An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains

Article abstract of DOI:10.1002/anie.201807825

Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl-lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule-based approach to covalently modify recombinant and endogenous bromodomain-containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in-gel visualization and pull-down of the desired bromodomains.

Full text of DOI:10.1002/anie.201807825

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201807825
German Edition: DOI: 10.1002/ange.201807825
Chemical Proteomics
An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved
Amino Acid Residues within Bromodomains
Melissa D’Ascenzio, Kathryn M. Pugh, Rebecca Konietzny, Georgina Berridge, Cynthia Tallant,
Shaima Hashem, Octovia Monteiro, Jason R. Thomas, Markus Schirle, Stefan Knapp,
Brian Marsden, Oleg Fedorov, Chas Bountra, Benedikt M. Kessler,* and Paul E. Brennan*
Abstract: Bromodomain-containing proteins are epigenetic
modulators involved in a wide range of cellular processes, from
recruitment of transcription factors to pathological disruption
of gene regulation and cancer development. Since the drugg-
ability of these acetyl-lysine reader domains was established,
efforts were made to develop potent and selective inhibitors
across the entire family. Here we report the development of
a small molecule-based approach to covalently modify
recombinant and endogenous bromodomain-containing pro-
teins by targeting a conserved lysine and a tyrosine residue in
the variable ZA or BC loops. Moreover, the addition of
a reporter tag allowed in-gel visualization and pull-down of the
desired bromodomains.
Despite its widespread application to several different
[
5]
[6]
enzymatic families including kinases, serine hydrolases,
[
7]
and cysteine/threonine proteases, only minor progress has
been made with reference to epigenetic proteins. The value of
developing activity-based probes directed against epigenetic
modulators finds its rationale in the emerging potential of
[
8]
epigenetic proteins as therapeutic targets, and it has been
emphasized by recently reported successful attempts to use
similar strategies in order to profile lysine methyl trans-
[9–11]
[12,13]
ferases,
or histone deacetylases.
However, a significant amount of the afore mentioned
studies rely on photo-affinity labelling techniques for the
development of efficient probes, mostly because of the lack of
activated or easily targetable nucleophilic amino acid residues
in the active sites of these proteins. While there are no reports
of effective ABPP probes against native bromodomains
(BRDs), Daguer et al. recently reported on the discovery of
small molecules that are able to engage with a conserved
cysteine residue in the binding pocket when incubated with
C
hemical proteomics methodologies such as activity-based
protein profiling (ABPP) have been developed to comple-
ment genetic manipulation and interrogate the proteome to
[1]
capture the functional state of proteins in cells and tissues.
Through the use of covalent inhibitors, ABPP has provided
quantitative readouts of the functional state of individual or
multiple enzymes, taking into account allosteric, intrasteric,
[14]
truncated bromodomains. These small reader domains that
specifically recognize the e-N-acetylated (KAc) lysine mark
on histone tails, have been identified as functional modules
within at least 46 human proteins that are known to be
involved in the regulation of a wide range of cellular events,
from transcription factors recruitment to chromatin remodel-
[2]
and post-translational control. Furthermore, thanks to their
broad spectrum of selectivity, ABPP probes have been used to
screen prospective inhibitors against a wide panel of proteins
[
3,4]
and show target engagement in the cellular environment.
[
15,16]
ing, and in the occurrence of several types of cancer.
Bromodomain-containing proteins constitute a class of epi-
genetic modulators that are particularly challenging to profile
by ABPP, since they lack enzymatic activity and are deficient
of activated amino acid residues in their ligand binding sites.
For this challenging task to be achieved, new activity-based
probe should be endowed with widespread activity across the
BRD family, in order to target most of the 61 bromodomains
encoded by the human proteome. Bromosporine (BSP) is
currently the most broad spectrum bromodomain inhibitor; it
is characterized by in vitro KD values in the nanomolar/low
micromolar range, especially against the BET family, and sub-
micromoal cellular activity against the first bromodomain of
[
*] Dr. M. D’Ascenzio, Dr. K. M. Pugh, G. Berridge, Dr. C. Tallant,
S. Hashem, O. Monteiro, Prof. S. Knapp, Dr. B. Marsden,
Dr. O. Fedorov, Prof. C. Bountra, Prof. P. E. Brennan
Structural Genomic Consortium (SGC)
University of Oxford
Oxford OX3 7DQ (UK)
E-mail: paul.brennan@sgc.ox.ac.uk
Dr. M. D’Ascenzio, Dr. K. M. Pugh, Dr. R. Konietzny, G. Berridge,
Dr. C. Tallant, O. Monteiro, Prof. S. Knapp, Dr. O. Fedorov,
Prof. B. M. Kessler, Prof. P. E. Brennan
Target Discovery Institute (TDI)
University of Oxford
Oxford OX3 7FZ (UK)
E-mail: benedikt.kessler@ndm.ox.ac.uk
[17]
BRD4, BRD7, BRD9, CREBBP, and CECR2. Therefore,
Dr. J. R. Thomas, Dr. M. Schirle
Novartis Institute for BioMedical Research (NIBR)
its scaffold was considered as a perfect starting point for the
rational design of a novel broad spectrum activity-based BRD
probe. Through a series of preliminary sequence alignment
and docking studies, we observed that the methyl sulfonamide
group of BSP pointed towards one of three lysine residues
that are fairly conserved in the BC loop of BRDs belonging to
different sub-families, and corresponded to residue 85, 90 or
180 Massachusetts Ave, Cambridge, MA 02139 (USA)
Prof. S. Knapp
Institute for Pharmaceutical Chemistry and Buchmann Institute for
Life Sciences, Johann Wolfgang Goethe-University
60438 Frankfurt am Main (Germany)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201807825.
9
1 in the first BRD of BRD4 (BRD4(1)) (Figure 1).
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The ability of bromotriazine 2 (BTZ), to covalently
modify BRDs characterized by the presence of a lysine
residues on the variable BC loop, was tested by incubating the
probe with recombinant BRDs in buffer at 378C. The
formation of covalent adducts was detected by mass spec-
trometry (Figure 3). Different BRD sub-families were used in
a preliminary screen with the aim to define the covalent
labelling potential of the newly synthesized probe: BRD4(1),
BRD4(2), BRD3(2), BRD2(1), BRD2(2), TAF1(1), TAF1-
(2), TAF1L(1), BRD1, ATAD2 (Figure 3).
Figure 1. Crystal structure of bromosporine and docking of bromotria-
zine in BRD4(1), and sequence alignment of BRDs belonging to
different families. The methylsulfonamide moiety of BSP was replaced
by a dichlorotriazine warhead in order to selectively target one of three
lysines (red K in red box) present in the variable BC loop of
bromodomains belonging to different families. The amino acid
sequences were aligned using highly conserved residues (columns
highlighted in green).
As lysines are potentially nucleophilic amino acids that
[18]
have been reported to be targetable by covalent labelling,
we believed it would be possible to develop a novel BRD
covalent probe by replacing the sulfonamide scaffold of BSP
with a reactive warhead, and subsequently introduce a click-
able alkyne handle on the solvent exposed ethyl carbamate
moiety to allow tagged protein pull-down and in-gel visual-
ization (Figure 2).
Figure 3. Bromotriazine covalent labelling across the bromodomain
family tree. A) Bromotriazine 2, a covalent probe inspired by the
structure of bromosporine, a pan-bromodomain inhibitor, was incu-
bated with truncated BRDs belonging to different subfamilies. The
degree of covalent modification was measured by RapidFire mass
spectrometry, as shown for BRD4(1); B) Bromotriazine efficiently
labels a wide range of BRDs belonging to different subfamilies by
covalently reacting with non-activated nucleophilic lysine and tyrosine
residues on the rim of their binding site.
Moreover, a series of BRDs that did not contain
a nucleophilic amino acid within reach were included in the
study as controls: PB1(5), SMARCA2B, TIF1a, BRPF1B,
and CREBBP. An accurate analysis of the data originated in
this assay highlighted that the reactivity profile of BTZ (2)
seemed to follow the activity profile observed with bromo-
sporine, where higher affinity of the reversible probe
corresponded to higher percentage of covalent modification
by the irreversible probe, thus suggesting that the reversible
binding played a pivotal role in driving the covalent labelling
(Figure 3). In fact, BTZ efficiently labelled all the BETs (14–
67%) and was particularly effective against BRD2(1). At the
same time, bromodomains that were weakly interacting with
bromosporine (IC ꢀ 1 mm) were only slightly modified (1–
Figure 2. Design of bromosporine derived covalent probe bromotria-
zine (BTZ). A clickable alkyne handle was added to the ethyl carbamate
moiety of bromosporine, while a dichlorotriazine electrophile was
introduced to replace the methylsulfonamide scaffold and generate
bromotriazine 2.
In the search for the most appropriate electrophile to use
as a warhead for our probe, we found that Shannon et al. had
reported the discovery of a dichlorotriazine reactive moiety
that was capable of selectively labelling a range of lysine
[
19]
residues within the proteome of HeLa cells. A series of
docking experiments suggested that the dichlorotriazine
moiety could be well tolerated by the target proteins
5
0
4%) by BTZ, despite presenting a targetable lysine residue in
the correct position (GCN5L2, ATAD2, BRD1). Negligible
levels of modification (0 to 5%) were detected in BRDs
whose sequence suggested they could not be modified by
(
Figure 1), and the synthesis of bromosporine was re-designed
in order to include the selected warhead and the clickable
handle (see Supporting Information).
2
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BTZ since they lacked nucleophilic amino acids on the rim of
their binding site.
To confirm the correct binding of bromotriazine within
the KAc site and provide evidence for the preference of the
reactive warhead for lysine residues, recombinant BRDs were
incubated with the probe before undergoing tryptic digestion
[
20,21]
and LC-MS/MS analysis.
In the case of BRD4(1) and
BRD3(2), this approach confirmed that the preferred sites of
modification were in fact the predicted lysine 91 in BRD4(1)
and the corresponding residue, lysine 336, in BRD3(2)
(
Figure 4; see Supporting Information for complete data).
These findings were compatible with the estimated binding
mode of BTZ 2 in BRD4(1).
Figure 5. Mapping the sites of covalent modification via crystallo-
graphic techniques and crystal structure of the covalent complex
formed by BTZ (2) and BRD9. The site of modification in this case
was found to be a tyrosine (Tyr106) at the end of the AZ loop. The
probe was reported to reversibly interact with the binding site of one
BRD and form a covalent bond with the nucleophilic residue on an
adjacent BRD, thus creating an interlocked dimer.
Based on this, we analysed all the BRDs amino acid
sequences in order to identify those containing a tyrosine
residue at the same position of the one targeted by BTZ 2 in
BRD9 (Tyr106). Seven BRDs including PCAF, GCN5L2,
TAF1 and TAF1L complied with this requirement, and they
also contain a targetable lysine in the BC loop.
The reactivity of BTZ against the former two BRDs was
limited (9% and 0%, respectively), consistent with the low
affinity of the non-covalent BSP for family I BRDs. However,
labelling increased to 45% against TAF1(1) and TAF1(2),
reaching 86% in the case of BRD7, a protein with high
structural similarity to BRD9. These findings suggested that
the presence of a tyrosine residue in proximity to the binding
site could endow the corresponding BRD with higher
reactivity against our probe when compared with lysine
bearing BRDs.
Figure 4. Mapping the sites of covalent modification via LC-MS/MS.
BTZ (2) was docked within the binding site of BRD4(1) (left) and then
superimposed to the same pocket in BRD3(2) (right). Two lysines, K91
and K336, were found to be near the reactive dichlorotriazine warhead,
thus suggesting they would be the expected site of covalent inter-
action. The predictions made by docking were subsequently confirmed
by LC-MS/MS peptide mapping.
Interestingly, the clearest covalent labelling was observed
when BTZ was reacted with CECR2, a sub-family I BRD
characterized by the presence of a tyrosine residue in place of
the corresponding lysine 91 in BRD4(1). Surprisingly, BRD9,
a IV BRD sub-family member, was covalently modified up to
To confirm this, we determined the kinetics of the
covalent reaction between BTZ and BRDs, characterized
by the presence of a targetable lysine (BRD4(1), BRD4(2),
BRD3(2), BRD2(1)) or tyrosine (BRD9, CECR2) residue on
the rim of the binding site.
7
7% after 1.5 hours incubation at 378C, despite that it lacked
a lysine near the KAc binding site. Since repeated attempts to
modify BRD9 resulted in consistently high percentages of
labelling but no reliable data could be produced by LC-MS/
MS peptide mapping, a crystallization experiment was set up
in order to determine the exact site of interaction. The crystals
obtained by seeding the purified fraction containing the
modified protein showed a peculiar interlocked structure, in
which one BTZ probe was located inside the binding site of
one BRD, yet it was covalently linked to another tyrosine
residue just outside of the binding pocket of another BRD in
the crystallographic unit cell. (Figure 5—Top and bottom left)
The resulting interlocked dimer is compatible with the
previously reported 2:1 protein-peptide stoichiometry of
The reaction between a protein and its irreversible
[23]
inhibitor should follow a second order kinetic model.
However, the order of the reaction was reduced by applying
[
24]
the isolation method,
where each BRD (1 mm) was
incubated with a high concentration of BTZ (100 mm) and
the decrease of unmodified protein was monitored over time
with the RapidFire MS system. Each experiment was
conducted in triplicate, as reported in Figure 6a for BRD4(1),
and the average of the experimental values plotted as linear
trends as shown in Figure 6b (for additional data see
Supporting Information—Section 5). Since the experimental
conditions did not allow the discrimination between rever-
sible and irreversible binding to the kinetics of the process,
a cumulative constant (kapp) was calculated for each reaction.
Comparing the different kapp values confirmed the dramatic
[
22]
BRD9 functional complexes. Moreover, the superimposi-
tion of the crystal structures of BTZ and bromosporine in the
binding pocket of BRD9 highlighted the high similarity of the
two binding modes (Figure 5—Bottom right).
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Figure 6. BTZ binding kinetics against BRDs belonging to different
sub-families a) BRD4(1) (1 mm) was incubated with BTZ (100 mm) and
the amount of unmodified protein monitored over time as a function
of the area (A) of the peak registered using a RapidFire low-injection
QToF mass spectrometer. The experiment was run in triplicate in order
to determine the kinetic rate constant of the overall process. b) Com-
paring the linear graphs obtained in the previous kinetic experiments
allowed us to rank the relative reactivity of lysine (BRD4(1), BRD4(2),
BRD3(2) and BRD2(1)) and tyrosine (BRD9 and CECR2) containing
BRDs, indicating preferential binding of BTZ to CECR2.
Figure 7. Fluorescent labelling and enrichment experiment conducted
effect that changing the reactive amino acid had on the
overall reaction rate (Figure 6b). It should be highlighted that
CECR2 (kapp = 12.6 ꢀ 10 s ) is characterized by the pres-
ence of two tyrosine residues, one in a position corresponding
to Lys91 in BRD4(1) and the other one in a position
corresponding to Tyr106 in BRD9.
Taken together, we describe a broad-spectrum probe that
could covalently modify BRDs belonging to different sub-
families.
on recombinant BRD4(1) using BTZ (2) and its non-clickable analogue
À1
(2b) Recombinant BRD4(1) (1 mgmL ) was incubated with BTZ
À4 À1
(100 mm) or its non-clickable analogue 2b (100 mm) for 1.5 hours at
3
5
78C. The obtained samples were reacted with an azido-dye (Cyanine
.5) (top) or biotin-azide (bottom) under copper-catalyzed alkyne
azide cycloaddition (CuAAC) conditions, in order to selectively visual-
ize the formed BRD4(1)-BTZ (2) covalent adduct under IR irradiation
(top) or to pull down the obtained complex on Streptavidin beads
(bottom).
In addition, insightful information on the amino acid
involved in the binding could be derived by co-crystallization
or LC-MS/MS mapping.
Encouraged by these in vitro results, chemical proteomics
To validate the applicability of BTZ (2) as a chemical
proteomics tool, recombinant BRD4(1) was incubated with
the covalent probe and the obtained covalent complex
experiments were conducted in K-562 chronic myelogenous
leukemia cell line and in THP-1 monocytic cell line, as they
both express BRD4 and BRD2 at a high endogenous level.
[26]
[25]
subjected to a copper-catalyzed click chemistry reaction.
Promising results were obtained in particular after pull down
experiments were conducted on the THP-1 cell line, where
whole cell lysates were incubated with increasing concen-
trations of probe 2 (10, 25 and 100 mm) at 48C and samples
were collected at 1 h, 4 h, 8 h, and 24 h time points; an
additional time point was taken after 5 minutes of incubation
to highlight any unspecific pull down that could be ascribed to
the probeꢁs intrinsic reactivity (see Supporting Information).
The analysis of the resulting protein pull-down on
NeutrAvidin beads by silver staining and Western blotting
highlighted a direct correlation between the total amount of
When an azido-dye (Cyanine 5.5-IR dye) was used as the
cycloaddition partner, the truncated protein appeared as
a bright spot on polyacrylamide gel. When biotin azide was
used in place of the azido-dye, BRD4(1) was successfully
immobilized on Streptavidin-coated beads and then eluted
from the resin by thermal denaturation. A covalent non-
clickable analogue of bromotriazine (2b) was used in both
experiments as a control since it lacked the alkyne functional
handle necessary to react with the azide moiety on the dye or
biotin tag (Figure 7).
4
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pull down, the incubation time, and the concentration of the
probes.
In a series of subsequent pull-down experiments, the
In conclusion, we designed and synthesized a covalent
probe, BTZ (2), against bromodomain containing proteins.
Our results highlight the feasibility of selectively targeting
non-catalytic amino acids on the rim of the ligand binding site
by correctly orienting the chosen electrophilic moiety towards
formation of BRD-BTZ (2) covalent adducts was competed
by preincubation of THP-1 whole cell lysates with 100 mm
BTZ non-clickable analogue (2b) or 100 mm JQ1(+),
the desired nucleophilic residue, as recently reported by Zhao
[
27]
[29]
a known BET bromodomains inhibitor.
Although the
et al. in the case of protein kinases.
In particular, an
MS/MS analysis of the competition experiments did not
show a significant enrichment of BRDs pull down in the non-
competed samples, possibly because of the generally low
levels of BRD pull down at low temperatures, these experi-
ments highlighted a series of potentially interesting BTZ off-
targets. In particular, Thymosin beta-4 (TMSB4X), a protein
involved in cell proliferation and migration identified as
a biomarker of malignancy in solid tumors, was enriched by
treatment with BTZ (2) and selectively competed by pre-
treatment with both 2b and JQ1(+), thus suggesting that BTZ
unprecedented selective labelling of non-catalytic and highly
conserved lysines of purified bromodomains was reported.
We also discovered the previously unreported ability of a 2,4-
dichlorotriazine electrophile to label tyrosine residues and the
surprising dimerization process induced by such covalent
interaction with BRD9 that broadens the prospective for
future design of covalent and selective inhibitors. This may
apply not only to the bromodomain family, but to alternative
cellular targets characterized by the presence of a lysine and/
or tyrosine residue in the proximity of the binding site.
[28]
(
2) selectively bound and labeled this protein (see Supporting
Information).
The initial attempts to selectively visualize the covalent Acknowledgements
BRD-BTZ adducts formed after incubation of K-562 cell
lysates with BTZ (2) using Cy 5.5 dye proved to be extremely
challenging, possibly due to the promiscuity of the probe (see
Supporting Information).
The SGC is a charity (number 1097737) funded by AbbVie,
Bayer Pharma AG, Boehringer Ingelheim, Canada Founda-
tion for Innovation, Eshelman Institute for Innovation,
Genome Canada, Innovative Medicines Initiative (EU/
EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck
KGaA Darmstadt, MSD, Novartis Pharma AG, Ontario
Ministry of Economic Development and Innovation, Pfizer,
S¼o Paulo Research Foundation-FAPESP, Takeda, and Well-
come [106169/ZZ14/Z]. We thank the Medical Research
Council for funding this research through the “Proximity to
Industry” grant Scheme and Dr. Suzanne Muller-Knapp for
insightful conversations.
However, the MS/MS analysis conducted on pull-down
samples obtained by incubating non-transfected K-562 cell
lysates for 1 hour at 378C with 5, 50, and 100 mm BTZ (2),
respectively (see Supporting Information for complete data),
and then appending a biotin handle on the formed covalent
complexes by CuAAC, showed a significant enrichment of
BRD4, BRD8 and BRD associated proteins (SMARCA4,
SUPT16H) in the higher concentration samples, compared to
the lowest concentration one (Figure 8). The identification of
BRD8 is of particular note as BRD8 has never been produced
recombinantly and although BTZ (2) was not tested for
binding BRD8 in vitro, its enrichment in cell lysate shows that Conflict of interest
BTZ is potentially a BRD8 inhibitor.
The authors declare no conflict of interest.
Keywords: activity-based protein profiling · bromodomain ·
chemical proteomics · covalent probes · epigenetics
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E. Weerapana, J. Am. Chem. Soc. 2014, 136, 3330 – 3333.
20] R. Fischer, B. M. Kessler, Proteomics 2015, 15, 1224 – 1229.
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Fischer, J. Proteome Res. 2017, 16, 1288 – 1299.
[
[
Manuscript received: July 9, 2018
Revised manuscript received: October 20, 2018
Version of record online: && &&, &&&&
6
www.angewandte.org
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2018, 57, 1 – 7
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Chemical Proteomics
M. D’Ascenzio, K. M. Pugh, R. Konietzny,
G. Berridge, C. Tallant, S. Hashem,
O. Monteiro, J. R. Thomas, M. Schirle,
S. Knapp, B. Marsden, O. Fedorov,
C. Bountra, B. M. Kessler,*
P. E. Brennan*
&&&& — &&&&
Bromodomain-containing proteins: Since
the druggability of these acetyl-lysine
reader domains was established, efforts
were made to develop potent and selec-
tive inhibitors across the entire family.
Here the development of a small mole-
cule-based approach to covalently modify
An Activity-Based Probe Targeting Non-
Catalytic, Highly Conserved Amino Acid
Residues within Bromodomains
recombinant and endogenous bromodo-
main-containing proteins by targeting
a conserved lysine and a tyrosine residue
in the variable ZA or BC loops is reported.
Angew. Chem. Int. Ed. 2018, 57, 1 – 7
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!