Discovery of novel [1,2,4]triazolo[4,3-a]quinoxaline aminophenyl derivatives as BET inhibitors for cancer treatment

Article abstract of DOI:10.1016/j.bmcl.2017.09.025

Bromodomain and extra-terminal (BET) proteins, a class of epigenetic reader domains has emerged as a promising new target class for small molecule drug discovery for the treatment of cancer, inflammatory, and autoimmune diseases. Starting from in silico screening campaign, herein we report the discovery of novel BET inhibitors based on [1,2,4]triazolo[4,3-a]quinoxaline scaffold and their biological evaluation. The hit compound was optimized using the medicinal chemistry approach to the lead compound with excellent inhibitory activities against BRD4 in the binding assay. The substantial antiproliferative activities in human cancer cell lines, promising drug-like properties, and the selectivity for the BET family make the lead compound (13) as a novel BRD4 inhibitor motif for anti-cancer drug discovery.

Full text of DOI:10.1016/j.bmcl.2017.09.025

Accepted Manuscript
Discovery of novel [1,2,4]triazolo[4,3-a]quinoxaline aminophenyl derivatives
as BET inhibitors for cancer treatment
Imran Ali, Jooyun Lee, Areum Go, Gildon Choi, Kwangho Lee
PII:
S0960-894X(17)30921-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.09.025
BMCL 25289
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Bioorganic & Medicinal Chemistry Letters
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21 July 2017
6 September 2017
12 September 2017
Please cite this article as: Ali, I., Lee, J., Go, A., Choi, G., Lee, K., Discovery of novel [1,2,4]triazolo[4,3-
a]quinoxaline aminophenyl derivatives as BET inhibitors for cancer treatment, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.09.025
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Discovery of novel [1,2,4]triazolo[4,3-a]quinoxaline
aminophenyl derivatives as BET inhibitors for cancer
treatment
Imran Alia,b, Jooyun Leea, Areum Goa,b, Gildon Choia,b*, Kwangho Leea,b*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Discovery of novel [1,2,4]triazolo[4,3-a]quinoxaline aminophenyl derivatives as
BET inhibitors for cancer treatment
Imran Alia,b, Jooyun Leea, Areum Goa,b, Gildon Choia,b*, Kwangho Leea,b*
aBio-Organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 34114, Republic of Korea.
bMedicinal Chemistry & Pharmacology, Korea University of Science & Technology, Daejeon 34113, Republic of Korea.
Tel: +82-42-860-7176, Fax: +82-42-860-7160, e-mail: gchoi@krict.re.kr, kwangho@krict.re.kr
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Bromodomain and extra-terminal (BET) proteins, a class of epigenetic reader domains has
emerged as a promising new target class for small molecule drug discovery for the treatment of
cancer, inflammatory, and autoimmune diseases. Starting from in silico screening campaign,
herein we report the discovery of novel BET inhibitors based on [1,2,4]triazolo[4,3-
a]quinoxaline scaffold and their biological evaluation. The hit compound was optimized using
the medicinal chemistry approach to the lead compound with excellent inhibitory activities
against BRD4 in the binding assay. The substantial antiproliferative activities in human cancer
cell lines, promising drug-like properties, and the selectivity for the BET family make the lead
compound (13) as a novel BRD4 inhibitor motif for anti-cancer drug discovery.
Keywords:
Epigenetic readers
bromodomains
BET
BRD4
Triazoloquinoxaline
[1,2,4]triazolo[4,3-a]quinoxaline
cancer
2009 Elsevier Ltd. All rights reserved
.
Activation of transcription is modulated by acetylation of the
lysine residues on histones. Lysine acetylation, a key post
translational modification, is mediated by histone
molecule inhibitors and a number of potent small molecule BET
inhibitors have been reported up to date (Figure 1) 12,13. The rapid
progress in the development of small molecule BET inhibitors
have greatly stimulated the drug discovery efforts both from
academia and pharmaceutical industry, and have led to more than
a dozen BET inhibitors into the human clinical trials. Despite this
enormous progress, there is still a need for new chemotypes with
improved pharmacokinetics and physicochemical properties
which will further increase our understanding of the therapeutic
acetyltransferases (HATs) and results in the neutralization of
positive charge on lysine side chains. Consequently, the histone-
DNA interactions are altered and the diminished nucleosome
compaction enhances the transcriptional activity by recruiting the
transcription and chromatin remodeling factors which is
mediated by bromodomain containing proteins (BRDs)1. BRDs
act as epigenetic readers by recognizing and binding to the
acetylated lysine residues of the histone tails, thus regulating the
key processes of gene transcription and gene expression2,3.
Bromodomain and extra-terminal (BET) proteins, a subfamily of
human BRDs, is composed of four members (BRD2, BRD3,
BRD4 and BRDT) in humans. BET proteins have two similar N-
terminal bromodomains and an extra-terminal protein interaction
motif at the C-terminus. Like all other BRDs, the BET family
recognizes and binds to the acetylated lysine residues in histones
H3 and H4 4-7. BET proteins have been evidenced to be
potential of the BET inhibitors for various human diseases14-18
.
Recently, JQ1 has been reportedly related to memory deficits in
mice and the drug resistance against JQ1-motif triazoloazepines
has been described19. These issues further provide a strong
demand for the development of additional chemotypes for BET
inhibitors. The new chemotypes are thus strongly desirable as
alternatives for the clinical developments.
In the present work, we unveil the discovery of novel
[1,2,4]triazolo[4,3-a]quinoxaline aminophenyl derivatives as
potent BET inhibitors. Discovery from virtual screening and the
medicinal chemistry optimization of the hit compound to the lead
has been described. Biological evaluation of a new class of potent
and orally bioavailable BET inhibitors as anticancer agents has
been thoroughly discussed as well.
implicated in several human diseases such as MLL1-fusion
leukemia, NUT midline carcinoma, various inflammatory
diseases, and cardiovascular disorders8-11
.
The structural design of the acetyl lysine binding pocket of
BET proteins makes them attractive druggable targets for drug
discovery campaigns. The architecture of the binding pocket has
been reported to be favorable for the development of small

Figure 1. Representative BET inhibitors
Pharmaceuticals have also described a series of BET inhibitors
based on triazolopyridazine scaffold28. Putting all together, the
newly discovered triazolo quinoxaline scaffold seemed to be a
good starting point for the search of more potent and selective
BET inhibitors.
N
N
N
N
N
OH
N
N
N
N
N
S
S
(S)
O
NH
N
O
N
O
NH
O
O
II (I-BET762, GSK-525762A)
III (OTX-015, MK8658)
I (JQ1)
O
Cl
NH2
N
Cl
Cl
N
Cl
Cl
N
NH
NH2NH2.H2O, EtOH
O
O
N
N
NH2
+
N
N
N
25 oC, 16 h
95%
100 oC, 1 h
91%
N
H2N
N
Cl
N
Cl
O
(1d,1e)
N
O
(1b)
(1a)
(1c)
S
OH
1d: para
1e: meta
O
N
O
NH2
N
N
NH2
N
N
N
O
O
NH
DIPEA, iPrOH
N
N
DMAP, Boc2
O
N
N
NH2
NH
O
60 oC, 4 h
81-87%
O
N
N
H
MC, 0 oC, rt, 1 h
78-93%
N
H
IV (CPI-0610)
V (TEN-010)
VI (RVX-208)
Cl
Cl
O
(1, 2)
(1f,1g)
O
H
N
N
O
N
N
N
O
O
DIPEA, iPrOH
N
N
N
N
N
H
N
H
O
+
N
H
O
N
O
N
S
N
N
O
O
60 oC, 3 days
86%
H2N
N
O
O
Cl
N
H
O
NH
N
H
O
S
(1c)
N
H
F
F
(3)
N
(1h)
O
IX (PFI-1)
VIII (I-BET151, GSK1210151A)
VII (ABBV-075)
Scheme 1. Syntheses of compound 1-3
Compound 1-3 (Table 1) were prepared by the synthetic route
described in Scheme 1. Addition of hydrazine monohydrate to
the commercially available 2,3-dichloroquinoxaline (1a),
followed by cyclization with triethyl orthoacetate afforded 4-
chloro-1-methyl triazoloquinoxaline core (1c) in excellent yield.
Base-catalyzed coupling of para-phenylenediamine (1d) and
meta-phenylenediamine (1e) with 1c produced intermediates 1f
and 1g, respectively. Installation of tert-butyloxycarbonyl (Boc)
group to 1f and 1 g furnished compounds 1 and 2, respectively.
Base catalyzed coupling of 4-(N-Boc-aminomethyl)aniline (ih)
with key intermediate 1c afforded compound 3 in 86% yield.
All the molecular modeling was carried out using the Maestro
v10.2 (Schrödinger, Inc.)20. In an effort to discover BET
inhibitors with a novel scaffold, the docking based high
throughput virtual screening was performed using the fragment-
like database21 with more than 800,000 compounds from ZINC22
and the X-ray crystal structure of BRD4 (PDB id: 4CLB)23.
Starting the molecular modeling, four water molecules located
within the binding pocket were considered to be conserved in the
majority of bromodomains. The structural defects of the protein
were fixed by the Protein Preparation Wizard. The low-energy
3D structures of compounds were generated by the LigPrep, and
then they were docked into the binding pocket of BRD4 using the
Glide in Standard Precision (SP) mode based on a grid box of 20
× 20 × 20 Å3 centered on the co-crystallized ligand. We used
constraints such as H-bond interactions with the side-chain of
Asn140 and water molecule. The docking results were ranked
and filtered by the Glide score. The top 2000 poses were
clustered according to structural similarity, and finally 8
fragment-like motifs were selected. 230 Compounds having 8
fragment-like motifs were obtained and tested for the inhibitory
activity against BRD4. The active BET inhibitors with
[1,2,4]triazolo[4,3-a]quinoxaline scaffold were docked into the
binding pocket of each BD1 (PDB id: 4CLB) and BD2 (PDB id:
2YEM24) of BRD4 using the Glide in Extra Precision (XP) mode.
Other options were the same as mentioned above. The protein-
ligand interactions were analyzed by the Discovery Studio
Modeling Environment v4.025 and the molecular models of the
docked compounds were displayed using the PyMOL software26.
Figure 2. Discovery of hit compound 1 by virtual screening
Scheme 2. Syntheses of compounds 4-15
Compounds 4-15 (Table 1 & 2) were prepared according to
Scheme 2. Various orthoesters were used to synthesize C-1
variants of the triazoloquinoxaline with chlorine at C-4 position
(1c, 2a, 2c, 2d). In case of CF3 analogue at C-1 position of the
triazoloquinoxaline ring, triflouroacetic acid was used under
reflux conditions, followed by chlorination of the resulting
intermediate 1d with phosphorous oxychloride provided the
desired C-1 analogue 2b. 3-Aminobenzonitrile was then coupled
The virtual screening hit compound 1 shown in Figure 2,
based on a novel [1,2,4]triazolo[4,3-a]quinoxaline motif, was
validated by IC50 measurement using the AlphaScreen binding
assay29. A careful survey of the literature revealed bromosporine,
a triazolopyridazine based pan bromodomain inhibitor with very
weak cellular potency27. A patent from Constellation

with 1c and 2a-2d under acid catalyzed conditions using 0.08 M
HCl in ethoxyethanol at 100 oC for one hour to furnish various
nitriles (3a-3e). The resulting nitriles were reduced to the
corresponding amines by employing palladium catalyzed
hydrogenation (10, 4a-4d). Transformation of the resulting
benzylamines to various carbamates and amides furnished
compounds 4-9 and 11-15, respectively.
carry out SAR studies at the amino phenyl ring attached at the C-
4 position of the triazoloquinoxaline scaffold. The SAR of C-1
position of the triazoloquinoxaline scaffold was explored as well
(Table 1). The meta-analogue 2 was significantly more potent
(>50 fold) in the biochemical assay but lacked cellular potency.
Introduction of methylene linker (entry 3) boosted the
biochemical potency but still lacked the cellular potency against
Ty-82, a human NUT midline carcinoma cell line. Delightfully,
the meta-analogue 4 exhibited BRD4 inhibitory activity
comparable with the reference compounds I-BET762 and OTX-
015 in biochemical assay, and showed appreciable in vitro
cellular potency against Ty-82 and THP-1 (human leukemic cell
line). These observations confirmed that the methylene linker is
essential for the potent BET inhibition. SAR exploration at the C-
1 position of the triazoloquinoxaline revealed that methyl was the
optimal substitution at this position. All other analogues having
H, CF3, ethyl, and phenyl group at C-1 position proved to be
inactive in biochemical assay (5-8).
N
N
NH2
NH
N
N
Cl
Cl
N
N
NH2NH2.H2O, EtOH
triethyl orthoacetate
N
H
N
O
+
H2N
100 oC, 1
79%
h
25 oC, 16 h
86%
Cl
N
Cl
O
(5a)
(5b)
(5c)
(5d)
N
N
DIPEA, iPrOH
N
N
H
N
O
100 oC, 4 h
64%
N
H
O
(16)
Scheme 3. Synthesis of 3-methyl-[1,2,4]triazolo[4,3-a]pyrazine 16
Table 2. Detailed C-4 SAR of triazoloquinoxaline scaffold
The synthesis of 3-methyl-[1,2,4]triazolo[4,3-a]pyrazine 16 is
described in Scheme 3. Addition of hydrazine monohydrate to
2,3-dichloropyrazine, followed by cyclization with triethyl
orthoacetate afforded 5c. The base catalyzed coupling of 5c with
5d produced the desired compound 16 in 64% yield.
N
N
N
H
N
N
N
R
H
aBRD4 IC50
(µM)
Ty-82 IC50
(µM)
THP-1 IC50
(µM)
Table 1. C-1 and C-4 SAR of triazoloquinoxaline scaffold
R
4
0.017
7.10
3.40
9
0.16
0.35
0.53
0.20
0.068
0.097
>10
>10
>10
7.60
2.50
>10
>10
ND
ND
ND
1.60
ND
aBRD4
IC50
Ty-82
IC50
THP-1
IC50
R1
R2
(µM)
(µM)
(µM)
10
11
12
13
14
H
1
Me
6.40
>10
ND
2
3
4
5
6
7
8
Me
Me
Me
H
0.12
2.70
0.017
>10
>10
>10
7.10
ND
ND
ND
3.40
ND
15
0.64
8.60
0.39
0.067
ND
CF3
Et
>10
ND
ND
I-BET762
OTX-015
-
0.061
0.016
0.29
0.033
>10
ND
ND
-
Ph
>10
ND
ND
a: BRD4 IC50 (BD1 + BD2)
ND: Not Determined
I-BET762
OTX-015
-
-
0.061
0.016
0.39
0.067
0.29
0.033
With compound 4 as a potent BRD4 inhibitor, the drug-like
properties of this lead compound were examined. Accordingly
the liver microsomal stability and pharmacokinetic parameters of
4 were investigated and it was observed that 4 have low liver
microsomal stability in mouse and rat while acceptable stability
in human liver microsomes in vitro (Table 4 & 5). The inferior
cellular potency and sub-optimal rat pharmacokinetic parameters
of the 4 demanded for further optimization.
-
-
a: BRD4 IC50 (BD1 + BD2)
ND: Not Determined
Compound 1, discovered by virtual screening, exhibited
modest BRD4 inhibitory activity of 6.40 µM in AlphaScreen
assay. In order to design more potent derivatives, we planned to

Further SAR analysis revealed that hydrophobic group is
essential at the benzylamine end (Table 2). When the bulky and
hydrophobic tertiary butyl carbamate (Boc) group was removed,
the potency was diminished and this observation was in
agreement with the docking studies (vide infra). Small-sized
substitutions such as acetyl and ethyl carbamates were not
tolerated. After thorough investigation, it was established that
isobutyryl amide 13 was the substitution of choice. Replacement
of the tertiary butyl carbamate (Boc) with isobutyryl amide not
only boosted the cellular potency but also improved the
pharmacokinetic parameters of the triazoloquinoxaline scaffold
(Table 4 & 5).
Figure 3. Western blot of the lysates from Ty-82 cells treated with
compound 13 for 24 hr. The expression levels of c-MYC were found to be
decreased in a dose-dependent manner upon the compound treatment
Table 3. Role of quinoxaline phenyl ring
aBRD4
IC50
Ty-82
IC50
Structure
(µM)
0.068
(µM)
2.50
Recent studies of BET bromodomain inhibition identified a
significant decrease in c-Myc expression with concomitant
depletion of BRD4 at the Myc promoter in several cancer cell
lines30-32. The oncogenic transcription factor c-Myc is well
known to control a gene expression program mediating cellular
proliferation, metabolic adaptation, and survival. Thus, we can
hypothesize that compound 13 treatment should decrease c-Myc
expression in Ty-82 cell line. In fact, when the cells were treated
with compound 13 for 24 h, c-Myc expression was found to be
decreased in a dose-dependent manner as revealed by Western
blot analysis33 (Figure 3).
4
16
1.50
>10
a: BRD4 IC50 (BD1 + BD2)
Finally, the role of the quinoxalinic phenyl ring was
investigated. Changing the core from the quinoxaline to benzene-
missing pyrazine 16 resulted in a considerable loss in
biochemical potency and cellular activity (Table 3). This
observation can be attributed to the loss of the protein-
triazoloquinoxaline ligand hydrophobic interactions and was
consistent with the docking studies (vide infra).
Figure 4. Cell cycle arrest induced by compound 13 in Ty-82 cells. DNA
content histograms were recorded after treating the cells with various
concentrations of compound 13 for 24 h. The G1 population (M2) was found
to be increased upon treatment of the compound from 73% (0 µM) to 88%
(10 µM). M1: sub-G1 phase, M2: G1 phase, M3: S phase, M4: G2/M phase,
M5: miscellaneous
Table 4. In vitro liver microsomal stability of compound 4 and 13
Compound
MLM
RLM
HLM
% remaina
% remaina
% remaina
0.12
0.19
27
4
0.024
5
92
13
a: percent remaining after 30 minutes of incubation.
Table 5. Pharmacokinetic parameters of compound 4 and 13 in rat
4
4
13
parameters
IV, 2mg/kg
PO, 5
mg/kg
PO, 20
mg/kg
1.3
Tmax (hr)
-
3.42
Cmax (mg/mL)
T1/2 (hr)
-
0.34
3.69
2.11
2.39
-
4.6
0.72
4.02
4.02
0.50
0.46
-
3
AUCt (ug.hr/mL)
AUC∞ (ug.hr/mL)
CL (L/kg/hr)
Vss (L/kg)
20.6
25.3
The eukaryotic cell cycle is traditionally divided into four
phases: G1, S, G2 and M. G1 represents the gap between M phase
(M for mitosis) and S phase (S for DNA synthesis), while G2 is
the gap between S phase and M phase. It has been reported that
genetic knockdown of BRD-NUT in NUT midline carcinoma cell
lines such as Ty-82 results in terminal squamous cell
differentiation and G1 cell cycle arrest. The cytotoxic effects of
compound 13 on Ty-82 cell line were investigated by cell cycle
assays34 whether the compound could inhibit the BRD4 activity
and induce G1 cell cycle arrest in the cell line. The cells were
-
-
-
-
F(%)
20.9

treated with compound 13 for 24 h and labeled by cell-permeable
nucleic acid stain for cell cycle analysis. The histograms in
Figure 4 showed that compound 13 induced accumulation of Ty-
82 cells in G1 phase. Cell population in G1 phase exposed to
compound 13 (81% for 1 µM, 89% for 3 µM, and 88% for 10
µM) was found to be significantly higher than the control cells
(73%), strongly indicating that the cells are undergoing G1 cell
cycle arrest due to BRD4 inhibition by the compound.
Compound 13 was screened for its bromodomain selectively
using BROMOscanTM service from DiscoverX. The TREEspotTM
interaction map for compound 13 at a concentration of 500 nM is
shown in Figure 6. Compound 13 TREEspotTM interaction map
revealed that compound 13 is selective for the BET family of the
bromodomains. 13 was found to be a pan inhibitor within the
BET family inhibiting both BD1 and BD2 binding domains of all
the four members of the family. This observation was confirmed
by the similar Kd values between the two bromodomains of
BRD4 (Kd values for BRD4(BD1) and BRD4(BD2) were 27 nM
and 9 nM, respectively). No significant interaction was observed
with other members of the bromodomain family at a
Figure 5. (A) Proposed binding mode of compound 13 (green stick) to the
BD1 of BRD4: The key residues are represented by sticks, which of colors
are different according to their interactions types such as hydrophobic
(salmon) and H-bond (cyan). The different residues between binding pockets
of BD1 and BD2 are represented by white sticks. The important waters are
represented by red spheres. The H-bond and hydrophobic interactions are
drawn as dashed red and gray lines, respectively. (B) Proposed binding mode
of compound 13 (green stick) to the BD2 of BRD4: The key residues are
represented by sticks, which of colors are different according to their
interactions types such as hydrophobic (salmon) and H-bond (cyan). The
different residues between binding pockets of BD1 and BD2 are represented
by white sticks. The important waters are represented by red spheres. The H-
bond and hydrophobic interactions are drawn as dashed red and gray lines,
respectively
concentration of 500 nM35.
Figure 6. Bromodomain selectivity profiling of compound 13 with
BROMOscanTM technology (DiscoverX, Inc.). The subclass II represents
BET family. Measurements were performed at a concentration of 500 nM of
the compound. The affinity was defined with respect to a DMSO control
In conclusion, we identified novel [1,2,4]triazolo[4,3-
a]quinoxaline scaffold as BET inhibitors. An extensive SAR of
the [1,2,4]triazolo[4,3-a]quinoxaline scaffold has been described.
Compound 4 exhibited comparable biochemical potency with
IBET-762 and OTX-015 with inferior cellular potency. Further
optimization of compound 4 afforded compound 13 with
improved cellular potency and pharmacokinetic parameters. An
insight into the binding mode of compound 13 with BRD4 has
been described. Novel [1,2,4]triazolo[4,3-a]quinoxaline scaffold
along with anti-proliferative activity and drug-like properties
makes compound 13 a promising lead for anticancer drug
discovery. Efforts toward further optimization of compound 13
are underway and will be reported in due course.
An insight of the binding interaction of 13 with BRD4 was
examined by molecular docking. Compound 13 was docked with
BRD4 and the docking result revealed the key interactions
between 13 and the acetyl-lysine binding pocket of the BRD4.
Compound 13 binds to the acetyl-lysine recognition pockets of
both BD1 and BD2 of BRD4, which means that compound 13
recognizes common features of two bromodomains. The binding
modes of compound 13 to BD1 and BD2 of BRD4 and their key
interactions revealed by docking studies are shown in the Figure
5(A) & 5(B), respectively. The N3 atom on the
[1,2,4]triazolo[4,3-a]quinoxaline forms H-bond interactions with
the side-chain of Asn140 and the N2 atom interact tightly into
nearby water molecule as depicted in Figure 5(A). The amine
group at the C4 position of [1,2,4]triazolo[4,3-a]quinoxaline also
forms the H-bond interaction with the side-chain of Asn140. The
methyl group at the C1 position is located in the small
hydrophobic pocket formed by Pro82, Phe83, Val87, and Ile146.
The [1,2,4]triazolo[4,3-a]quinoxaline scaffold lies in the
hydrophobic pocket formed by Pro82, Val87, Leu92, Cys136,
and Ile146, and accepts alkyl-π interactions with the residues.
The benzene ring of amino benzene substituent at the C4 position
makes an alkyl-π interaction with Leu94. In particular, the
isobutyryl amide group on the meta-position of benzene ring
locates adjacent to the hydrophobic region named as the WPF
shelf including Trp81, Pro82, Phe83, Ile146 and Met149, which
improves its inhibitory activity against BRD4. Compound 13
binds to the acetyl-lysine recognition pocket of BD2 as well. The
binding mode and key interactions of compound 13 in the BD2 is
identical with those of compound 13 in the BD1 except an
additional interaction between the amino benzene ring at the C4
position of [1,2,4]triazolo[4,3-a]quinoxaline scaffold and His437
as shown in Figure 5(B). As a result, compound 13 recognizes
common features of two bromodomains.
Acknowledgments
The authors greatly acknowledge the generous financial support
from the Bio & Medical Technology Development Program of
the National Research Foundation funded by the Korean
government (NRF-2012M3A9C1053340), the Korea Research
Institute of Chemical Technology (KK1703-B01) and Korea
Chemical Bank (SI1707-04).
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Discovery. 2012;11:384-400.
3. Belkina AC, Denis GV. BET domain co-regulators in obesity,
−77.
inflammation and cancer. Nat Rev Cancer. 2012;12:465
4. Filippakopoulos P, Picaud S, Mangos M, et al. Histone
recognition and large-scale structural analysis of the human
bromodomain family. Cell. 2012;149(1):214-31.

5. Müller S, Knapp S. Discovery of BET bromodomain inhibitors
and their role in target validation. Med Chem Comm.
2014;5(3):288-96.
29. Measurement of the interaction between BRD4 and histone
peptide was performed by utilizing the AlphaScreen technology
provided by Perkin-elmer. Purified BRD4 proteins containing two
bromodomains were incubated with the tetraacetylated histone
peptide (Anaspec., Inc.). The final concentrations of BRD4 and
histone peptide were all 100 nM. BRD4 proteins and the histone
peptide were added to the wells and incubated on ice for 2 hrs.
Subsequently, nickel chelate acceptor beads and streptavidin
coated donor beads (PerkinElmer) were added to the mixture and
incubated for the additional 30 minutes at room temperature. The
changes in alpha signal were read in alpha mode using a Fusion
alpha-FP (Packard).
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