Chemical Proteomic Profiling of Bromodomains Enables the Wide-Spectrum Evaluation of Bromodomain Inhibitors in Living Cells

Article abstract of DOI:10.1021/jacs.9b02738

Bromodomains, epigenetic "readers" of lysine acetylation marks, exist in different nuclear proteins with diverse biological functions in chromatin biology. Malfunctions of bromodomains are associated with the pathogenesis of human diseases, such as cancer. Bromodomains have therefore emerged as therapeutic targets for drug discovery. Given the high structural similarity of bromodomains, a critical step in the development of bromodomain inhibitors is the evaluation of their selectivity to avoid off-target effects. While numerous bromodomain inhibitors have been identified, new methods to evaluate the inhibitor selectivity toward endogenous bromodomains in living cells remain needed. Here we report the development of a photoaffinity probe, photo-bromosporine (photo-BS), that enables the wide-spectrum profiling of bromodomain inhibitors in living cells. Photo-BS allowed light-induced cross-linking of recombinant bromodomains and endogenous bromodomain-containing proteins (BCPs) both in vitro and in living cells. The photo-BS-induced labeling of the bromodomains was selectively competed by the corresponding bromodomain inhibitors. Proteomics analysis revealed that photo-BS captured 28 out of the 42 known BCPs from the living cells. Assessment of the two bromodomain inhibitors, bromosporine and GSK6853, resulted in the identification of known as well as previously uncharacterized bromodomain targets. Collectively, we established a chemical proteomics platform to comprehensively evaluate bromodomain inhibitors in terms of their selectivity against endogenous BCPs in living cells.

Full text of DOI:10.1021/jacs.9b02738

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Article
Chemical Proteomic Profiling of Bromodomains Enables the Wide-
Spectrum Evaluation of Bromodomain Inhibitors in Living Cells
Xin Li, Yizhe Wu, Gaofei Tian, Yixiang Jiang, Zheng Liu, Xianbin Meng,
Xiucong Bao, Ling Feng, Hongyan Sun, Haiteng Deng, and Xiang D. Li
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 27 Jun 2019
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Chemical Proteomic Profiling of Bromodomains Enables the Wide-
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†,#
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‡
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Xin Li, Yizhe Wu, Gaofei Tian, Yixiang Jiang, Zheng Liu, Xianbin Meng, Xiucong Bao, Ling
Feng,§ Hongyan Sun,§,⊥ Haiteng Deng, and Xiang David Li
,⊥
‡
*,†
†Departments of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China
‡Center of Biomedical Analysis, Tsinghua University, Beijing 100084, China
§Department of Chemistry and Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong,
83 Tat Chee Avenue, Kowloon, Hong Kong, China
⊥Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of
Hong Kong, Shenzhen 518057, China
Supporting Information Placeholder
bromodomains of BET (bromo and extraterminal domain) family
ABSTRACT: Bromodomain, an epigenetic ‘reader’ of lysine
members (e.g., BRD4), representing a hallmark of targeting the
bromodomains as a novel therapeutic strategy. The development of
bromodomain inhibitors in the last decade has provided useful tools
to probe the biological significances of these epigenetic modules in
the maintenance of normal cell physiology and the pathogenesis of
diseases. A set of bromodomain inhibitors are even in different
stages of clinical trials for the treatment of cancer, atherosclerosis,
acetylation marks, exists in different nuclear proteins with diverse
biological functions in chromatin biology. Malfunctions of
bromodomains are associated with the pathogenesis of human
diseases, such as cancer. The bromodomains have therefore
emerged as therapeutic targets for drug discovery. Given the high
structural similarity of the bromodomains, a critical step in the
development of bromodomain inhibitors is the evaluation of their
selectivity to avoid off-target effects. While numerous
bromodomain inhibitors have been identified, new methods to
evaluate the inhibitor selectivity toward endogenous
bromodomains in living cells remain needed. Here we report the
development of a photoaffinity probe, photo-BS, that enables the
wide-spectrum profiling of bromodomain inhibitors in living cells.
Photo-BS allowed light-induced cross-linking of recombinant
bromodomains and endogenous bromodomain-containing proteins
(BCPs) both in vitro and in living cells. The photo-BS-induced
labeling of the bromodomains was selectively competed by the
corresponding bromodomain inhibitors. Proteomics analysis
revealed that photo-BS captured 28 out of the 42 known BCPs from
the living cells. Assessment of the two bromodomain inhibitors,
bromosporine and GSK6853, resulted in the identification of
known as well as previously uncharacterized bromodomain targets.
Collectively, we established a chemical proteomics platform to
comprehensively evaluate bromodomain inhibitors in terms of their
selectivity against endogenous BCPs in living cells.
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as well as diabetes.
The human genome encodes 61 bromodomains that can be
divided into 8 subfamilies based on their sequence and structure
similarity, which exist in 46 bromodomain-containing proteins
2
(BCPs). While varying in primary sequences, the bromodomains
share a high structural similarity. It is therefore important to
examine the selectivity of an inhibitor toward its authentic target
against other bromodomains to avoid off-target effects. A variety
of approaches have been applied to assess the selectivity of
inhibitors using recombinant bromodomains in vitro, such as
differential scanning fluorimetry (DSF), surface plasmon
resonance (SPR), bio-layer interferometry (BLI), AlphaScreen, and
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fluorescence polarization (FP).
However, the results obtained
from the in-vitro assays can be different from the real situations of
how the inhibitors engage with the bromodomains in the cellular
environment. The BCPs are usually multi-domain proteins and
function as members of various nuclear complexes. The
conformation of a bromodomain can be altered by the interactions
with the adjacent domains in a same BCP or with other
biomolecules. In turn, the inhibitor engagement with the
bromodomain can also be affected.
INTRODUCTION
Fluorescence recovery after photobleaching (FRAP)14 and
Bromodomains are epigenetic ‘readers’ of lysine acetylation (Kac)
on histones and non-histone proteins. These Kac-recognition
Nanoluc luciferase-based bioluminescence resonance energy
transfer (NanoBRET) are two commonly used methods to
1-2
15
motifs, in collaboration with other epigenetic regulators, play
important roles in chromatin biology and the regulation of gene
expression. The bromodomains have emerged as ‘hotspots’ for
drug discovery as their malfunctions often lead to human diseases
investigate the interactions of bromodomain inhibitors with their
targets in living cells. Although working in the cellular context,
these two methods are still dependent on the recombinant
bromodomains instead of the endogenous ones, as they require
overexpression of the bromodomains of interest in fusion with a
fluorescent protein or luciferase for the detection. In addition, the
set-up of FRAP or NanoBRET normally allows the test of only one
bromodomain at one time. These methods are suitable for the
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such as cancer. The discovery of small molecular ligands that
block the bromodomain of KAT2B (or PCAF) marked the start of
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bromodomain inhibitor development. Later endeavors led to the
identification of JQ1 and I-BET7629 that inhibit the
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validation of the target engagement of bromodomain inhibitors
The synthesis of photo-BS followed a 13-step route (Scheme S1)
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rather than to examine inhibitor selectivity. The methods that allow
the assessment of bromodomain inhibitors against multiple
endogenous bromodomains in living cells are of great value for the
development of bromodomain inhibitors. Toward this end, attempts
to profile endogenous bromodomains by developing chemical
proteomics approaches has been reported. For example, a series of
BET bromodomain inhibitor GW841819X (a JQ1 analog)-based
photoaffinity probes were employed to examine the proteome-wide
with the overall yield of 0.3%.
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target selectivity of the inhibitor
, while the high specificity of
GW841819X toward the BET bromodomains prevent the
applications of these probes in the evaluation of other
bromodomains. Recently, an activity-based probe has been
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reported to covalently capture bromodomains. However, this
probe could only enrich less than 10 BCPs from cell lysates in vitro,
which limits its scope of application in broad-spectrum evaluation
of bromodomain inhibitors. More importantly, the probe has not
been shown suitable for the use in living cells. Here we describe
the development of a useful chemical proteomics approach, which
combines the use of a photoaffinity probe that promiscuously
captures endogenous bromodomains and the stable isotope labeling
with amino acids in cell culture (SILAC)-based quantitative mass
spectrometry, to profile bromodomains and evaluate their inhibitor
selectivity in living cells.
RESULTS
Development of
a
photoaffinity probe to profile
bromodomains. A wide-spectrum evaluation of bromodomain
inhibitors requires a tool to label as many as possible, if not all,
cellular bromodomains. Recently, a series of tricyclic or dicyclic
triazolo fused ring derivatives were developed to target a broad
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range of bromodomains.
Structural optimization has led to the
Figure 1. Design of photo-BS as a photoaffinity probe for
bromodomain profiling. (A) Structure of bromosporine and photo-
BS. (B) Superimposition of the structures of BRD4(1), BRPF1,
BRD9, and TAF1L(2) in complex with bromosporine.
Bromosporine molecules are depicted as sticks and proteins are
shown as ribbons in indicated colors. (CF) Surface views of
bromosporine bound to BRD4(1) (C), BRPF1 (D), BRD9 (E), and
TAF1L(2) (F) to show that the solvent-exposed C8 carbamate ethyl
tail. Red arrows indicate potential steric hindrance for further
derivatization. Proteins are colored in light grey. Bromosporine is
colored in salmon. Figures were created using Pymol.
identification of bromosporine (Figures 1A and S1) as a non-
selective inhibitor with nanomolar to micromolar binding affinity
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toward a collection of bromodomains covering all 8 subfamilies.
Considering its high target promiscuity, we reasoned that
bromosporine could be a good prototype to design chemical probe
for capturing the bromodomains. In our design, bromosporine was
armed by a bifunctional moiety carrying both a diazirine group (for
photo-cross-linking) and a terminal alkyne (for bioorthogonal
reaction). We expected that such derivatization could facilitate the
covalent labeling of the bromodomains upon UV activation and
subsequent conjugation of the target proteins to fluorescent or
affinity tags for visualization or enrichment, respectively. In
bromosporine, the C8 carbamate and C3’ sulfonamide are two
positions that the bifunctional moiety can be attached to without
significantly changing the molecule skeleton or complicating the
synthesis of the probe (Figure 1A). Importantly, the incorporation
of the moiety should not interfere with the inhibitor-bromodomain
interactions. By analyzing the reported co-crystal structures of four
bromodomains, BRD4(1) (PDB code: 5IGK), BRPF1 (PDB code:
Photo-BS labels bromodomains in vitro. We first examined if
photo-BS can label the bromodomains. 21 recombinant
bromodomains in all the 8 subfamilies, including ASH1L, ATAD2,
BAZ2B, BRD2(2), BRD3(2), BRD4(1), BRD4(2), BRD9,
BRWD1(2), CECR2, CREBBP, EP300, KAT2A, KAT2B,
PBRM1(1), PBRM1(5), PBRM1(6), TAF1(2), TRIM24, TRIM28,
and ZMYND8, were respectively incubated with increasing
concentrations of photo-BS. After UV irradiation, the probe-
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C7N), BRD9 (PDB code: 5IGM), and TAF1L(2) (PDBcode:
IGL), in complex with bromosporine, we found that all the
labeled proteins were conjugated to rhodamine-azide (Rho-N ) via
‘click chemistry’, followed by SDS-PAGE and in-gel fluorescence
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bromodomains accommodated bromosporine in a similar pattern
Figure 1B), in which the dicyclic heteroaromatic core of
(
scanning. We observed a dose-dependent labeling of all the 21
bromodomains by photo-BS (Figures 2A and S2). The labeling
efficiencies of the tested bromodomains by photo-BS seemed to
follow a similar trend with the binding affinities of the parent
compound bromosporine toward the bromodomains , as the more
tightly bromosporine bound to a bromodomain, the higher
efficiency photo-BS labeled the bromodomain (Figure 2B). In
addition, the presence of excessive bromosporine suppressed the
photo-BS-mediated labeling of all tested bromodomains (Figure
3A), indicating that the labeling was bromosporine-bromodomain
interaction-dependent instead of nonspecific photo-cross-linking.
bromosporine was buried deeply in the binding pockets, the C8
carbamate and C3’ sulfonamide were located at the pockets’ entry
regions (Figures 1C1F). While the C3’ sulfonamide pointed to the
open space in the BRPF1 and TAF1L(2) complexes, there could be
steric clash if a larger group is introduced to this position in the
BRD4(1) and BRD9 complexes. In contrast, the ethyl tail of
bromosporine at the C8 carbamate was solvent accessible in all the
four complexes, and thereby suitable to introduce substituents.
Guided by these analyses, we designed a photoaffinity probe called
photo-bromosporine (photo-BS, Figure 1A) by replacing the ethyl
of the C8 carbamate in bromosporine with the bifunctional moiety.
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the presence of three bromodomain inhibitors, bromosporine ,
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JQ1 , and PFI-1 (Figure S1), respectively. As expected,
bromosporine efficiently blocked the labeling of its known targets
BRD4(1), BRD4(2), CECR2, and TAF1(2), while little
competition was observed on ASH1L, a poor binder of
bromosporine. The JQ1 and PFI-1 treatment led to selective signal
reduction on both BRD4(1) and BRD4(2), which were
characterized with high affinities to these two inhibitors, while the
fluorescence intensities of the rest three bromodomains that were
inert to JQ1 and PFI-1 left unaffected (Figure 3B). Furthermore, we
varied the concentration of PFI-1 and JQ1 in the photo-BS
mediated cross-linking reactions of BRD4(1) and BRD4(2),
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respectively. From the competition curves (Figures 3C and S3), the
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apparent K
i
of PFI-1 and JQ1 toward BRD4(1) and BRD4(2) were
in line with the reported their corresponding dissociation constants
(Figure 3D), indicating that photo-BS could quantitatively
differentiate the inhibitory activities of a compound against
different bromodomains.
Photo-BS labels endogenous BCPs in living cells. To examine
whether photo-BS could be used to label bromodomains in
complex proteomes, we added the recombinant CECR2 and
BRD4(1) into the lysates of HEK293T cells and performed photo-
cross-linking reactions using photo-BS. As shown in Figure 4A,
both bromodomains were robustly labeled by the probe and the
cross-linking efficiencies were comparable to that using the single
proteins. Remarkably, the labeling of CECR2 and BRD4(1) in the
lysates was also selectively competed by their corresponding
inhibitors, suggesting that photo-BS-based cross-linking assay
could be used to evaluate bromodomain inhibitors in complex
proteome samples.
Encouraged by the in-vitro studies, we next sought to explore the
application of photo-BS in living cells. HEK293T cells were
incubated with the different concentrations of photo-BS. After UV
irradiation, the cells were lysed, and the lysates were ‘clicked’
with Rho-N
linking was dose-dependent (Figure 4B) and saturated between
020 M (Figure S4). We further showed that the co-incubation
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. In-gel fluorescence scanning revealed that the cross-
1
with bromosporine greatly inhibited the labeling (Figure 4C). On
the other hand, when we used photo-BS to label the lysate derived
from the HEK293T cells, the fluorescence intensity of the labeled
proteins kept increasing and was not saturated even at the
concentration of 50 M of photo-BS (Figure S5). In addition, we
noticed the different protein labeling patterns between the lysate
and the live cell samples (Figure 4D), suggesting that the target
engagement profiles of photo-BS in cells were significantly
different from that in vitro.
We next asked if the endogenous BCPs could be labeled by
photo-BS in the living cells; and more importantly, if the labeling
could be selectively competed by the bromodomain inhibitors. To
this end, HEK293T cells were pre-incubated with or without the
Figure 2. Photo-BS labels bromodomains in vitro. (A) In-gel
fluorescence and concentration dependence curves of photo-BS-
induced photo-cross-linking toward representative recombinant
bromodomains. For all the photo-cross-linking experiments,
protein concentration used was 5 g/mL. After UV irradiation, the
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bromodomain inhibitors bromosporine, JQ1, and GSK2801
(Figure S1), respectively. Photo-BS was then added to conduct
photo-cross-linking in living cells. After lyzing the cells, the photo-
BS-labeled proteins were conjugated to biotin-azide, followed by
streptavidin enrichment. The resulted protein mixtures were
resolved by SDS-PAGE and analyzed by immunoblotting using
antibodies against BRD4, BAZ2A, and KAT2A. As shown in
Figure 4E, photo-BS successfully enriched all the three selected
BCPs from the cells. Bromosporine and JQ1 dramatically
attenuated the enrichment of BRD4 that contains bromodomains
targeted by these two inhibitors, while the enrichment of BAZ2A
was only impeded by GSK2801, a selective BAZ2A bromodomain
inhibitor. In contrast, the enrichment level of KAT2A was not
significantly affected by the three tested inhibitors, as it is known
that the KAT2A bromodomain is not the target of these inhibitors.
This result demonstrated the ability of photo-BS in the enrichment
photo-BS-labeled proteins were conjugated to Rho-N
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and
visualized by in-gel fluorescence scanning. The fluorescence
intensity of each band was quantified by ImageJ. Data are reported
as mean  s.d. (n = 2). (B) Comparison of bromosporine binding
affinity (K
bromodomains. The K
d
) and photo-BS labeling efficiency (EC50) toward the
(data acquired from Ref. 20) and EC50 (data
d
see Figure S2) are displayed on the human bromodomain
phylogenetic tree as spheres with indicated sizes and colors.
We next tested if the photo-BS-induced labeling of
bromodomains could be selectively competed by the inhibitors
with defined target specificities. To this end, BRD4(1), BRD4(2),
CECR2, TAF1(2), and ASH1L were cross-linked with photo-BS in
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of endogenous BCPs, and its potential to evaluate the selectivity of
the bromodomain inhibitors.
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Figure 3. Bromodomain inhibitors selectively compete the photo-BS-induced labeling. (A) The photo-BS-induced labeling toward 21
selected recombinant bromodomains could be competed in the presence of excessive bromosporine. (B) Bromosporine, JQ1, and PFI-1
selectively competed the photo-BS-induced labeling toward BRD4(1), BRD4(2), CECR2, TAF(2), and ASH1L. (C) In-gel fluorescence and
competition curves of PFI-1 against BRD4(1) and BRD4(2). The fluorescence intensity of each band was quantified by ImageJ. All curves
were normalized between 100% and 0% at the highest and lowest fluorescence intensities, respectively. Data are reported as mean  s.d. (n
=
2). (D) Summarization of the K
d
, IC50 and apparent K
i
values of PF1-1 and JQ1 toward BRD4(1) and BRD4(2), respectively. a: K
d
values
. For details of
of PFI-1 and JQ1 toward each bromodomain were obtained from Refs. 21 and 8, respectively. b: Calculated apparent K
calculation, see SI. BS: bromosporine.
i
Photo-BS leads to wide-spectrum profiling of BCPs. We next
identified dozens of known BCPs that lay in the regions above the
used photo-BS, coupled with SILAC-based quantitative
thresholds (Figures 5A5B).
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proteomics , to profile the endogenous BCPs in cells. In a forward
We then manually picked up the BCPs for further analysis
Figures 5C5D). Specifically, 26 BCPs were detected in the
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SILAC experiment, ‘heavy’ (grown in medium containing C,
(
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N-substitued arginine and lysine) and ‘light’ (grown in medium
samples derived from HEK293T cells. While BRPF3 was enriched
by 2.9 folds in the forward sample, no signal in the reverse one was
obtained. The rest 25 BCPs gave both forward and reverse SILAC
ratios higher than 2. From the U2OS cells, 31 BCPs were detected,
in which 26 of these BCPs matched the thresholds for
bromosporine-interacting proteins. The proteins BRWD3,
CREBBP, EP300, TRIM28 and TRIM33 were either unmet to the
SILAC ratio criteria, or only detected in one SILAC experiment. In
total, 28 BCPs showed more than 2-fold enrichment from both
SILAC experiments in HEK293T and U2OS cells. More
specifically, 23 of these BCPs were shared by both cell lines.
KAT2B and TRIM33 were only enriched from HEK293T, and the
unique enrichment of BPTF, BRPF3, and SP100 was obtained from
U2OS. These results revealed a good coverage of the photo-BS
toward the endogenous BCPs (two-thirds of all the 42 BCPs were
enriched).
with arginine and lysine in natural isotope abundance forms)
HEK293T cells were shone by UV light in the presence and
absence of photo-BS (10 M), respectively. After cell lysis, the
‘heavy’ and ‘light’ lysates were pooled. The captured proteins were
biotinylated via ‘click chemistry’, followed by streptavidin-based
affinity purification, gel separation, and in-gel trypsin digestion.
The resulting peptide mixtures were analyzed by liquid
chromatography coupled with tandem mass spectrometry (LC-
MS/MS). To ensure the data reliability, a reverse SILAC
experiment, in which the ‘light’ but not the ‘heavy’ cells were
cross-linked with photo-BS, was performed in parallel. The same
set of SILAC experiments were also performed in U2OS cells. We
designated the proteins with SILAC ratio heavy/light (H/L) > 2 in
the forward experiment and L/H > 2 in the reverse experiment as
bromosporine-interacting proteins. In both the cell lines tested, we
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Figure 4. Photo-BS enriches endogenous BCPs in living cells. (A) Recombinant bromodomains could be labeled by photo-BS (5 M) in the
presence of complex proteome, and the labeling was selectively competed by corresponding bromodomain inhibitors. (B) Concentration-
dependent labeling of photo-BS in living cells. (C) Pre-incubation of the cells with bromosporine reduced the photo-BS-induced labeling
intensity. (D) Photo-cross-linking in living cells resulted in distinct labeling pattern compared with in cell lysate (1 mg/mL), protein bands
with different intensities are indicated by red arrows. For all the photo-cross-linking experiments, after UV irradiation, cells were lyzed (for
cell-based assays), the photo-BS-labeled proteins were conjugated to Rho-N and visualized by in-gel fluorescence scanning. (E) Photo-
3
cross-linking pull-down in living cells with photo-BS (2 μM) in the absence or presence of different bromodomain inhibitors (5 M). Samples
without photo-BS were prepared as negative control. After photo-cross-linking and cell lysis, the photo-BS-labeled proteins were conjugated
3
to biotin-N and enriched by streptavidin. The eluted protein mixtures were analyzed by immunoblotting against indicated antibodies. The
blotting is representative of three independent experiments. BrD: bromodomain. BS: bromosporine.
Evaluation of target selectivity of bromodomain inhibitors in
living cells. The wide-spectrum profile of BCPs by photo-BS
promoted us to examine whether this probe can be used to evaluate
the target selectivity of bromodomain inhibitors in living cells. To
this end, both the ‘light’ and ‘heavy’ cells were photo-cross-linked
with the probe, and either the ‘heavy’ (forward experiment) or the
inhibitor-untreated/inhibitor-treated in both forward and reverse
experiments would be considered as the targets of the inhibitor
(Figure 6A). The logarithmic (Log ) SILAC ratios (H/L) of the
identified BCPs in the forward (x axis) and reverse (y axis)
experiments would be plotted in 2-dimentional plots. The target
BCPs of the tested inhibitors would stand out as the top-left outliers
in the plots.
2
‘light’ cells (reverse experiment) were pre-incubated with the
inhibitors of interest. BCPs that showed the high SILAC ratios of
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Figure 5. Photo-BS leads to wide-spectrum profile of BCPs. (A and B) SILAC ratio plots for total proteins identified in HEK293T (A) and
U2OS (B) cells. Forward: ‘heavy’ cell with photo-BS (10 M), ‘light’ cell with DMSO as control. Reverse: ‘light’ cell with photo-BS (10
M), ‘heavy’ cell with DMSO as control. BCPs were highlighted as red dots. See Datasets S1 and S2 for SILAC ratios. (C) Heat map shows
the SILAC ratios of BrD obtained from A and B. Proteins with SILAC ratios photo-BS-treated/photo-BS-untreated > 2 in both forward and
reverse experiments were defied as photo-BS-enriched ones. Names of BCPs enriched by photo-BS in both cell lines are labeled in red,
enriched in either one cell line are labeled in black, enriched in neither cell line are labeled in grey. See Table S1 for SILAC ratios. (D) Venn
diagram of the enriched BCPs from the SILAC experiments in HEK293T (red) and U2OS (blue) cells.
We first examined the selectivity of bromosporine toward
endogenous BCPs in both the HEK293T and U2OS cells. As shown
in Figure 6B, five BCPs, BRD2, BRD3, BRD4, BRD7 and BRD9,
were identified to bind bromosporine in the HEK293T cells.
Similarly, BRD2, BRD3, BRD4, and BRD7 were also found to be
targeted by bromosporine in the U2OS cells (Figure 6C). The
identification of the three BET family members, BRD2, BRD3, and
BRD4, as the endogenous targets of BS was in line with the
previous studies that showed that the bromosporine treatment led
to BET inhibition signatures of transcription in a number of
experiments, while other identified BCPs all had the ratio close to
1 (Figure 6E). This is consistent with the reported high specificity
of GSK6853 toward BRPF1. Together, these data suggest that our
photo-BS-based proteomics method is suitable for comprehensive
analysis of target profiles of bromodomain inhibitors in living cells.
DISCUSSION
The evaluation of inhibitors, especially for their selectivity, is a
critical step during drug discovery. The development of chemical
20
proteomics approaches has provided powerful methods26-27 to
leukemia cell lines. The engagement of bromosporine with BRD7
or BRD9 in living cells, however, has not been characterized. To
validate if BRD7 and BRD9 were the targets of bromosporine,
photo-BS was incubated with HEK293T and MV4;11, a leukemia
cell line, in the presence or absence of bromosporine. As expected,
both BRD7 and BRD9 were enriched by photo-BS from both the
cell lines tested. The addition of bromosporine indeed dampened
the enrichment of BRD7 and BRD9, but not KAT2A, a BCP that
showed a SILAC ratio around 1 (Figure 6D). These data indicate
that bromosporine can engage with endogenous BRD7 and BRD9
in living cells.
examine the selectivity of an inhibitor against dozens or even
thousands of proteins in one experiment, particularly for the
assessments toward a family of structurally and functionally related
proteins. Most of the reported chemical proteomics studies for
2
8-32
inhibitor assessment have been focusing on enzymes
, for
example kinases that transfer a phosphate from an adenosine
triphosphate (ATP) to the substrates. Early examples include ATP-
3
3-34
derived probes
and beads immobilized with kinase inhibitors
3
5
(kinobeads) to profile kinases in cell lysates. In a recent study, the
development of sulfonyl fluoride-based probes, which covalently
captured up to 133 endogenous kinases (~ 25% of the human
kinome) from a single cell line, enabled the cell-based assessment
of the kinase inhibitors. The bromodomains have emerged as
hotspots’ for inhibitor development, highlighting the significance
of the methods used for the assessment of bromodomain inhibitors.
Using this photo-BS-based chemical proteomics approach, we
2
5
evaluated another bromodomain inhibitor, GSK6853 (Figure S1),
that selectively targets BRPF1 bromodomain. Remarkably, only
BRPF1 showed high SILAC ratios in both the forward and reverse
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Photo-BS developed in this study represents, to the best of our
knowledge, the first chemical probe that captures wide-spectrum
endogenous bromodomains and enables the evaluation of
bromodomain inhibitors in living cells.
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Figure 6. Target selectivity evaluation of bromodomain inhibitors in living cells. (A) Schematics illustrating the chemical proteomics method
for the evaluation of bromodomain inhibitors using photo-BS. (B and C) Two-dimensional plots showing the Log SILAC ratios of BCPs
for the evaluation of bromosporine in HEK293T (B) and U2OS (C) cells. (D) Photo-cross-linking pull-down in living cells with photo-BS
in the absence or presence of bromosporine to validate the engagement of bromosporine with endogenous BRD7 and BRD9. After photo-
2
cross-linking and cell lysis, the photo-BS-labeled proteins were conjugated to biotin-N
mixtures were analyzed by immunoblotting against indicated antibodies. The blotting is representative of three independent experiments.
BS: bromosporine. (E) A two-dimensional plot showing the Log SILAC ratios of BCPs for the evaluation of GSK6853 in HEK293T cells.
3
and enriched by streptavidin. The eluted protein
2
For all the SILAC experiments, both ‘heavy’ and ‘light’ samples were treated with photo-BS (10 M). In forward experiments, ‘heavy’ cells
with bromosporine (10 M) or GSK6853 (5 M). In reverse experiments, ‘light’ cell with bromosporine (10 M) or GSK6853 (5 M). BCPs
as top-left outliers were highlighted in red. See Figures S6, S7, and S8 for two-dimensional plots of other identified proteins. See Datasets
S3S5 for SILAC ratios.
In recent years, photo-cross-linking has become a powerful
approach to examine protein-protein and small molecule-protein
approach, for the identification of specific targets of bromodomain
inhibitors, even in the presence of high photo-cross-linking
background. In addition to the BCPs, the other three proteins
(PI4K2B, SDR39U1, and RPUSD2) that were specifically
competed by bromosporine are potentially interesting ‘off-targets’
of this inhibitor at whole-proteome level, which need further
validation and characterization.
37
interactions. Given the nature of the method that convert non-
covalent interactions to covalent chemical bonds, photo-cross-
linking enables capture of not only strong binding partners but also
weak and transient ones that are usually considered as ‘non-
specific’ binders. In addition, the commonly used photo-cross-
linkers (e.g, benzophenone and diazirine) are non-polar and
hydrophobic. They could therefore mediate non-specific protein
interactions, which often results in ligand-independent non-specific
While the photo-BS provided a good BCP coverage (two-thirds
of the BCPs were enriched), 14 BCPs were not enriched by the
probe in our SILAC experiments (Figures 5C5D). By analyzing
these 14 BCPs, we noticed that the enrichment failure could be due
to the limited expression levels of the BCPs. For example, in
HEK293T and U2OS cells, the abundances of BRDT, which is a
testis specific BET family member, and SP140, which is mainly
expressed in leukemia and lymphoma cell lines, are too low to
ensure a robust detection by mass spectrometry (the expression data
38-39
photo-cross-linking.
Not surprisingly, the application of photo-
BS in living cells captured many other proteins other than the
BCPs, (Figure 5A and 5B). We reasoned that most of these
captured proteins should be involved in either weak interactions
with the bromosporine scaffold or with the diazirine moiety. As a
common practice, the competitive photo-cross-linking experiment
using a relatively low concentration (10 M) of bromosporine as
the competitor was performed to distinguish the strong or ‘specific’
protein binders from the ‘non-specific’ including the weak ones.
Consistent with our hypothesis, among thousands of proteins
captured by photo-BS, only 6 of them showed competition by
bromosporine in either HEK293T (Figure S6) or U2OS (Figure S7)
cells. Notably, 5 (in HEK293T) and 4 (in U2OS) of the 6 proteins,
respectively, were BCPs known or validated to bind bromosporine.
This result demonstrated the robustness and feasibility to use
photo-BS, in combination of competitive chemical proteomics
40
was obtained from the Human Protein Atlas ). We believe that
more BCPs will be added to the enrichment list if the photo-BS-
based SILAC experiments were performed in additional cell lines.
On the other hand, the low binding affinity of some BCPs toward
bromosporine could also be the reason for why these proteins were
undetected or unmet the enrichment criteria in our SILAC
experiments. For example, the acquired EC50 values of photo-BS-
induced labeling towards ASH1L, BAZ2B, and TRIM28
bromodomains were all larger than 20 M (Figure S2). It is
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M.; Munro, S.; McKeown, M. R.; Wang, Y.; Christie, A. L.; West, N.;
therefore not surprising that 10 M of photo-BS used in the SILAC
experiments failed to enrich these BCPs. One solution is to use
higher concentration of photo-BS in the SILAC experiments or
develop new photoaffinity probes based on other bromodomain
inhibitors with target profiles different from bromosporine, which
will be an important next step.
Cameron, M. J.; Schwartz, B.; Heightman, T. D.; La Thangue, N.; French,
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of BET bromodomains. Nature 2010, 468 (7327), 1067-73.
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Theodoulou, N. H.; Tomkinson, N. C.; Prinjha, R. K.;
ASSOCIATED CONTENT
Supporting Information
Humphreys, P. G., Clinical progress and pharmacology of small molecule
bromodomain inhibitors. Curr Opin Chem Biol 2016, 33, 58-66.
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The Supporting Information is available free of charge via the
Internet at http://pubs.acs.org.
Filippakopoulos, P.; Picaud, S.; Keates, T.; Felletar, I.; Ciulli, A.; Knapp,
S.; Heightman, T. D., Bromodomain-peptide displacement assays for
interactome mapping and inhibitor discovery. Mol Biosyst 2011, 7 (10),
899-908.
12.
2
Additional experimental data, including experimental details,
synthesis, and characterization (PDF).
Ciulli, A., Biophysical screening for the discovery of small-
molecule ligands. Methods Mol Biol 2013, 1008, 357-88.
3. Zolotarjova, N. I.; Wynn, R., Binding Assays for Bromodomain
1
AUTHOR INFORMATION
Proteins: Their Utility in Drug Discovery in Oncology and Inflammatory
Disease. Curr Protoc Pharmacol 2018, 80 (1), 3 16 1-3 16 14.
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Strain-Damerell, C.; Burgess-Brown, N. A.; Gingras, A. C.; Knapp, S.;
Muller, S., Assessing cellular efficacy of bromodomain inhibitors using
fluorescence recovery after photobleaching. Epigenetics Chromatin 2014,
7, 14.
Corresponding Author
*xiangli@hku.hk
Philpott, M.; Rogers, C. M.; Yapp, C.; Wells, C.; Lambert, J. P.;
Author Contributions
#X.L. and Y.W. contributed equally to this work.
1
5.
Machleidt, T.; Woodroofe, C. C.; Schwinn, M. K.; Mendez, J.;
Notes
Robers, M. B.; Zimmerman, K.; Otto, P.; Daniels, D. L.; Kirkland, T. A.;
Wood, K. V., NanoBRET--A Novel BRET Platform for the Analysis of
Protein-Protein Interactions. ACS Chem Biol 2015, 10 (8), 1797-804.
The authors declare no competing financial interest.
1
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Li, Z.; Wang, D.; Li, L.; Pan, S.; Na, Z.; Tan, C. Y.; Yao, S. Q.,
"
Minimalist" cyclopropene-containing photo-cross-linkers suitable for live-
cell imaging and affinity-based protein labeling. J Am Chem Soc 2014, 136
(28), 9990-8.
ACKNOWLEDGMENT
17.
Pan, S.; Jang, S. Y.; Wang, D.; Liew, S. S.; Li, Z.; Lee, J. S.;
We acknowledge support from the Hong Kong Research Grants
Council Collaborative Research Fund (CRF C7029-15G to
X.D.L.), the Areas of Excellence Scheme (AoE/P-705/16 to
X.D.L.), the General Research Fund (GRF 17126618, 17125917
and 17303114 to X.D.L.), the Early Career Scheme (ECS) (HKU
Yao, S. Q., A Suite of "Minimalist" Photo-Crosslinkers for Live-Cell
Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors.
Angew Chem Int Ed Engl 2017, 56 (39), 11816-11821.
18.
D'Ascenzio, M.; Pugh, K. M.; Konietzny, R.; Berridge, G.;
Tallant, C.; Hashem, S.; Monteiro, O.; Thomas, J. R.; Schirle, M.; Knapp,
S.; Marsden, B.; Fedorov, O.; Bountra, C.; Kessler, B. M.; Brennan, P. E.,
An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved
Amino Acid Residues within Bromodomains. Angew Chem Int Edit 2019,
7
09813P to X.D.L.), and the National Natural Science Foundation
of China (21572191 and 91753130 to X.D.L). We thank Q. Zhao
for useful discussions. We thank M.G. Lee for providing plasmid
of the ZMYND8 bromodomain. We thank A.Y.-H. Leung for
providing the MV4;11 cell line.
5
8 (4), 1007-1012.
9. Fedorov, O.; Lingard, H.; Wells, C.; Monteiro, O. P.; Picaud, S.;
1
Keates, T.; Yapp, C.; Philpott, M.; Martin, S. J.; Felletar, I.; Marsden, B.
D.; Filippakopoulos, P.; Muller, S.; Knapp, S.; Brennan, P. E.,
[
1,2,4]triazolo[4,3-a]phthalazines: inhibitors of diverse bromodomains. J
Med Chem 2014, 57 (2), 462-76.
0. Picaud, S.; Leonards, K.; Lambert, J. P.; Dovey, O.; Wells, C.;
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