An optimized BRD4 inhibitor effectively eliminates NF-κB-driven triple-negative breast cancer cells

Article abstract of DOI:10.1016/j.bioorg.2021.105158

Acetylation of NF-κB's RelA subunit at lysine-310 (AcLys310) helps to maintain constitutive NF-κB activity in cancers such as triple-negative breast cancer (TNBC). Bromodomain-containing factor BRD4 binds to acetylated RelA to promote the activity of NF-κB. Hence, interfering with the acetylated RelA-BRD4 interaction is a potential strategy for treating NF-κB-driven TNBC. Here, a new compound 13a was obtained by structural optimization and modification of our previously reported compound. In comparison with the well-known BRD4 inhibitor (+)-JQ1, 13a showed more potent anticancer activity in NF-κB-active MDA-MB-231 cells. Mechanistically, 13a antagonized the protein–protein interaction (PPI) between BRD4 and acetylated RelA, decreased levels of IL-6, IL-8, Snail, Vimentin, and ZEB1, induced cell senescence and DNA damage, and weakened the adhesion, metastasis, and invasion ability of TNBC cells. Our results provide insights into avenues for the further development of potent BRD4-acetylated RelA PPI inhibitors. Moreover, our findings highlight the effectiveness and feasibility of blocking the interaction between BRD4 and acetylated RelA against NF-κB-active cancers, and of screening antagonists of this PPI.

Full text of DOI:10.1016/j.bioorg.2021.105158

Bioorganic Chemistry 114 (2021) 105158
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
An optimized BRD4 inhibitor effectively eliminates NF-κB-driven
triple-negative breast cancer cells
,
,
,
,*
Guan-Jun Yanga 1, Ying-Qi Songa 1, Wanhe Wangb, Quan-Bin Hanc *, Dik-Lung Mab
,
,d,*
Chung-Hang Leunga
a State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, 999078, Macao
b Department of Chemistry, Hong Kong Baptist University, Kowloon Tong 999077, Hong Kong, China
c School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong 999077, Hong Kong, China
d Department of Biomedical Sciences, Faculty of Health Sciences, University of Macau, 999078, Macao
A R T I C L E I N F O
A B S T R A C T
Keywords:
Acetylation of NF-κB’s RelA subunit at lysine-310 (AcLys310) helps to maintain constitutive NF-κB activity in
cancers such as triple-negative breast cancer (TNBC). Bromodomain-containing factor BRD4 binds to acetylated
RelA to promote the activity of NF-κB. Hence, interfering with the acetylated RelA-BRD4 interaction is a po-
tential strategy for treating NF-κB-driven TNBC. Here, a new compound 13a was obtained by structural opti-
mization and modification of our previously reported compound. In comparison with the well-known BRD4
inhibitor (+)-JQ1, 13a showed more potent anticancer activity in NF-κB-active MDA-MB-231 cells. Mechanis-
tically, 13a antagonized the protein–protein interaction (PPI) between BRD4 and acetylated RelA, decreased
levels of IL-6, IL-8, Snail, Vimentin, and ZEB1, induced cell senescence and DNA damage, and weakened the
adhesion, metastasis, and invasion ability of TNBC cells. Our results provide insights into avenues for the further
development of potent BRD4-acetylated RelA PPI inhibitors. Moreover, our findings highlight the effectiveness
and feasibility of blocking the interaction between BRD4 and acetylated RelA against NF-κB-active cancers, and
of screening antagonists of this PPI.
BRD4
NF-κB
Acetylated RelA
Protein-protein interaction
TNBC
1. Introduction
Nuclear factor-κB (NF-κB) plays an important role in the initiation,
development, metastasis and resistance of human cancer. Because of the
Triple-negative breast cancer (TNBC) is a subtype of heterogeneous
and clinically aggressive breast cancer without targeted agents [1],
while metastasis is a leading factor of morbidity and mortality in TNBC
patients. Current chemotherapeutic strategies for TNBC treatment are
limited by drug resistance and serious side effects such as organ damage,
which increases the suffering of TNBC patients. Therefore, there is an
urgent necessity to advance new remedies to treat this type of cancer.
Bromodomain and extra-terminal (BET) proteins are a family of
epigenetic “readers” that regulate gene activity via their ability to
recognize acetylated lysine motifs in histones as well as non-histone
substrates, including transcription factors. Bromodomain-containing
factor BRD4, a BET family member, has a key function in controlling
the gene transcription and the cell cycle, and is also linked with
tumorigenesis. BRD4 contains BD1, BD2 bromodomains, and an extra-
terminal domain. BRD4 recognizes acetylated lysine motifs in histones
or transcription factors using its BD1 and BD2 domains [2].
inflammatory microenvironment and various oncogenic mutations,
many human cancers exhibit constitutive NF-κB activity, which pro-
motes tumor cell proliferation, increases angiogenesis, inhibits
apoptosis, and also induces the epithelial-mesenchymal transition
(EMT) and distant metastasis [3]. Acetylation of NF-κB at lysine-310
(AcLys310) promotes NF-κB target gene expression [4–9] and helps to
maintain constitutive NF-κB activity in cancers [3,10,11]. BRD4 binds to
AcLys310 and functions as a coactivator regulating the transcriptional
activity of NF-κB [3]. Thus, targeting BRD4 may embody a new strategy
for treating cancers with constitutively active NF-κB [12–16].
(+)-JQ1, the first and most extensively used BET inhibitor, has been
documented to inhibit the proliferation and transformation potential of
A549 lung cancer cells and suppress the tumorigenicity of A549 cells in
severe combined immunodeficiency mice [3], (+)-JQ1 also was found to
inhibit constitutively active NF-κB in cancer cells and to relieve viral-
induced airway inflammation via disturbing the BRD4-acetylated NF-
* Corresponding authors.
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.105158
Received 23 May 2021; Received in revised form 2 July 2021; Accepted 5 July 2021
Available online 9 July 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

G.-J. Yang et al.
Bioorganic Chemistry 114 (2021) 105158
Fig. 1. Chemical structures for screening BRD4-acetylated RelA PPI inhibitors in this paper. The red parts are modified groups compared to compound N6.
Scheme 1. Synthesis of 4-methyl-2-(4-methyl-2-oxo-2H-chromen-7-yloxy) pentanoic acid 5(a & b). Reagents and conditions: (a) NaNO2/HBr (48%), 0 ◦C to RT,
Yield = 98% (2a) & 95% (2b); (b) Con. H2SO4/MeOH, Yield = 90% (3a) & 92% (3b); (c) Cs2CO3/CH3CN, Yield = 78% (4a) & 75% (4b); d) LiOH, THF/H2O (1:1), 2
h, 0 ◦C to RT, Yield = 93% (5a) & 90% (5b).
κB PPI mediated signaling [3]. CPI-061, a BET antagonist with prefer-
ence for the first bromodomain, has advanced into Phase II human
clinical trials for myelofibrosis [17]. RVX-208, a BET antagonist selec-
tive for the second bromodomain, has entered into Phase III human
clinical trials for cardiovascular diseases [18,19].
value of 0.062 μM and 0.081 μM, respectively. Mechanistically, com-
pound 13a (Fig. 1D) blocked the interaction between BRD4 and RelA,
leading to the deactivation of NF-κB and reducing the expression of NF-
κB target genes. Compound 13a is an attractive scaffold for the further
development of anticancer agents that can suppress metastasis and
induce senescence in NF-κB-driven cancer cells.
Our group have previously identified two single-domain BRD4 in-
hibitors with good anti-melanoma activity [20,21]. However, while a
number of BRD4 inhibitors are under preclinical or clinical trials
[22,23], no BRD4 inhibitor has yet been approved in clinic. Most re-
ported BRD4 inhibitors are pan-BET inhibitors that lack selectivity
[22,24]. Thus, there is an urgent need to develop a more novelty,
effective and selective BRD4 inhibitor.
The key reaction intermediates 4-methyl-2-(4-methyl-2-oxo-2H-
chromen-7- yloxy) pentanoic acid (5) and (S)-tert-butyl 3-(2-amino-3-
methoxy-3-oxopropyl)-5-hydroxy-1H-indole-1-carboxylate (10) were
synthesized in four steps from enantiopure isoleucine (1a and 1b)
(Scheme 1) and (S)-tryptophan 6, respectively (Scheme 2).
Compounds 5 were then reacted separately with compound 10 to
generate the diastereomers 13 (Scheme 3). Alternatively, compound 10
could be reacted first with enantiopure 14 to introduce a guanidine
group, before subsequent reaction with 5 to generate diastereomers 17,
respectively (Scheme 4). All the synthetic reaction intermediates and
targeted compounds were characterized using 1H NMR, 13C NMR, high-
resolution mass spectrometry (HRMS), and elemental analysis
(Fig. S1–S8).
2. Results and discussion
(+)-JQ1 (Fig. 1A–B) exhibits excellent in vitro and in cellulo activity
against BET proteins [3]. Based on the X-ray crystal structure of BRD4
BD1 with (+)-JQ1 (PDB: 3MXF), our previous study identified (R)-3-(5-
hydroxy-1H-indol-3-yl)-2-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)
acetamidopropanoic acid (N6) as a BRD4 inhibitor (Fig. 1C) by virtual
screening and hit validation [21]. This compound exhibited potent
inhibitory activity against BD1 and BD2 domain of BRD4 with an IC50
Enantiomers of chiral drugs can exhibit marked differences in bio-
logical activities such as pharmacokinetics, pharmacology, metabolism,
and toxicology [25]. The enantiomers of JQ1 also exhibited different
2

G.-J. Yang et al.
Bioorganic Chemistry 114 (2021) 105158
Scheme 2. Synthesis of (S)-tert-butyl 3-(2-amino-3-methoxy-3-oxopropyl)-5-hydroxy-1H-indole-1-carboxylate 10. Reagents and conditions: (a) SOCl2 /MeOH, 0 ◦C
to RT, Yield = 98%; (b) Fmoc-Cl/10% solution of Na2CO3, 1,4-dioxane, Yield = 95%; (c) Boc2O, DMAP (cat)/CH3CN, Yield = 75%; (d) DBU (1 eq), DCM, RT, Yield =
quantitative.
Scheme 3. Synthesis of compound 13 (a & b). Reagents and conditions: (a) EDCI/HOBt /DCM, 0 ◦C to RT, Yield = 78% (11a) & 80% (11b); (b) LiOH, THF/H2O
(1:1), 2 h, 0 ◦C to RT, Yield = 94% (12a) & 97% (12b); (c) TFA, DCM Yield = 93% (13a) & 95% (13b).
activity as BET inhibitors [3,26,27]. Hence, we designed two pairs of
enantiomers based on the structure of N6, a BRD4 inhibitor identified in
our previous work [21]. Compared with N6, compounds 13a/13b
contains an additional isobutyl group to increase hydrophobicity, which
could potentially improve engagement of hydrophobic pockets in BRD4.
Meanwhile, an arginine residue was added in diastereomers 17a/17b
(Fig. 1D and E) to increase the number of hydrogen bonds that could
potentially be formed with the target.
h treatment, the levels of downstream genes of the BRD4-acetylated
RelA PPI were detected using real-time quantitative PCR (Fig. 2A–F),
enzyme-linked immunosorbent assay (Fig. 2G, H), and Western blotting
(Fig. 2I). Compound 13a exhibited the highest BRD4 inhibitory activity
at a concentration of 3 μM, as indicated by decreased transcriptional and
translational levels of downstream genes (IL-6, IL-8, Snail, Vimentin,
and ZEB1) of BRD4-NF-κB signaling, with little effect on c-Myc, a gene
regulated by the interaction BRD4 and its histone substrates.
The inhibitory activity of the compounds against BRD4 in vitro was
assessed by a TR-FRET assay that measures the binding of BRD4 to its
acetylated substrates. The results showed that all the derivatives
generally exhibited better inhibitory activity against BRD4 than N6
(Table 1). Compound 13a was the top dual-domain BRD4, with IC50
The cytotoxicity of five candidate BRD4 inhibitors against four breast
cancer cell lines (MDA-MB-231, MCF-10A, T47D, and MCF-7) and a
human normal liver cell line (LO2) was also tested (Table 1). 13a
exhibited the greatest cytotoxicity against MDA-MB-231 cells (IC50
=
0.36 μM), which constitutively express NF-κB. For the other cell lines
values of 0.023
μ
M and 0.016
μ
M for BRD4 BD1 and BD2, respectively.
which do not have constitutively active NF-κB, IC50 values were
increased by at least 22.5-fold (Table 1). Moreover, as shown using a
luciferase reporter assay, 13a significantly reduced NF-κB-driven ac-
tivity in a dose-dependent fashion in MDA-MB-231 cells and was
significantly more potent than (+)-JQ1 at the same concentration
Interestingly, just as with JQ1, the diastereomers of each pair also
exhibited different activity to each other.
Then, the in cellulo activity of five hit compounds (including N6) was
further investigated in a human TNBC cell line (MDA-MB-231). After 24
3

G.-J. Yang et al.
Bioorganic Chemistry 114 (2021) 105158
Scheme 4. Synthesis of compound 17 (a and b). Reagents and conditions: (a) EDCI/HOBt /DCM, 0 ◦C to RT, Yield = 68% (15 Fmoc protected); (b) DBU (1 eq), DCM,
RT, Yield = quantitative for (15 amine); (c) EDCI/HOBt /DCM, 0 ◦C to RT, Yield = 75% (16a) & 80% (16b); (d) LiOH, THF/H2O (1:1), 2 h, 0 ◦C to RT, Yield = 95%
(16a) & 93% (16b); (e) TFA, DCM Yield = 70% (17a) & 73% (13a).
Table 1
The 50% inhibiting concentration (IC50, μM) of N6 and its derivatives in in vitro and in cellulo. The inhibitory activity of our compounds on the binding ability of the two
BDs of BRD4 with their acetylated substrates were detected by TR-FRET assay in vitro, while the cytotoxicity of these five compounds were analyzed by MTT assay.
Compounds (
μ
M)
BRD4 BD1
BRD4 BD2
MDA-MB-231
MCF-7
MCF-10A
T47D
LO2
N6
0.062
0.023
0.054
0.073
0.032
0.081
0.016
0.013
0.049
0.014
4.68
0.36
8.73
6.15
9.26
1.21
8.16
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
13a
13b
17a
17b
(Fig. S1). The cytokines IL-8 and IL-6 directly regulated by NF-κB
signaling induce a self- and cross-reinforcing inflammatory environment
which promotes tumorigenesis in breast cancer cells [28]. Interestingly,
13a not only lead to cytotoxicity but also impaired the release of IL-8
and IL-6 (Fig. 2G and 2H), which suggested that 13a may, at least in
part, mediate the cytotoxicity via BRD4-NF-κB signaling.
chromatin immunoprecipitation assay and RT-qPCR were conducted to
test the ability of 13a interfere with the binding activity of BRD4 to
promoters of its downstream genes (primers in Supplementary
Table S1). The results showed that 13a weakened the binding ability
between BRD4 and the promoters of the IL-6, IL-8, Snail, Vimentin, and
ZEB1 (Fig. 4B–F), thus downregulating those genes at both the tran-
scriptional and translational levels (Fig. 4G–I and K–O). To further
explore the effect of acetylated site of RelA on the interaction between
BRD4 and acetylated RelA, p300 protein, an acetyltransferase that
acetylates RelA at lysine-310 [29–31], was knocked down. The results
indicated that both knocking down p300 and 13a treatment the BRD4-
acetylated RelA PPI, and moreover, p300 knockdown impaired the
inhibitory effect of 13a his PPI (Fig. 4J). Taken together, these data
suggested that 13a exhibited its anticancer activity via blocking the
interaction between BRD4 and its substrate acetylated RelA K310Ac,
resulting in the transcriptional repression of downstream genes (IL-6, IL-
8, Snail, Vimentin, and ZEB1).
To further investigate if BRD4 is directly targeted by 13a in MDA-
MB-231 cells, the cellular thermal shift assay was performed (Fig. 3A).
13a stabilized BRD4 in cell lysates (ΔTm: 5.7 ◦C) relative to DMSO-
treated controls (Fig. 3B), while it had little effect on the thermal sta-
bility of acetylated RelA (Fig. 3C). This suggested that 13a directly and
selectively engaged BRD4 (rather than its partner acetylated RelA) in
vitro and in cellulo to inhibit NF-κB signaling. BRD4 binds to acetylated
lysine-310 of RelA to function as a coactivator of NF-κB signaling, and
this helps to maintain constitutive NF-κB activity in cancers [3–11]. The
mode of action of 13a was studied using co-immunoprecipitation ex-
periments. The results revealed that 13a blocked the interaction be-
tween BRD4 and acetylated RelA without affecting BRD4 and acetylated
RelA levels in MDA-MB-231 cells (Fig. 4A). Meanwhile, the levels of
acetylated RelA were sharply decreased due to degradation of nuclear
RelA by the ubiquitin system (Fig. S4), which is in accordance with a
previous report [3].
BRD4 is overexpressed in MDA-MB-231 cells, contributing to
constitutive NF-κB activity and high metastatic potential [32–34].
Upregulation of Snail, Vimentin, and ZEB1 genes are hallmark features
of the epithelial-mesenchymal transition, which maintains the metas-
tasis phenotype of epithelial cancer cells [18]. To further investigate the
anticancer mechanism of 13a, its effects on the adhesion, metastasis,
and invasion abilities of MDA-MB-231 cells were investigated. The re-
sults showed that 13a weaken the adhesion (Fig. S2A), metastasis
The BRD4-acetylated RelA PPI increases cellular adhesion, metas-
tasis, and invasion by occupying the promoters of IL-6, IL-8, Snail,
Vimentin, and ZEB1, and increasing their expression [3–8]. The
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G.-J. Yang et al.
Bioorganic Chemistry 114 (2021) 105158
Fig. 2. Compound 13a selectively inhibits BRD4-NF-κB signaling. (A) The effect of the five hit compounds and JQ1 on gene levels of BRD4-NF-κB signaling and c-
Myc level was determined by RT-qPCR assay. (B-F) The effect of the five hit compounds and JQ1 on downstream gene levels of BRD4-NF-κB signaling was determined
by RT-qPCR assay. (G-I ) The effect of the five hit compounds and JQ1 on downstream protein levels of BRD4-NF-κB signaling by ELISA and Western blotting.
(Fig. S2B and S2C), and invasion (Fig. S2D and S2E) abilities of MDA-
MB-231 cells. BRD4 also insulates chromatin from DNA damage
signaling via promoting the secretion of cytokines IL-6 and IL-8 [30,31].
Hence, the effect of 13a on DNA damage was evaluated using immu-
nofluorescence and western blotting. The results showed that both 13a
and JQ1 induced DNA damage in MDA-MB-231 cells as indicated by the
accumulation of γ-H2AX based on immunofluorescence assay (Fig. 5A
and B) and western blotting (Fig. 5C), a biomarker positively correlated
with DNA damage [35]. Importantly, 13a showed more potent effects at
inducing DNA damage than JQ1 at the same concentration. At the same
time, DNA damage often triggers senescence in cancer cells [36], while
BRD4 relieves cell senescence via downregulating p21 levels in head and
neck cancer [37].
MB-231 cells. We anticipate that the discovery of 13a may provide a
novel molecular scaffold for the future development of more efficacious
and selective BRD4 inhibitors or theranostic probes against NF-κB-
driven cancers.
4. Experimental section
4.1. Reagents and cell lines
Normal cell lines (HEK293T and LO2) and breast cancer cell lines
(MDA-MB-231, MCF-10A, T47D, and MCF-7 cells) used in this paper
were cultured at 37 ◦C with 5% CO2 in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/
streptomycin. Compounds N6, 1a, 1b, 6, 14, and (+)-JQ1 were pur-
chased from J&K Scientific Ltd. (Hong Kong, China) and dissolved in
DMSO. Two TR-FRET assay kits for single bromodomain of BRD4 were
bought from Cayman Chemical (Carlsbad, CA, USA). The resources of all
the antibodies and related reagents used here were shown in this paper.
In this study, 13a induced cell senescence in MDA-MB-231 cells in a
dose-dependent fashion, as revealed by an increase of β-galactosidase
(SA-β-gal), a senescence-associated biomarker (Fig. S3A and B), and the
upregulation of p21 (Fig. S3C). Taken together, the anticancer activity of
13a could be associated, at least in part, with its ability to inhibit the
BRD4-acetylated RelA PPI thus leading to cell senescence.
4.2. Molecular modeling
3. Conclusion
The initial model of BRD4 BD1 in complex with (+)-JQ1 and BRD4
BD2 in complex with A-1457066 were derived from the X-ray crystal
structures (PDB: 3MXF and 5UVV, respectively) using the molecular
conversion procedure implemented in the ICM-pro 3.6-1d program
(Molsoft, San Diego, CA, USA). The molecular conversion procedure and
high throughput molecular docking were administrated as previous
In summary, we have identified compound 13a as a new BRD4 in-
hibitor. Compound 13a inhibited NF-κB signaling via blocking the
interaction between BRD4 and acetylated RelA, thus impeding the
transcriptions of downstream genes. Moreover, compound 13a induced
senescence, and impaired adhesion, metastasis, and invasion of MDA-
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Bioorganic Chemistry 114 (2021) 105158
Fig. 3. Compound 13a increases the thermal stability of BRD4 in cell lysates. (A) Lysates of MDA-MB-231 cells were treated with 13a at 3.0
μM. Proteins in the
soluble fraction were visualized by Western blotting (B–C). Densitometry analysis of BRD4 and Rel-Ac content. Data are represented as mean ± SD.
reports [38,39].
a control for normalization. Each sample was conducted in triplicate in a
BeyoFast™ SYBR Green qPCR Mix (Beyotime, Shanghai, China). The
reaction procedures and results were analyzed following previous
methods [40–42], and the related primers for these genes were docu-
mented in Table S1.
4.3. Time resolved fluorescence resonance energy transfer (TR-FRET)
The effect of tested compounds on the interaction between single
domain of BRD4 and its acetylated ligand peptides was detected using
kits according to the manufacturer’s protocols (Cayman Chemical,
Carlsbad, CA, USA) [20]. The results were read using a multimode plate
reader (PerkinElmer, Waltham, MA, USA) via detecting the dual emis-
sions at 620 nm and 670 nm using an excitation at 340 nm. Data analysis
was conducted using the TR-FRET ratio (670 nm emission/620 nm
emission).
4.7. Co-immunoprecipitation (co-IP) assay
The co-IP assay was performed as previous procedures [40,43].
Briefly, 1 × 106 MDA-MB-231 cells were plated into a 6-well plate and
incubated for 24 h with the indicated concentrations of 13a. Cells were
lysed and the protein samples were extracted. The protein concentration
of each sample was measured using the Bradford method. 30
μ
g total
protein for each sample was incubated overnight with 10
μL anti-BRD4
4.4. Cellular thermal shift assay
(Abcam, ab128874, Cambridge, MA, USA) magnetic beads according to
the manufacturer’s procedures. The complex was washed five times to
remove non-specific and non-cross-linked antibodies. Then, the precip-
itated proteins were separated by SDS-PAGE and detected by western
blotting with anti-H4K5Ac (Abcam, ab51997, USA) anti-Twist1 (Absin
abs136367, Shanghai, China), and anti-Acetylated-lysine antibody (CST
9441, Danvers, MA, USA).
The cellular thermal shift assay was conducted to monitor BRD4
engagement in MDA-MB-231 cells using BRD4 antibody as described
previously [40].
4.5. Western blotting analysis
MDA-MB-231 cells were lysed using RIPA lysis buffer containing 1%
PMSF (100 mM). 20 mg of extracted total cellular proteins were loaded
into each well and separated using SDS-polyacrylamide gel electro-
phoresis. After electroblotting, the membranes were successively incu-
bated with corresponding primary antibodies and horseradish
peroxidase-conjugated second antibody (1:1000), and developed with
the enhanced chemiluminescent method. GAPDH protein was used as an
international reference protein.
4.8. Enzyme-linked immunosorbent assay (ELISA)
The release of cytokines IL-8 and IL-6 in MDA-MB-231 cells after
treatment with compound was detected by ELISA using human IL-6
ELISA kit and human IL-8 ELISA kit (NeoBioscience, Shenzhen, China)
[33,34].
4.9. Gene knockdown assay
4.6. Real-time quantitative PCR assay
MDA-MB-231 cells were seeded in 10 cm dishes at 80% confluence in
DMEM medium with 10% fetal calf serum and a mixture of penicillin
RT-qPCR assay was conducted in a ViiA 7™ Real-Time PCR system
(Applied Biosystems, Foster City, CA, USA). After extraction xfrom
MDA-MB-231 cells using RNAiso Plus reagents (TaKaRa, Dalian, China),
the total RNA transcribed into the cDNA using a First-stand cDNA
Synthesis kit (Beyotime, Shanghai, China). The transcriptional changes
of target genes were detected and the housekeeping GAPDH was used as
and streptomycin (1%) for 24 h. After 5 min preincubation with 250 μL
serum-free medium, Lipo3000 reagent, BRD4, p300 or control siRNA
were gently mixed and incubated at room temperature for 20 min. Then,
the growth medium was removed from the wells and replaced with 0.5
mL of fresh medium. Then, the knockdown mixture was added to each
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G.-J. Yang et al.
Bioorganic Chemistry 114 (2021) 105158
Fig. 4. 13a impairs expression of downstream gene levels of NF-κB signaling by blocking the interaction between BRD4 and acetylated RelA in MDA-MB-231 cells.
Fig. 5. The effect of 13a on DNA damage of MDA-MB-231 cells. (A, B) The effect of 13a on DNA damage in the MDA-MB-231 cells. (C) The effect of 13a on γ-H2AX
levels. Data are represented as mean ± SD. * p < 0.05, * p < 0.01 versus DMSO-treated groups.
7

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Bioorganic Chemistry 114 (2021) 105158
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incubator before further study.
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CRediT authorship contribution statement
Guan-Jun Yang: Investigation, Validation, Project administration,
Data curation, Writing - review & editing. Ying-Qi Song: Writing -
original draft, Project administration. Wanhe Wang: Project adminis-
tration. Quan-Bin Han: Writing - review & editing. Dik-Lung Ma:
Compound synthesis, Funding acquisition, Resources, conceptuation,
methods, Writing - review & editing. Chung-Hang Leung: Supervision,
Funding acquisition, Resources, conceptuation.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgments
This work is supported by Hong Kong Baptist University, the Health
and Medical Research Fund, National Natural Science Foundation of
China (22077109 and 21775131), Hong Kong Baptist university Cen-
tury Club Sponsorship Scheme 2020, Interdisciplinary Research
Matching Scheme (RC-IRMS/15-16/03), the Science and Technology
Development Fund, Macau SAR, China (File no. 0072/2018/A2 and
0007/2020/A1); SKL-QRCM(UM)-2020-2022; the University of Macau,
China (MYRG2019–00002–ICMS), and the Science and Technology
Project of Shenzhen (No. JCYJ20160531193812867).
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105158.
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