,
viral
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Journal Name
1
4
15, 16
17
infection, diabetes
and fibrosis. As such, there is a need for
Table 1. Structures, cLogP and LSD1 inhibitory activity for 3,5-
diaminotriazoles 6-20. Each data point is the average of 3
determinations + standard error of the mean.
Residual
potent epigenetic modulators that do not cause overt cytotoxicity.
To date, a number of small molecule inhibitors of LSD1
have been described, as shown in Figure 1. Effective LSD1
inhibitors include tranylcypromine-based analogues
oligoamines such as verlindamycin 2
isosteric ureas and thioureas related to
amidoximes such as 4,
such as 5.
inhibitors are structurally based on the clinically used
antidepressant tranylcypromine, which has an IC value of 20.7
3
, 18-20
% LSD1
Activity
such
and related
small-molecule
and peptide based LSD1 inhibitors
Many of the most potent and selective LSD1
Structure
Cmpd.
cLogP
2
1
22
as
1
,
([I] 10µM)
2
3, 24
3
,
2
5
2
6, 27
6
3.73
15.1 ± 4.7
5
0
µM for LSD1. Because tranylcypromine is a potent inhibitor of
monoamine oxidases and other flavin-dependent amine
oxidases, there is a potential for undesired off-target effects in
7
8
3.58
4.46
25.1 ± 1.9
72.3 ± 9.0
tranylcypromine-based LSD1 inhibitors.
In addition,
compounds built on a tranylcypromine scaffold rely on
covalent, irreversible adduct formation with FAD to inactivate
the enzyme. Herein we describe a novel scaffold for a new
series of reversible, competitive inhibitors of LSD1, the 3,5-
diaminotriazole moiety. The 3,5-diaminotriazole scaffold was
used to produce histone demethylase inhibitors exhibiting
increased potency for LSD1 (IC50 1-2 µM), higher specificity
when compared to monoamine oxidase A and B (IC50 values >
9
1
4.46
3.21
69.4 ± 6.4
97.8 ± 6.9
0
1
00 µM), and reduced cytotoxicity. These analogues have great
therapeutic potential for treatment of cancer, and importantly,
for use in other epigenetically driven disorders where
cytotoxicity is not a desired endpoint.
11
2.90
103.1 ± 7.8
RESULTS
1
2
2.49
95.4 ± 6.9
81.9 ± 8.2
Virtual screen for novel LSD1 inhibitors
Potential new scaffolds for small-molecule LSD1 inhibitors
were identified through a virtual screen of the Maybridge
2
5
13
3.648
Hitfinder 5 compound library, as previously described. The
crystal structure of LSD1/CoREST (PDB 2V1D) was prepared
using PrepWizard, and SiteMap was then used to assess
efficient binding within the LSD1 histone-binding pocket.
Lowest energy conformers of 3D compounds were determined
and docked in the LSD1 active site using Glide. A total of 10
hits were identified with Glide scores lower than -7.5 kcal/mol.
The synthesis and biological evaluation of other lead
compounds identified in this screen have been previously
1
1
1
4
5
6
3.53
3.21
3.08
93.1 ± 7.5
69.1 ± 7.7
103 ± 12.7
2
5
published. In the present study, two hits from the virtual
screen, compounds and (Figure 1), were identified, and
6
7
these compounds, as well as related analogues 8-20, were
purchased and evaluated (Table 1).
1
1
7
8
3.03
2.62
80.1 ± 8.4
77.9 ± 7.3
In vitro activity against recombinant LSD1/CoREST
The ability of compounds
recombinant LSD1 was measured using a commercially
available fluorescence-based assay kit (Cayman Chemicals
6-20 to inhibit purified
#
700120). Initially, all compounds were tested at
a
concentration of 10 µM in phosphate-buffered saline (PBS)
containing <1% DMSO (Table 1). The screen was performed as
suggested by the supplier and modified as previously
1
2
9
0
1.27
69.2 ± 5.7
79.2 ± 5.0
2
3, 25
described.
Compounds 6 and 7 were the most effective
1.795
2
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DOI: 10.1039/C4MD002 K
O
inhibitors of LSD1 (%LSD1 activity remaining 15.1 ± 4.7 and ng/µL of LSD1 at 37 C prior to addition of increasing amounts
5.1 ± 1.9, respectively). For comparison, tranylcypromine of the H3K4me2 peptide substrate (concentrations between 0
TCP) and compound reduced LSD1 activity to 72.6 and and 100 µM). Initial rates were determined by linear least-
5.8%, respectively. Compounds and were then subjected to squares fit, and K and V values were determined using the
2
(
2
1
6
7
m
max
titration analysis to determine the in vitro IC50 of each GraphPad Prism 6 software package. The Vmax remained
compound against LSD1 (Figure 2, Panel A . Compound constant (30.62 0.8 unit/min) indicating competitive
had an inhibition, and the K for was determined to be 2.20 M.
)
6
±
possessed an IC50 value of 1.19 µM while compound
IC50 value of 2.22 µM.
7
6
µ
i
To assure that 3,5-diaminotriazoles were bound to LSD1
with 1:1 stoichiometry, nanoisothermal titration calorimetry
(
ITC) was performed (Figure 2, Panel C) using compound
and purified LSD1 (Enzo Life Sciences, #BML-SE544-0050).
Titration of compound to LSD1 resulted in an independent
binding isotherm signifying significant heat release on binding
6
Panel A
6
2
of
6 to LSD1. The R of heat released and the molar ratio was
found to be 0.96.
Amine oxidase selectivity of 3,5-diaminotriazoles
LSD1 and other amine oxidases, such as the monoamine
oxidases (MAO), use the cofactor FAD to reoxidize molecular
oxygen to produce H O . Tranylcypromine and derivatives
Panel B
Panel C
2
2
often show significant off-target efficacy against MAO/A and
MAO/B. commercial recombinant luminescent assay
Promega Corporation, #V1401) was performed as described by
the manufacturer to assess the ability of 3,5-diaminotriazole
and to inhibit monoamine oxidase (MAO) A and B isoforms.
Compounds and were diluted in 1% DMSO prior to
measuring the activity of MAO A and B. Compounds and
both exhibited IC50 values greater than 100 M against both
MAO isoforms, while tranylcypromine inhibited MAO A and B
A
(
6
7
6
7
6
7
µ
Panel D
with IC50 values of 4.2 µM and 5.8 µM, respectively (Figure 2D).
Molecular modeling studies for compound 6
In silico molecular modeling (GOLD software package,
version 5.1, Cambridge Crystollagraphic Data Center,
Cambridge, UK) was performed to predict key residues
interacting with compound
6 (Figure 3) and compound 7 (data
Figure 2. Potency, inhibition kinetics, binding and selectivity of
,5-diaminotriazoles 6 and 7. Panel A. Dose-response of LSD1
following treatment with compounds 6 and 7. Compound 6 IC50 = 1.19
µM; compound 7 IC50 = 2.22 µM Panel B. Competitive enzyme
inhibition kinetics for LSD1 treated with compound 6 at 0, 0.375, 0.75,
and 1µM concentrations over a range of substrate concentrations
not shown). The LSD1 active site (PDB #3ZMT, LSD1-
CoREST in complex with a peptide with the sequence
PRSFLV) was defined as a sphere enclosing residues within
3
1
0Å around the crystallographic peptide ligand. Prior to energy
minimization, proteins were protonated and the pH was set to
7.4. Initial screens focused on using the PRSFLV peptide for
defining potential key interaction sites. The 3D inhibitor
structures were energy minimized using the MM94x force field
for 1000 iterations and a convergence value of 0.001
kcal/mol/Å as the termination criterion. Initial docking results
yielded 60 poses of each structure bound in the active site of
LSD1. The top 5 poses that yielded the lowest E-score were
chosen for further analysis. The best fit for binding was
analyzed for interacting residues. Key interactions with
i
between 0 and 100 µM. K for 6 = 2.2 µM Panel C. Isothermal
calorimetry trace for LSD1 showing a release of heat upon titration
2
a
with compound 6. K = 48.89 nM, R =0.96 Panel D. MAO A and
MAO B enzyme activity for 3,5-diaminotriazoles 6 and 7 compared to
the known MAO inhibitor tranylcypromine (TCP). TCP IC50= 4.2 µM
and 5.8 µM for MAO A and MAO B, respectively). Compounds 6 and
7
exhibited IC50 values > 100µM for both MAO A and B. For Panels
A. B and D, each data point is the average of 3 determination that in
each case differed by 3% or less.
compound
6 include two hydrogen bonds with aspartate 555
Compound 6 is a reversible, competitive inhibitor of LSD1
and another hydrogen bond with the carbonyl of alanine 539.
In addition, the compound participates in pi-stacking with the
flavin ring of the FAD cofactor within 2.98Å. Thus, compound
The fluorescence-based assay method described above was
then used to determine Michaelis-Menten kinetics for
shown in Figure 2, Panel B. In brief, compound
6
, as
was
6
6
shows close association with the active site and effectively
incubated at 0, 0.375, 0.75 and 1.0 µM for 30 mins with 15
prohibits substrate binding. A 2-dimensional rendition of the
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Journal Na3Kme
Figure 3. In silico analysis of compound 6 in the LSD1/CoREST catalytic site (PDB file 3ZMT). The aromatic portion of the o-phenoxy substituent lies
.98Å from the FAD cofactor.
2
binding of
Figure S1.
6
to LSD1/CoREST appears in the supplement, were dissolved in DMSO and then further diluted with culture
medium. The cells were exposed to DMSO concentrations of
less than 1%, and 1% DMSO was used as a negative control for
Cytotoxicity, Cellular Effects, and Global H3K4me2 Levels
cell growth. The known LSD1 inhibitor verlindamycin 2 was
Calu-6 human lung adenocarcinoma cells were purchased used as a positive control. Cells were fixed with 4% PFA,
from ATCC (HTB-56), and cultured in EMEM
growth medium containing 10% (v/v) fetal bovine serum and
permeabilized with 1% Triton-X100 and stained with DAPI at
1:1000 with in PBS. As shown in Figure 4, compounds and
6
7
o
5
% penicillin and streptomycin. Cultures were grown at 37 C in were not cytotoxic to cultured Calu-6 lung tumor cells (IC50
a humidified environment containing 5% CO . The cells were values > 100
µ
M), while
M. TCP was also not cytotoxic in the Calu-6 cell line at
seeded at 1,000 cells/ well. After attachment, the cells were concentrations up to 100 M, and in fact did not cause
2 was cytotoxic with an IC50 value of
2
plated and maintained in a clear bottom, 96 well plate and
5 µ
µ
significant cytotoxicity at much higher concentrations (Figure
S2).
The cytotoxicity of
6 and 7 was examined in 5 additional
cell lines including CA46 Burkitt’s lymphoma, the PC3 human
prostate cancer cell line, the PANC1 human pancreatic cell line,
the MDA-MB-231 estrogen receptor-negative cell line and the
MCF-10A human breast epithelial cell line. LSD1 has been
shown to be overexpressed in the PC3, PANC1 and MDA-MB-
2
31 cell lines. Compounds
cytotoxicity in the CA46 and MCF-10A lines, although
an IC50 of 52 M in the MCF-10A line. In the PC3 line, both 6
and 7 exhibited IC50 values near 74 M, compared to , which
produced 85% growth inhibition at 8 M. In the PANC1 and
MDA-MB-231 lines, was not an effective growth inhibitor,
with IC50 values of 80 and 55 M, respectively. Interestingly,
compound was an effective growth inhibitor in the PANC1
and MDA-MB-231 cell lines, exhibiting IC50 values of 19 and
6
and
7
produced no significant
6
had
µ
µ
2
µ
Figure 4. Cell viability and compound cytotoxicity in Calu-6. Cells
3
were seeded at 3x10 cells/well and treated 48-72hrs with increasing
7
concentrations of TCP, 2, 6 or 7. Cell viability was determined by
colormetric CellTiter 96 Aqueous MTS (Promega, #G3580).
µ
6
exposed to varying concentrations of the drug, while being kept
in a humidified environment for 48 or 72 hours. Compounds
1
2
µ
M, respectively. The mechanism underlying this activity
4
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Figure 5. Global methylation changes by immunofluorescent staining. Calu-6 human lung adenocarcinoma cells were plated at 1x10 cells/well and
treated with 30µM TCP, 1- or 10µM compound 4, and 1- or 10µM compound 5 for 48hrs. Cells were stained for nucleus (DAPI), F-actin (Alexa Fluor 594
Phalloidin), and dimethyl-H3K4 (Alexa Fluor 488 Secondary Antibody). Fluorescent intensity on cell-by-cell basis was obtained by Hermes WiScan
(
IDEA Biomedical) and graphed as a frequency distribution histogram of relative cell count at specific intensities. Two-way ANOVA; *** P-value <
.001, representative of 3 experiments with n=4-6 wells each.
0
remains to be determined; however, it can be generally stated of signal per cell (Hermes WiScan, Idea Biomedical). The
that
vitro.
6
and
7
produce little cytotoxicity in multiple cell lines in imaging system is able to generate 10-40x images as well as
quantify average intensity on a per-well basis, eliminating any
To measure the cellular effects of 3,5-diaminotriazoles
, treated cells from the cytotoxicity assay above were
stained for immunofluorescence (IF) imaging using the that was comparable to the fluorescence promoted by 30
appropriate fluorescently labelled secondary antibodies. In a tranylcypromine (Figure 5), indicating a significant increase in
6-well plate, 5% BSA was added to specific wells and the H3K4me2 levels. The H3K4me2 levels were quantified and
6
bias towards IF staining. Compounds
6 and 7 at both 1 and 10
and
7
µ
M developed green fluorescence in the nucleus at 48 hours
µ
M
9
plate was allowed to stand for 2 hours. The plate was then graphed as cell count vs. average intensity (RFU) as shown in
o
incubated at 4 C overnight with the primary antibody for the histogram (Figure 5). The mean population intensity (MPI)
4
H3K4me2 (Cell Signaling #2139S) diluted in 1% bovine serum ± SEM for the vehicle was 1.44x10 ± 71 RFU. By way of
4
albumin (BSA). Fluorescent secondary antibodies were then comparison, the MPI for 30
added to each well at 1:500 dilutions in 1% BSA for 2 hours. (1.3-fold increase), for 10
µ
µ
M TCP was 1.86x10 ± 136 RFU
4
M compound 6 1.82x10 ± 105
Cells were washed, suspended in PBS and viewed for intensity RFU (1.26-fold increase), and for 10 µM compound 7 1.91x10
4
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Jou al Name
rn
±
124 RFU (1.33-fold increase). Thus, compounds
6
and
7
at LSD1 inhibitors at 10
µ
M than the known LSD1 inhibitor 2,
1
.0
µ
M were as effective as 30
µ
M TCP at increasing while 11 of the 16 compounds measured inhibited LSD1 by
H3K4me2 levels in Calu-6 cells in vitro.
50% or more. The small number of analogues shown in Table 2
are insufficient for the development of structure/activity
Synthesis of 3,5- diaminotriazole analogues
In order to produce additional analogues in the 3,5- and 39 suggests that activity is retained with the addition of
diaminotriazole library, a synthesis of was developed. as small electron releasing groups to the phenoxy aromatic ring at
shown in Scheme 1. Condensation of phenol 21 and 2-cyano- positions R -R . Compounds 37 and 39, which have LSD1
relationships, however, the activity associated with 6, 32, 35, 37
6
1
4
o
3
-chlorofluorobenzene 22 (K CO , microwave, 190 C, 6 min) inhibitory activity comparable to 6, have been selected for
2 3
resulted in the phenoxyphenyl intermediate 23. The cyano further study, and are being subjected to the bioevaluation
group in compound 23 was then reduced (LiALH ) to afford studies described above. In addition, we are continuing to
4
primary amine 24, which was reacted with 25 (microwave, synthesize analogues in this series for the purpose of refining a
o
4
0 C, 5 min) to yield 26. Intermediate 26 was then treated with structure/activity model for 3,5-diaminotriazole-based LSD1
o
hydrazine (microwave, 90 C, 10 min) to produce the desired inhibitors. These results will be reported in a subsequent
,5-diaminotriazole . This route was used to synthesize the publication along with the complete biological characterization
previously unreported 3,5-diaminotriazoles of the general of 37 and 39
structure shown in Scheme 1, compounds 27 42 (Table 2).
Each compound was evaluated as an inhibitor of recombinant Discussion
LSD1 as described above at a concentration of 10 M (Figure
37 and 39) were more effective
3
6
.
-
µ
Newly identified roles for epigenetic modulation involving
LSD1 continue to emerge, both in cancer and in other disease
states, and thus, has become a promising target for therapeutic
intervention. The TCP-based LSD1 inhibitors are the most
advanced chemical class with respect to drug development, and
the first clinical trial for a tranylcypromine-based LSD1
inhibitor for treatment of acute myeloid leukemia began earlier
6
). Six compounds (6, 7, 32, 35,
Table 2. Structures, cLogP and LSD1 residual activity for 3,5-
diaminotriazoles 2, 6, 7 and 27-42 at 10 µM. Each data point is the
average of 3 determinations + standard error of the mean. Compounds 34
and 36 were insoluble and thus were not screened for LSD1 inhibition.
TCP = tranylcypromine
2
8
this year. TCP is a moderately potent, irreversible inhibitor of
LSD1 and continues to provide a popular scaffold for the
design of clinically relevant LSD1 inhibitors. However, it is
challenging to design a TCP-based LSD1 inhibitor for clinical
use that is specific, has low toxicity, and is devoid of the many
biological responses to TCP itself. Thus, these analogues have
the potential to produce off-target effects mediated through
%
LSD1
#
X
R
1
R
2
R
3
R
4
cLogP
Activity
10µM)
(
TC
P
-
-
-
-
--
--
--
64.2 + 6.1
-
-
-
-
-
-
2
6
7
--
H
--
--
H
--
--
H
--
--
33.9 + 6.7
15.1 ± 4.7
25.1 ± 1.9
H
--
3.73
3.58
2
2
7
8
O
O
H
H
H
H
CF
3
O
H
H
5.47
4.94
61.4 + 5.6
40.7 + 6.1
CH
3
(
CH
H
3
)
2
C
2
3
3
9
0
1
O
O
O
H
H
CH
H
3
5.97
4.36
6.27
43.7 + 3.2
55.4 + 5.1
41.8 + 8.8
H
CH
3
O
H
(
CH )
3 3
H
H
H
C
3
3
3
3
3
3
3
3
4
4
4
2
3
4
5
6
7
8
9
0
1
2
O
O
O
O
O
O
O
O
O
S
H
H
H
CH
3
H
H
CH
3
5.44
6.21
6.11
6.19
6.11
4.51
4.45
5.39
4.41
4.74
4.40
32.1 + 8.8
55.1 + 4.6
insolb.
CF
3
CF
3
H
S-CF
3
H
CF
3
H
Br
H
32.2 + 5.4
insolb.
H
H
S-CH
3
H
CH
3
O
H
CH
3
H
15.3 + 3.2
36.7 + 8.6
19.4 + 1.9
34.2 + 6.4
39.2 + 7.9
46.8 + 7.4
H
CH
3
O
H
CH
3
O
Figure 6. LSD1 inhibition assay. Percent LSD1 activity remaining after
treatment with 3,5-diaminotriazoles 6, 7, 27-33, 35, 37-42 at 10 µM. Each
data point is the average of 3 determinations ± SEM (see Table 2).
CH
3
CH
3
H
H
H
O-CH
2
-O
H
H
H
H
H
H
S
H
CH
3
O
3
CH O
6
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9
other flavin-dependent amine oxidase enzymes.
As such,
there is a continuing need to identify novel small-molecule
scaffolds for inhibitors of LSD1 that can be used to design Notes and references
highly specific LSD1 inhibitors. The 1,2,4-triazole moiety
a
Department of Drug Discovery and Biomedical Sciences, College of
Pharmacy, Medical University of South Carolina, 70 President St.,
Charleston, SC 29425.
described in this manuscript could be used as such a scaffold,
and the preliminary studies described herein support the
contention that potent, non-toxic LSD1-specific inhibitors can
be designed in this chemical class.
§
These authors contributed equally to the research described in this
manuscript.
During the preparation of this manuscript, a series of 1,2,3-
3
0-32
Electronic Supplementary Information (ESI) available: complete
experimental section, including synthetic details and a description of all
biological procedures.
triazole-based LSD1 inhibitors were described.
In these
analogues, the triazole ring system is derived from click
chemistry used in their preparation, and is regioisomeric to the
1
,2,4-triazoles described in this manuscript. Although the 1,2,3-
1
1
.
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activation and/or covalent attachment to the FAD cofactor for
inhibition of LSD1, and our enzymatic kinetic studies have
revealed that these compounds act as potent competitive
inhibitors (Figure 2B
first reversible small molecule compounds that possess
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