Identification of PR-SET7 and EZH2 selective inhibitors inducing cell death in human leukemia U937 cells

Article abstract of DOI:10.1016/j.biochi.2012.06.003

Chemical manipulations undertaken on some bis(bromo- and dibromo-phenol) compounds previously reported by us as wide-spectrum epigenetic inhibitors let us to identify bis (bromo- and dibromo-methoxyphenyl) derivatives highly selective for PR-SET7 and EZH2

Full text of DOI:10.1016/j.biochi.2012.06.003

Biochimie 94 (2012) 2308e2313
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Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
Identification of PR-SET7 and EZH2 selective inhibitors inducing cell death
in human leukemia U937 cells
,
,
,
,
Sergio Valente a 1, Ilaria Lepore b 1, Carmela Dell’Aversana b c, Maria Tardugno a d, Sabrina Castellano d,
Gianluca Sbardella d, Stefano Tomassi e, Salvatore Di Maro e, Ettore Novellino e, Roberto Di Santo a,
Roberta Costi a, Lucia Altucci b c, Antonello Mai a
,
,
*
a Istituto Pasteur e Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, P.le A. Moro 5, 00185 Roma, Italy
b Dipartimento di Patologia Generale, Seconda Università degli Studi di Napoli, vico L. De Crecchio 7, 80138 Napoli, Italy
c IGB-CNR, Institute of Genetics and Biophysics, Via P. Castellino, 80100 Napoli, Italy
d Dipartimento di Scienze Farmaceutiche e Biomediche, Epigenetic Med Chem Lab, Università degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA), Italy
e Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli “Federico II”, Via D. Montesano, 49, 80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical manipulations undertaken on some bis(bromo- and dibromo-phenol) compounds previously
reported by us as wide-spectrum epigenetic inhibitors let us to identify bis (bromo- and dibromo-
methoxyphenyl) derivatives highly selective for PR-SET7 and EZH2 (compounds 4, 5, 9, and 10).
Western blot analyses were carried out in U937 cells to determine the effects of such compounds on the
methyl marks related to the tested enzymes (H3K4me1, H3K9me2, H4H20me1, and H3K27me3). The
Received 8 February 2012
Accepted 5 June 2012
Available online 16 June 2012
Keywords:
Epigenetics
Epi-drugs
Bis(bromo- and dibromo-phenol)
compounds
1,5-bis(3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one 4 (EC50 vs EZH2 ¼ 74.9
cells at 50 M, induced massive cell death and 28% of granulocytic differentiation, highlighting the
potential use of EZH2 inhibitors in cancer.
mM), tested in U937
m
Ó 2012 Elsevier Masson SAS. All rights reserved.
Histone methylation
Leukemia
1. Introduction
bromo domains to recognize specific modified residues, are the
readers of epigenetic information. The writers/erasers-driven
chemical modifications display different effects on chromatin,
depending on the type of modification. Lys acetylation is usually
related with transcription activation, while deacetylation typically
lead to gene silencing. Differently, Lys methylation can be associated
to transcription activation or repression depending on the specific
Lys residue and the extension of the resulting methylation (mono-,
di-, or tri-methylation). Accordingly, methylation of histone H3 at
Lysresidues 9 or 27 (H3K9 or H3K27) orhistoneH4 at Lys 20 (H4K20)
is associated with gene silencing, while H3K4, H3K36 and H3K79
methylation correlates with gene activity. An altered expression
and/orfunction of writers, readers orerasers of epigenetic marks can
lead to the development of tumors, by activation of oncogenes or by
silencing tumor-suppressor proteins [4e8].
Reversible covalent modification of histones is one of the
epigenetic mechanisms involved in control of gene expression and
transcription. In particular, acetylation or methylation reactions can
occur at the lysine (Lys) residues on histone tails, to switch chro-
matin from its transcriptionally silent (heterochromatin) to its
transcriptionally active (euchromatin) form, and vice versa [1].
Enzymes involved in chromatin remodeling can add, remove, or
read such covalent modifications, and are now classified into
“writers”, “erasers”, or “readers” [2,3]. Histone acetyltransferases
(HATs) and histone Lys methyltransferases (HKMTs), able to add
acetyl and methyl groups, respectively, at histone tails are writer
enzymes. Histone deacetylases (HDACs) and histone Lys demethy-
lases (HKDMs), catalyzing the opposite reaction, representexamples
of erasers. Another group of proteins, which possess effector
domains including plant homeodomain (PHD), tudor, chromo or
From the report by Cheng et al. indicating some dye-like small
molecules as inhibitors of protein arginine methyltransferases
(PRMTs) and/or HKMTs [9], we chose AMI-1 as PRMT1-selective
prototype to develop a novel series of PRMT-targeted inhibitors
[10,11], and AMI-5 (Fig. 1) as unselective lead compound to design
new simplified analogs to test against PRMTs and HKMTs [12]. We
* Corresponding author. Tel.: þ39 06 4991 3392; fax: þ39 06 4969 3268.
E-mail address: antonello.mai@uniroma1.it (A. Mai).
1
These authors contributed equally to this work.
0300-9084/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.biochi.2012.06.003

S. Valente et al. / Biochimie 94 (2012) 2308e2313
2309
and 8 (Fig. 2), led to compounds inactive against PRMT1, CARM1,
and SET7, or endowed with slight CARM1 inhibiting activity
(compound 5) [12]. Thus, we prepared the 2,6-bis(3-bromo- and
3,5-dibromo-4-methoxybenzylidene)cyclohexanones 9 and 10 as
constrained analogs of 4 and 5, and we tested the derivatives 4e10
against PR-SET7, G9a, and SET7/9. In addition, selected bis(bromo-
or dibromo-methoxyphenyl) compounds 4, 5, 9, and 10 were tested
against Enhancer of Zeste Homolog 2 (EZH2), to ascertain their
capability to inhibit its enzymatic activity. EZH2 catalyzes di- and
trimethylation of H3K27 residues, is the enzimatically active
component of the multiprotein Polycomb Repressor Complex 2
(PRC2), and is involved in stem cell identity, pluripotency, and
cancer [22e24]. In cancer, EZH2 mediates epigenetic silencing of
tumor-suppressor genes in many contexts, and has been found
overexpressed in breast and prostate cancers [25e27], Ewing’s
sarcoma [28], glioblastoma [29], and neuroblastoma [30].
In addition to enzymatic assays, compounds 4, 5, 9, and 10 were
tested in human leukemia U937 cells to determine their effects on
some methylation marks, H4K20me1, H3K9me2, H3K4me1, and
H3K27me3, related to PR-SET7, G9a, SET7/9, and EZH2 activity,
respectively. Moreover, such compounds have been tested in the
same cellular model to study their outcome on cell cycle, cell death
induction and granulocytic differentiation.
Fig. 1. Bromo-hydroxy-containing compounds acting as epi-MLs or non specific PRMT/
HKMT inhibitors.
were intrigued by the presence of two dibromo-hydroxy-phenyl
moieties in the AMI-5 structure, and we postulated these frag-
ments to be crucial for PRMT and/or HKMT inhibiting activity. Thus
we prepared a large series of compounds bearing two bromo- or
dibromo-hydroxyphenyl portions (or their analogs) separated by
variously substituted spacers, and we tested them against two
PRMTs, PRMT1 and CARM1, and against SET7 as an example of
HKMT. In addition, since the structure of the designed compounds
reminded of chemical features typical of HAT and SIRT modulators,
we tested some of them against p300 HAT and SIRT1/2 enzymes
[13e15]. From these assays, it resulted that compounds carrying
two 3,5-dibromo-4-hydroxyphenyl moieties linked through
2. Materials and methods
2.1. Chemistry
2.1.1. General
Melting points were determined on a Buchi 530 melting point
apparatus and are uncorrected. Infrared (IR) spectra (KBr) were
recorded on a PerkineElmer Spectrum One instrument. 1H-NMR
spectrawere recorded at 400 MHz on a Bruker AC 400 spectrometer;
chemical shifts are reported in d (ppm) units relative to the internal
reference tetramethylsilane (Me4Si). Electronic impact mass spec-
trometry (EI-MS) was performed on a Finnigan LCQ DECA Termo-
Quest (San Josè, California, USA) mass spectrometer. All compounds
were routinely checked by TLC and 1H NMR. TLC was performed on
aluminum-backed silica gel plates (Merck DC, Alufolien Kieselgel 60
F254) with spots visualized by UV light or using a KMnO4 alkaline
solution. All solvents were reagent grade and, when necessary, were
purified and dried by standard methods. Concentration of solutions
after reactions and extractions involved the use of a rotary evapo-
rator operating at reduced pressure of ca. 20 Torr. Organic solutions
were dried over anhydrous sodium sulfate. Analytical results are
within 0.40% of the theoretical values (see Supplementary material).
All chemicals were purchased from Aldrich Chimica, Milan (Italy), or
from Lancaster Synthesis GmbH, Milan (Italy), and were of the
highest purity. As a rule, samples prepared for physical and biolog-
a
penta-1,4-dien-3-one, 2,6-dimethylene(hetero)cycloalkanone,
1,1-(1,3-phenylene)diprop-2-en-1-one, or hepta-1,6-diene-3,5-
dione spacer behaved as epigenetic multiple ligands (epi-MLs),
inhibiting at the same time all the tested PRMT, HAT, and SIRT
enzymes, as well as SET7 [14].
Among the 1,4-diphenylpenta-1,4-dien-3-one derivatives, those
showing either a 3-bromo-4-hydroxy/3,5-dibromo-4-hydroxy or
a bis(3,5-dibromo-4-hydroxy) substitution at the phenyl rings
(compounds 1 and 2, Fig. 1) were epi-MLs, while the bis(3-bromo-
4-hydroxy) analog (3) showed PRMT1, CARM1, and SET7 inhibition,
but was totally inactive against HATs and SIRTs (Fig. 1).
To further investigate the effect of such compounds against
HKMTs, we tested 1e3 against PR-SET7, G9a, and SET7/9, three
HKMTs different from SET7. In particular, PR-SET7 is a H4K20 lysine
methyltransferase highly involved in cell cycle regulation and
progression [16,17], G9a [18] acts on H3K9 and has been found
expressed in aggressive lung cancer cells, with its elevated
expression related to poor prognosis [19], and SET7/9 [20] (with its
epigenetic mark H3K4me1) has been associated to inflammatory
diseases and diabetes [21]. As expected, 1e3 confirmed their wide
inhibitory spectrum against epigenetic targets, they being able to
ical studies were dried in high vacuum over P2
O5 for 20 h at
temperatures ranging from 25 to 110 ꢀC, depending on the sample
melting point. Syntheses and chemical and physical data of 4e8
have been previously described [12].
2.1.2. General Procedure for the synthesis of the 2,6-bis(3-bromo-
and 3,5-dibromo-4-methoxybenzylidene)cyclohexanones (9 and
10). example: synthesis of 2,6-bis(3-bromo-4-methoxybenzylidene)
cyclohexanone (9)
inhibit the three enzymes in the range 4.3e62.5
mM (Table S1 in
Supplementary material). Then, with the aim to identify selective
HKMT inhibitors among this library of compounds, we noticed that
analogs of derivatives 1e3 which bear methoxy instead of hydroxy
group on the two phenyl wings of the penta-1,4-dien-3-one scaf-
fold (4 and 5, Fig. 2) as well as simplified products such as the
Cyclohexanone (1.15 mmol, 0.12 mL) was added to a suspension
of barium hydroxide octahydrate (4.6 mmol, 1.45 g) in methanol
(20 mL), and the mixture was stirred for 5 min. Then a solution of 3-
bromo-4-methoxybenzaldehyde (2.3 mmol, 0.5 g) in methanol
(10 mL) was added, and the resultant mixture was stirred for 2 h at
room temperature. The precipitate was filtered, washed with water,
dried and recrystallized by acetonitrile to afford the pure product.
bis(3,5-dibromo-4-hydroxyphenyl)methanone
6 and the 4-(3-
bromo- and 3,5-dibromo-4-hydroxyphenyl)but-3-en-2-ones
7

2310
S. Valente et al. / Biochimie 94 (2012) 2308e2313
Fig. 2. Bromo-hydroxy- and emethoxy-phenyl derivatives.
Melting point: 170e172 ꢀC; yield: 82%; 1H NMR (400 MHz, DMSO-
d6) 1.72e1.75 (m, 2H, cyclohexanone protons), 2.86e2.89 (m, 4H,
S-adenosyl-L
-[methyl-3H]methionine. Enzyme: 100 nM EZH2
d
complex. Reaction Procedure: prepare indicated substrate in freshly
prepared Reaction Buffer; deliver EZH2 into the substrate solution
and mix gently; deliver compounds in DMSO into the EZH2 reaction
mixture by using Acoustic Technology (Echo 550, LabCyte Inc. Sun-
nyvale, CA) in nanoliter range, incubate for 15 min; deliver 3H-SAM
into the reaction mixture to initiate the reaction; incubate for 1 h at
30 ꢀC; deliver reaction mixture to filter-paper for detection.
cyclohexanone protons), 3.90 (s, 6H, OCH3), 7.19e7.21(d, 2H,
benzene protons), 7.55e7.59 (m, 4H, benzene protons and
PhCH ¼ CCO), 7.78 (s, 2H, benzene protons) ppm; 13C NMR
(400 MHz, DMSO-d6)
d 25.1, 26.1 (2C), 55.1 (2C), 112.1 (2C), 112.2
(2C), 128.4 (2C), 129.2 (2C), 130.9 (2C), 132.2 (2C), 137.1 (2C), 156.2
(2C), 190.4 ppm; m/z [M]þ: 491.9759.
2.1.3. 2,6-bis(3,5-dibromo-4-methoxybenzylidene)cyclohexanone
(10)
2.3. Cellular assays
Recrystallized by: acetonitrile; melting point: 204e206 ꢀC;
yield: 78%; 1H NMR (400 MHz, DMSO-d6)
d 1.73e1.76 (m, 2H,
2.3.1. Compounds
All compounds were dissolved in DMSO (SigmaeAldrich) and
cyclohexanone protons), 2.84e2.87 (m, 4H, cyclohexanone
protons), 3.84 (s, 6H, OCH3), 7.51 (s, 2H, PhCH ¼ CCO), 7.83 (s, 4H,
used at 25
mM or 50 mM.
benzene protons) ppm; 13C NMR (400 MHz, DMSO-d6)
d 25.1, 26.1
2.3.2. Cell lines
(2C), 60.8 (2C), 118.3 (4C), 129.9 (4C), 131.9 (2C), 132.2 (2C), 137.1
(2C), 154.2 (2C), 190.4 ppm; m/z [M]þ: 649.7949.
U937 (human leukemic monocyte lymphoma cell line-ATCC)
were grown in RPMI 1640 medium (Euroclone) supplemented
with 10% heat-inactivated FBS (Euroclone), 1% glutamin (Lonza), 1%
penicillin/streptomycin (Euroclone) and 0.1% gentamycin (Lonza),
at 37 ꢀC in air and 5% CO2.
2.2. Biochemical assays
2.2.1. PR-SET7, G9a, and SET7/9 inhibitory assays
Assays were performed essentially as described previously for
PR-SET7 [16], G9a [18], and SET7/9 [20]. Briefly, the samples were
incubated at 30 ꢀC for 10e60 min in a reaction buffer containing
2.3.3. Cell cycle analysis
2.5 ꢁ 105 U937 cells were collected by centrifugation after 30 h
stimulation with compounds at 25 mM and 50 mM. The cells were
50 mM TriseHCl (pH 8.5), 5 mM MgCl2, 4 mM DTT, and 1
mM S-
-[methyl-3H]methionine
resuspended in 500
mL of hypotonic buffer (0.1% NP-40, 0.1% sodium
adenosyl- (Amersham Pharmacia
L
citrate, 50 g/mL PI, RNAse A) and incubated in the dark for 30 min.
m
Biotech). Two micrograms of octamer, oligonucleosomes, or mon-
onucleosome were used as substrates. The total volume of the
reaction mixture was adjusted to 25 mL. The reaction was stopped
by addition of SDS sample buffer and then fractionated on 15% SDS-
PAGE. Separated histones were then transferred onto an
Immobilon-P membrane (Millipore) and visualized by CBB staining.
The membrane was sprayed with EN3HANCE (NEN), and exposed
to Kodak XAR film overnight.
The analysis was performed by FACS-Calibur (Becton Dickinson)
using the Cell Quest Pro software (Becton Dickinson) and ModFit LT
version 3 software (Verity). The experiment was performed in
triplicate.
2.3.4. Granulocytic differentiation analysis
2.5 ꢁ 105 U937 cells were collected by centrifugation after 30 h
stimulation with compounds at 25 mM and 50 mM. The cells were
2.2.2. EZH2 inhibitory assay
2.2.2.1. Reagent. Reaction buffer; 50 mM TriseHCl (pH 8.0), 50 mM
NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1% DMSO.
Table 1
Inhibitory activities of compounds 4e10 against PR-SET7, G9a, and SET7/9.a
Compd
PR-SET7
EC50, mM
G9a
SET7/9
2.2.2.2. Reaction conditions. EZH2: Complex of human EZH2 (Gen-
Bank Accession No. NM_004456), (amino acids 2-end) with
N-terminal His tag, MW ¼ 86 kDa, human EED (NM_003797) (a-a
2-end) with N-terminal Flag tag, MW ¼ 51 kDa, human SUZ12
(NM_015355) (a-a 2-end) with N-terminal His tag, MW ¼ 87 kDa,
Human AEBP2 (NM_153207) (a-a 2-end) with N-terminal His tag,
MW ¼ 53 kDa, and human RbAp48 (NM_005610) (a-a 2-end) with
N-terminal His tag, MW ¼ 48 kDa, co-expressed in baculovirus
4
5
6
7
8
9
10
9.0 ꢂ 0.4
3.3 ꢂ 0.2
38.8 ꢂ 3.0
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
164.4 ꢂ 11.0
>250
10.2 ꢂ 0.5
2.6 ꢂ 0.1
a
expression system. Substrate: 5
mM Histone H3. Methyl donor: 1
mM
Values are means ꢂ SD determined from at least three experiments.

S. Valente et al. / Biochimie 94 (2012) 2308e2313
2311
Scheme 1. Synthesis of compounds 9 and 10; reagents and conditions: Ba(OH)2, CH3OH, room temperature, 2 h, 78e82%.
washed with PBS and incubated in the dark at 4 ꢀC for 30 min with
PR-SET7, the bis(3,5-dibromo-4-methoxyphenyl) analogs 5 and 10
displayed the highest potency (also higher than 1e3), while the
benzophenone 6 was the less potent.
10
10
m
m
L of PE-conjugated anti-CD11c surface antigen antibody or with
L of PE-conjugated IgG, in order to define the background
signal. At the end of the incubation the samples were washed again
and resuspended in 500 L of PBS containing 0.25 g/mL PI. The
analysis was performed by FACS-Calibur (Becton Dickinson) using
the Cell Quest Pro software (Becton Dickinson). The experiment
was performed in triplicate and PI positive cells were excluded
from the analysis.
Selected compounds 4, 5, 9, and 10 were then tested against
EZH2 (Table 2). In this assay, the human 5-component PRC2 (con-
taining EZH2, EED, SUZ12, RBAP48, and AEBP2) was used as the
enzyme source, and histone H3 was used as a substrate. S-adeno-
m
m
syl-L-homocysteine (SAH), a known methyltransferase inhibitor,
was used as a reference drug. Against EZH2, the bis(3-bromo-4-
methoxyphenyl) derivatives 4 and, to a lesser extent, 9 were the
most effective inhibitors, suggesting that in this case the bis(3,5-
dibromo) substitution, respect to the bis(monobromo) substitu-
tion, is detrimental for the inhibiting activity.
2.3.5. Histone extraction
After stimulation with compounds, the cells were collected by
centrifugation and washed two times with PBS. Then the samples
were resuspended in Triton extraction buffer (PBS containing 0.5%
Triton X 100 (v/v), 2 mM PMSF, 0.02% (w/v) NaN3), and the lysis was
performed for 10 min at 4 ꢀC. Next, the samples were centrifugated
at 2000 rpm for 10 min at 4 ꢀC, and the pellets were washed in TEB
(half the volume). After a new centrifugation under the same
conditions, the samples were resuspended in 0.2 N HCl and the acid
histone extraction was carried out overnight at 4 ꢀC. The superna-
tant was recovered by centrifugation and the protein content was
ensured by BCAÔ Protein Assay (Pierce).
3.3. Western blot analysis
Western blot analyses were performed with 4, 5, 9, and 10 at
50 mM in human leukemia U937 cells treated for 24 h (Fig. 3). H3
methylation marks, H3K4me1 H3K9me2 and H3K27me3, and
H4K20me1 have been evaluated using specific antibodies. Con-
cerning H4K20me1 compound 5 and, to a lesser extent, 9 and 10
displayed a signal reduction, in agreement with the PR-SET7
inhibitory data. Differently, H3K9me2 and H3K4me1 expression
levels appear unmodified after treatment with 4, 5, 9, and 10,
according to their lack of G9a and SET7/9 inhibitory activity. On the
other hand, H3K27me3 strongly decreased after treatment with 4
and 9, and was less evident with 5 and 10, in accordance with their
different degree of EZH2 inhibition.
2.3.6. Western blot
5 mg of histonic extracts were loaded on 15% polyacrylamide gel.
The histone H3 methylation was assessed with anti-dimethyl-K9-
histone H3 antibody (Abcam), with anti-monomethyl K20 histone
H4 (Diagenode), with anti-monomethyl K4 histone H3 (Dia-
genode), and with anti-trimethyl-K27-histone H3 antibody (Dia-
genode). To check for equal loading, Ponceau Red (Sigma) staining
and the anti-histone H4 antibody (Abcam) were used.
3.4. Cell cycle and differentiation effects on human leukemia U937
cells
3. Results
Compounds 4, 5, 9, and 10 were tested at 25 and 50 mM in
human leukemia U937 cells for 30 h, to determine their effect on
cell cycle, cell death (pre-G1 peak), and granulocytic differentiation
(Fig. 4).
3.1. Chemistry
The 2,6-bis(3-bromo- and 3,5-dibromo-4-methoxybenzylidene)
cyclohexanones 9 and 10 were prepared by condensation of
cyclohexanone with 2 equivalents of the properly substituted
benzaldehyde in the presence of barium hydroxide in methanol at
room temperature (Scheme 1).
In the tested conditions, only derivatives 4 (at 25
and 50 M) affected cell cycle, inducing a slight increase of the cells
at the G1 phase. At 50 M it was not possible to analyze the cell cycle
effect of 4 due to the extensive cell death caused. As regards cell
death induction (pre-G1 peak), at 50 M compound 4 displayed
mM) and 5 (at 25
m
m
m
a massive effect (near 100%, we considered a cut-off of 90%), and 5
induced 41% of cell death. Granulocytic differentiation was evaluated
by determining the number of CD11c positive cells with subtraction
3.2. Histone methyltransferase (PR-SET7, G9a, SET7/9, and EZH2)
inhibition
Compounds 4e10 (Fig. 2) were tested against PR-SET7 using
nucleosome as a substrate, and against G9a and SET7/9 using in
both cases the histone octamer as a substrate.
Table 2
Inhibitory activity of 4, 5, 9, and 10 against EZH2.a
Compd
EC50 (mM) or % inhibition at 75 mM
The resulting EC50 (effective compound concentration able to
inhibit 50% of the enzyme activity) values are reported in Table 1.
The bis(bromo- and dibromo-methoxyphenyl) derivatives 4, 5, 9,
and 10 as well as the bis(3,5-dibromo-4-hydroxyphenyl)meth-
anone 6 were able to selectively inhibit PR-SET7, whereas the
4-phenylbut-3-en-2-ones 7 and 8 were totally ineffective. Against
4
5
9
10
SAH
74.9 ꢂ 4.0
8.7%
313.8 ꢂ 15.0
6.2%
66.8 ꢂ 3.5
a
Values are means ꢂ SD determined from at least three experiments.

2312
S. Valente et al. / Biochimie 94 (2012) 2308e2313
of the propidium iodide (PI) positive cells (% CD11cþ/PI- cells).
In such assay, compounds 4 and 10 at 50 M showed high
m
differentiation effects, with 28 and 20% of CD11c positive cells.
4. Discussion
Fig. 3. Western blot analyses for compounds 4, 5, 9, and 10 in U937 cells: levels of
H3K4me1, H3K9me2, H4K20me1, and H3K27me3 methylation. Ponceau Red (PR)
staining of histones or total histone H4 were used for equal loading. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
In conclusion, while bromo- and dibromo-hydroxyphenyl
substituents at the penta-1,4-dien-3-one scaffold (compounds
1e3) led to unselective inhibitors of epigenetic targets including
PR-SET7, G9a, and SET7/9, bromo-methoxy substitution at either
the 1,5-diphenylpenta-1,4-dien-3-one or 2,6-bis(benzylidene)
cyclohexanone scaffold (compounds 4 and 9) afforded specific PR-
SET7 and EZH2 inhibitors. The insertion in such compounds of
additional bromine atoms (5 and 10) increases their inhibitory
action against PR-SET7 and decreases the effect against EZH2. In
Western blot analyses (U937 cells, 24 h) at 50 mM compound 5 and,
to a lesser extent, 9 and 10 reduced the signal for H4K20me1,
according to their inhibition of PR-SET7, and 4 and 9 strongly
decreased the level of H3K27me3, confirming their EZH2 inhibi-
tion. Methylation levels of H3K4me1 and H3K9me2, used as
markers of the SET7/9 and G9a enzymatic activity, respectively,
remained unchanged respect to the control, in agreement with the
lack of inhibition observed with the tested compounds. In leukemia
U937 cell assays, compounds 4 and 5 showed a slight increase of
the cells at the G1 phase, in part due to their PR-SET7 (4 and 5)
and/or EZH2 (4) inhibition. In addition, 4 tested at 50 mM in U937
cells induced massive cell death, and gave a considerable increase
of CD11c positive cells, of about 28%, thus validating the use of EZH2
inhibitors as apoptotic and/or cytodifferentiating agents in cancer.
Acknowledgments
We thank Patrick Trojer (Constellation Pharmaceuticals, Sidney
St., Cambridge, MA, USA), Jin Zhang and Danny Reinberg (Howard
Hughes Medical Institute, Department of Biochemistry, New York
University School of Medicine, New York, USA) for critical reading
of the manuscript and helpful discussion. This work was supported
by AIRC, PRIN 2009PX2T2E, FIRB RBFR10ZJQT, and FP7 Project
BLUEPRINT/282510.
Appendix A. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.biochi.
2012.06.003.
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