A Suite of “Minimalist” Photo-Crosslinkers for Live-Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors

Article abstract of DOI:10.1002/anie.201706076

Affinity-based probes (AfBPs) provide a powerful tool for large-scale chemoproteomic studies of drug–target interactions. The development of high-quality probes capable of recapitulating genuine drug–target engagement, however, could be challenging. “Mini

Full text of DOI:10.1002/anie.201706076

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Accepted Article
Title: A Suite of "Minimalist" Photo-Crosslinkers for Live-Cell Imaging
and Chemical Proteomics: Case Study with BRD4 Inhibitors
Authors: Shao Q. Yao, Sijun Pan, Se-Young Jang, Danyang Wang, Si
Si Liew, Zhengqiu Li, and Jun-Seok Lee
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A Suite of “Minimalist” Photo-Crosslinkers for Live-Cell Imaging
and Chemical Proteomics: Case Study with BRD4 Inhibitors
Sijun Pan, Se-Young Jang, Danyang Wang, Si Si Liew, Zhengqiu Li, Jun-Seok Lee* and Shao Q. Yao*
Abstract: Affinity-based probes (AfBPs) provide a powerful tool for
large-scale chemoproteomic studies of drug-target interactions. The
development of high-quality probes capable of recapitulating genuine
drug-target engagement however could be challenging. “Minimalist”
photo-crosslinkers, which contain an alkyl diazirine group and a
chemically tractable tag, could alleviate such challenges, but few are
currently available. Herein, we have developed new alkyl diazirine-
containing photo-crosslinkers with different bioorthogonal tags. They
were subsequently used to create a suite of AfBPs based on
GW841819X (a small molecule inhibitor of BRD4). Through in vitro
and in situ studies under conditions that emulated native drug-target
interactions, we have obtained better insights into how a tag might
photo-reactive groups, alkyl diazirines have shown superior
protein-labeling efficiency with low nonspecific labeling.[16] On the
other hand, given its small size and chemical stability, a terminal
alkyne is an excellent tractable tag in the probe design, but the
use of toxic Cu catalysts during Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) makes it ill-suited for live-cell applications.
The past decade has witnessed a remarkable expansion of
tractable tags enabled by Cu-free bioorthogonal chemistries.[
Notwithstanding, such tags are uncommon in AfBPs, presumably
due to the need of an additional photo-reactive group.[
Despite several elegant studies on the reactivity and selectivity of
various bioorthogonal reactions,[20-22] how significant a tag might
affect the overall performance of an AfBP under “in situ drug
17]
18-20]
affect
the
probe’s
performance.
Finally,
SILAC-based
chemoproteomic studies have led to the discovery of a novel off-target, profiling” settings has not been explored. We anticipated different
APEX1. Further studies showed GW841819X binds to APEX1 and
caused up-regulation of endogenous DNMT1 expression under
normoxia conditions.
bioorthogonal pairs would affect the proteome reactivity profiles
of a probe, providing both drug- and tag-specific labeling patterns.
Herein, we report the comprehensive cell-based proteome
studies of GW841819X (a small molecule inhibitor of the
epigenetic reader protein BRD4[23]), by using a newly developed,
diverse set of AfBPs containing a common alkyl diazirine and
different tractable tags (Figure 1). We were hopeful that biased
tag-specific signals caused by individual probes may be reduced,
ultimately resulting in the emergence of true cellular targets (on
and off) of the parent drug as common signals from all probes.
This process was further combined with modern techniques in
quantitative mass spectrometry (qMS), namely stable isotope
labeling with amino acids in cell culture (SILAC) and neutron-
encoded isobaric mass tag labeling (TMT10),[24,25] to integrate
In drug discovery, lacks of efficacy and safety are two major
causes of drug candidate attrition, which may be reduced by a
more integrated understanding of the drug candidate in terms of
its exposure, target engagement and pharmacological
activities.[ Strategies based on small molecule probes have
been introduced to facilitate studies of target engagement and
identification, but they are not without major limitations.[2-4] Ideally,
such strategies should recapitulate drug-target interactions in situ
1,2]
(
e.g. live cells) and allow for subsequent proteome-wide target
enrichment/identification in vitro (from cell lysates).[5] We recently
named such approaches “in situ drug profiling”, which could trace
its root to activity-based protein profiling (ABPP).[6,7] For ABPP in
which a reversible activity-based probe is used, since a photo-
reactive moiety is needed in the probe design, such a probe is
also called an “affinity-based probe” (AfBP).[8] Given that non-
covalent small molecules constitute > 90% of FDA-approved
drugs, the development of high-quality AfBPs suitable for drug-
target interaction studies in living cells is of paramount
importance.[3-14] In order to retain the original pharmacological
properties of a non-covalent drug, chemical modifications on the
parent compound should be made as small as possible.[5,15]
Based on recent systematic studies of AfBPs containing various
various probes within
experimental errors associated with sample preparation and MS
analysis (Figure S1).
We previously reported the first-generation “minimalist” photo-
crosslinkers which consist of a small alkyl diazirine flanked by a
terminal alkyne and various functional groups.[26] They could be
a single experiment and reduce
used to modify different bioactive compounds. Recently, one of
these photo-crosslinkers was used to label GW841819X
[27]
(
renamed as WT in current study; Figure 1A), giving BD-3.
When compared to a bulky photo-crosslinker (i.e. linker used in
[
9]
BD-4 ), such a “minimalist” design was shown to improve the
performance of AfBPs. Subsequent attempts to replace the
terminal alkyne with cyclopropene-containing tags, i.e. BD-1 and
BD-2, further enabled the in situ bioimaging capability by using
the well-known tetrazine-cyclopropene ligation through an inverse
electron demand Diels–Alder reaction (IEDDA).[17] Despite being
small in size and chemically accessible from the corresponding
terminal alkyne, the relative chemical instability of cyclopropenes
caused severe tag-specific background labeling in subsequent
proteomic studies.[17,27] Nevertheless, we found such imaging-
and proteome profiling-enabled, dual-purpose probes could help
in refining target identification by taking advantage of the
subcellular distribution information attainable from imaging
experiments. In the current study, we reasoned such issues may
be further alleviated with additional AfBPs that contain different
tractable tags in our chemoproteomic workflow (Figure S1).
[
*]
*]
S. Pan, D. Wang, S. S. Liew, Prof. Dr. S. Q. Yao
Department of Chemistry, National University of Singapore,
3
Science Drive 3, Singapore 117543 (Singapore)
E-mail: chmyaosq@nus.edu.sg:
S.-Y. Jang, Prof. Dr. J.-S. Lee
Molecular Recognition Research Center, Bio-Med Program of KIST-
School UST, Korea Institute of Science & Technology,
Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, 136791 (Korea)
E-mail: junseoklee@kist.re.kr
[
Prof. Dr. Z. Li
College of Pharmacy, Jinan University, Guangzhou, 510632 (China)
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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Figure 1. (A) Structures of various AfBPs based on GW841819X (WT), a recently discovered PPI inhibitor of BRD4,[23] and (B) summary of their performance in
various assays (in vitro labeling, live-cell imaging, in situ labeling & TMT10 qMS). Names of the “click” reactions and their fluorophore/biotin-containing reporters
are shown (see Figures S2-S5 for full details). n.a. = Not available.
Similar to previously developed “minimalist” photo-crosslinkers,
the new linkers (D8, N8 and T6 in Figure S3) consist of three key
components: 1) a functional group (e.g. NH ) that could be readily
2
cell-based assays with a focus on target binding and labeling, and
results are summarized in Figure 1B. By comparing these AfBPs
in various settings relevant to “in situ drug profiling”, we showed
the choice of tags in an AfBP indeed played a significant role in
determining the probe’s performance both in vitro (Figures S6-S8)
and in live-cell environments (Figures 2 & S9).
In vitro labeling experiments with BRD4-overexpressing
bacterial lysates showed UV-dependent, positive target labeling
with all probes except BD-5 at 0.2 µM (Figure S6). Addition of WT
(10x) abolished the labeling. Negative BRD4 labeling by BD-5
was attributed to a much lower reactivity between tetrazine and a
simple alkene compared to other click reactions.[17] BD-5 was
therefore not investigated further. Isothermal titration calorimetry
(ITC) experiments to measure direct probe-target binding
attached to a bioactive compound; 2) an alkyl diazirine, which is
the smallest photo-reactive moiety that possesses excellent UV-
induced protein labeling properties;[12] and 3) a small chemically
tractable tag for on-demand click chemistry. These components
were joined together with short carbon chains. The tags in the
current “minimalist” design, e.g. a terminal alkene, an aliphatic
azide and a tetrazine, were chosen on the basis of their small
sizes, chemical stability, robust bioorthogonality and scarcity in
AfBPs. Terminal alkenes could be clicked to a tetrazine reporter
via tetrazine-alkene ligation, although in general electron-rich or
strained alkenes are needed.[17,19] An aliphatic azide, despite
initial concerns that it might be slowly reduced by intracellular
thiols, was chosen because of its small size as well as wide
applications in metabolic engineering.[17] It could be clicked to
either a terminal alkyne reporter via CuAAC, or a strained
d
indicated the K values of all probes were within 3-5 folds of that
of WT, with the exception of BD-4. Of note, in addition to the linker
size as a primary concern, modifications of the ester linkage (to
an amide) and the benzyl moiety (to an alkyl linker) might affect
the physicochemical properties (e.g., hydrophobicity, H-bonding
ability) of the probes and hence their binding affinities. We
reasoned that these limitations could be outweighed by the
convenient preparation of small AfBPs by using our “minimalist”
linkers. We next investigated the cellular effects of these probes
on endogenous BRD4 activities. Inhibition of BRD4 was
previously shown to cause transcriptional down-regulation of c-
Myc.[23,29] As shown in Figure 2A, cells treated with WT or one of
the AfBPs showed apparent inhibition of c-Myc with WT being the
most effective inhibitor, followed by BD-7 > BD-3 ≈ BD-6 > BD-2
≈ BD-4. This order was similar but clearly not identical to the
relative BDR4-binding affinity obtained from in vitro ITC results.
More pronounced differences in cellular activities of the AfBPs
might have resulted from additional differences in their cell
permeability, subcellular distribution and off-targeting. To more
directly probe the on-target interaction of our AfBPs in live cells,
in situ proteome labeling followed by PD/Western blotting (WB)
were performed (Figures 2B & S9); the 152-kDa BRD4 band was
successfully detected in probe-labeled cells but not in cells pre-
treated with WT (10x). BD-4 showed very weak in situ BRD4
cyclooctyne
cycloaddition (SPAAC) in live cells.
via
Cu-free
strain-promoted
azide-alkyne
[
17,28]
Finally, a small tetrazine
tag was chosen over TCO because of its more favorable chemical
and cellular stability.[17] The resulting linkers, together with the
previously reported bulky photo-crosslinker,[9] were used to
synthesize four new BRD4-targeting AfBPs, BD-4, BD-5, BD-6,
and BD-7. They were then combined with two previously reported
probes (BD-2 and BD-3[27]), to create a suite of AfBPs for
comprehensive proteome profiling studies (Figure 1).
BRD4, a bromodomain-containing protein that recognizes
acetylated lysine (Kac) residues such as those located on
histones, is an important epigenetic “reader” involved in
numerous critical cellular processes.[29] Previous proteomic
studies, with either an immobilized probe and cells lysates or
AfBPs (i.e. BD-2 and BD-3) under in situ conditions,[23,27] could be
ineffective due to the aforementioned reasons. With all six BRD4-
targeting AfBPs in hand, including BD-2/BD-3 (BD-1 was not
used due to its poor labeling property[27]) and the newly
synthesized BD-4/BD-5/BD-6/BD-7, the overall performance of
this suite of probes was evaluated through a panel of in vitro and
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Figure 2. Probe performance in live-cell conditions. (A) Western blotting (WB) analysis of endogenous c-Myc expression level in MV4-11 cells treated with WT/probe
at indicated concentrations (for 24 h). (Bottom gels): GAPDH loading control. (B) PD/WB studies of endogenous BRD4 (i.e. the 152-kDa bands) from live HepG2
cells. Cells were in situ-labeled with a probe (5 µM for 3 h, + 10x WT). (C) Live-cell imaging with BD-2/BD-6/BD-7 (5 µM, 37 °C for 3 h + 10x WT) in HepG2 cells.
Upon probe treatment/UV irradiation, live-cell click reaction with a cell-permeable reporter (FL-TZ1, FL-DBCO or FL-TCO, respectively, 25 M for 1 h) was done
followed by image acquisition under indicated reporter channel. Where applicable, immunofluorescence (IF) and nuclear staining were carried out. Nu = Hoechst
channel; IF was done with anti-BRD4 antibody. Scale bar = 10 μm. (D) In-gel fluorescence scanning profiles of live HepG2 cells labeled with an indicated probe (5
µM for 3 h, ± WT). (Lane 11): overlaid picture of lanes 1/3/5/7/9.
Labeling, presumably due to its bulky photo-crosslinker (Figure
S9). These results, together with earlier findings that BD-4 had
weak cellular activities, highlight the need to carefully consider the
size of a photo-crosslinker in an AfBP design. Encouragingly, we
found two newly developed probes,BD-6 and BD-7, were
effective AfBPs for in situ labeling of endogenous BRD4, and
therefore suitable in subsequent proteome profiling experiments.
We previously showed BD-2, but not BD-3, was suited for live-
cell imaging. BD-6 and BD-7 were designed to have similar
capabilities as their tags may be tracked by Cu-free bioorthogonal
chemistries (Figure 1B). As shown in Figure 2C, similar to BD-2,
which upon being clicked to FL-TZ1 could be used to image
nuclear-localized BRD4 in live cells, comparable results were
obtained with BD-6/FL-DBCO and BD-7/FL-TCO pairs; probe-
treated cells showed strong fluorescence throughout cell nuclei
WT; lane 11 in Figure 2D), which could be treated as a reasonable
representation of the complete drug-target engagement profile of
WT from this suite of AfBPs. The overlaid gel was similar to that
of BD-3, indicating BD-3 might be one of the most suitable AfBPs
for subsequent target identification.
Having obtained a comprehensive picture of various AfBPs, we
summarized their performance in Figure 1B with the following
recommendations: (1) for in vitro and in situ proteome profiling,
where click chemistry is carried out post-UV irradiation under in
vitro conditions, terminal alkyne- and azide-containing photo-
crosslinkers (e.g. in BD-3/BD-6) are ideal, as both tags are small
and give minimum background labeling. Probes such as BD-2
and BD-7 might be suitable, but their performance could be
compromised by nonspecific labeling; (2) for live-cell imaging,
3
photo-crosslinkers having a suitable cyclopropene, N or tetrazine
(
panels 1/3/5), and BD-6 particularly colocalized well with signals
(e.g. in BD-2/BD-6/BD-7) may be used to provide additional
information on drug-target interaction at subcellular levels; (3) in
cases where both live-cell imaging and in situ proteome profiling
are carried out, N - containing AfBPs might be the most ideal (e.g.
3
BD-6), providing the best compromise in size, stability and
bioorthogonality.
from immunofluorescence (IF) experiments using anti-BRD4
antibody (panels 3/9). Interestingly, each probe displayed distinct
nuclear-localized staining patterns; while BD-2 signals were
evenly distributed throughout the entire nucleus (panel 1), signals
from BD-6 (panel 3) and BD-7 (panel 5), in sharp contrast,
avoided and concentrated in the region of nucleoli, respectively.
It should be noted that the nucleolus-avoiding BD-6 signals were
akin to those from IF experiments (compare panels 3 & 9). This
indicates BD-6 might be the best-performing BRD4-imaging
probe. Finally, we performed gel-based in situ proteome profiling
Unlike standard LC-MS/MS experiments,[27] quantitative mass
spectrometry (qMS) such as SILAC and TMT could minimize
intrinsic variations during sample preparation and MS analysis,
thus delivering more accurate protein hits in a chemoproteomic
workflow. The TMT10 approach can analyze up to 10 MS samples
in a single run, but requires labeling of post-PD peptides at the
final stage prior to MS analysis. In the current work, although it
was well-suited for simultaneous comparison of our AfBPs (Figure
S10), we found significant discrepancies in the resulting MS data
when compared to live-cell imaging results. Nevertheless, we
were able to conclude that, as earlier discussed, BD-3/BD-6 were
the most suitable AfBPs for target identification (Figure 1B).
SILAC on the other hand, despite its limited multiplicity, introduces
isotopes into proteins at the start of the workflow, and thus is well-
suited for subsequent in vitro PD/MS analysis which requires
multi-step sample preparations. Therefore, BD-3 and BD-6 were
(
Figure 2D); similar WT-dependent proteome labeling profiles
from all four probe-treated cells were obtained. As previously
observed with BD-3,[27] the fluorescent labeling of endogenous
BRD4 was not distinct in BD-2/BD-6/BD-7 treated samples,
presumably due to its low expression level. Among the probes,
the labeling profile of BD-7 appeared most distinct (lane 9), while
BD-3 displayed the lowest non-specific labeling (compare lanes 1
&
2). We further performed in vitro lysate labeling experiments to
compare background labeling profiles arising from various
reporters (Figure S9). Finally, aggregate labeling patterns were
generated by overlaying the various proteome labeling profiles (-
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Figure 3. (A) Venn diagram and SILAC plot of preferred hits, with “SILAC Hi” and “SILAC Lo” shown in blue and orange, respectively. (B) Nuclear localization and
functional classes of “SILAC Hi” and “SILAC Lo” in (A). (C) PD/WB studies of endogenous candidate proteins in HepG2 cells, in situ labeled with BD-3/BD-6 (5 µM),
with or without WT. (D) Same as (C) except BD-3 concentration was varied (0.2 or 1 µM). (E) Docked structure of WT (yellow) in the active site of the DNA repair
domain in APEX1 (PDB ID: 5DG0). (F) Same as (C) by using BD-3 (5 µM), and with either WT, (+)-JQ1 or E3330 as a competitor (10X). (G) WB analysis of
endogenous DNMT1 expression level (top gels) of HepG2 cells upon treated with WT or E3330 at an indicated concentration (0, 1, 3, 10 µM) for 24 h under normoxia
or hypoxia conditions. (Bottom gels): β-tubulin as loading control. (H) Same as (G) except the cells were transfected with one of the three APEX1 siRNAs (see
Supplementary Information for details) or treated with an inhibitor (10 µM) (top gels). (Bottom gels): effects on APEX1-knockdown cells without and with treatment
of WT (10 µM).
chosen in the following SILAC experiments. Following previously
established protocols,[30] a protein was designated as a hit if: 1) it
exhibited SILAC ratios ≥ 1.5 in both Forward and Reverse
experiments, or 2) the mean value of the two SILAC ratios ≥ 1.5
with coefficient of variation (CV) < 0.67 (Figure S10). Such filters
revealed 197 and 161 protein hits enriched by BD-3 and BD-6,
respectively, with 69 common targets designated as “SILAC Hi”
enzyme’s pocket bordered by Arg177/Phe266/Trp280 (Figure 3E).
Other binding sites on APEX1 (e.g. the redox domain) however
could not be excluded for possible WT interactions. Further
competitive PD/WB studies confirmed that WT indeed occupied
the same binding site on APEX1 as E3330, as well as (+)-JQ1
(another BRD4 inhibitor that is analogous to WT; Figure 3F). To
further substantiate direct WT-APEX1 engagement in cells, which
might lead to modulation of the enzyme’s cellular activities, we
carried out a cell-based assay to monitor endogenous DNMT1
expression. APEX1 was previously shown to be inhibited by
E3330 leading to DNMT1 up-regulation in epithelial stem cells.[
Similar cellular effects were observed in HepG2 cells treated with
WT, E3330 or (+)-JQ1 (Figure 3G & S11); both WT and E3330
caused an apparent increase in DNMT1 expression at 10 µM
under normoxia conditions. Such an effect was attenuated under
hypoxia conditions. Our finding thus suggests WT could mimic the
cellular activities of E3330, presumably through inhibition of
endogenous APEX1. Further siRNA knockdowns of APEX1
showed a similar increase in DNMT1 expression, which was
consistent with previous studies (Figure 3H);[ the effect was
directly relevant to the level of APEX1 down-regulation, with more
complete knockdown of APEX1 resulting in a higher DNMT1
expression. Finally, addition of WT to the APEX1-knocked down
cells further enhanced the up-regulation of DNMT1, presumably
through the effective inhibition of the remaining APEX1 activities
that were responsible for DNMT1 regulation.
(
Figure 3A). The remaining 220 protein hits enriched by only one
of the two AfBPs were designated as “SILAC Lo”. Further analysis
of both sets of hits indicates 94% and 73% of them are found in
the nucleus (Figure 3B), which corroborates with earlier live-cell
imaging results. Further functional class analysis of “SILAC Hi”
hits revealed most hits belong to transcription factors and
regulators. Finally, we found 11 of the “SILAC Hi” hits and 12 of
the “SILAC Lo” hits possess structural similarities with BRD4, with
many known to interact with BRD4 and/or its inhibitors (Table S1).
Further ranking identified four highest-confidence, nuclear-
localized hits as likely true off-targets of WT (Table S2).
Preliminary validation on these hits showed all of them were
labeled by both probes in an activity-based manner in live cells
33]
33]
(
Figure 3C). Among them, APEX1 (an enzyme containing a redox
and a DNA repair domain) immediately caught our attention due
to its well-documented therapeutic potential for various
diseases.[
31]
We first ensured that endogenous APEX1 was
successfully labeled by BD-3 at a physiologically relevant probe
concentration (Figure 3D); with 0.2 µM of BD-3, stronger labeling
of endogenous APEX1 over BRD4 was detected, indicating in the
same drug-treated cells, WT might engage APEX1 preferentially
over its intended target BRD4. This is a clear sign of off-targeting.
A well-known small molecule inhibitor of APEX1, E3330 (Figure
S11), was found to bind to the enzyme’s repair active site.[32]
Docking results showed WT also fits snugly in the repair active
site of APEX1, with the triazolodiazepine moiety located inside the
Our pursuit of “minimalist” photo-crosslinkers has led to the
successful development of new alkyl diazirine-containing linkers
with a variety of bioorthogonal tags, which were subsequently
used to generate a suite of AfBPs. By conducting comprehensive
studies under conditions that emulated native drug-target
interactions, we have obtained a clear view of how a tag might
affect the overall performance of a probe. By employing
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quantitative mass spectrometry with select probes, we have
conducted chemoproteomic studies for GW841819X, and
successfully identified APEX1 as a novel off-target. Subsequent
cell-based assays indicated GW841819X was able to bind to
APEX1 in HepG2 cells and caused up-regulation of endogenous
DNMT1 expression under normoxia conditions.
[14] J. Park, M. Koh, S. B. Park, Mol. BioSyst. 2013, 9, 544-550.
[
15] S. Ziegler, V. Pries, C. Hedberg, H. Waldmann, Angew. Chem. Int. Ed.
013, 52, 2744-2792.
2
[
16] P. Kleiner, W. Heydenreuter, M. Stahl, V. S. Korotkov, S. A. Sieber,
Angew. Chem. Int. Ed. 2017, 56, 1396-1401, and references cited
therein.
[
[
[
[
17] D. M. Patterson, L. A. Nazarova, J. A. Prescher, ACS Chem. Biol. 2014,
9, 592-605.
18] K. S. Yang, G. Budin, T. Reiner, C. Vinegoni, R. Weissleder, Angew.
Chem. Int. Ed. 2012, 51, 6598-6603.
Acknowledgements
19] C. Li, T. Dong, Q. Li, X. Lei, Angew. Chem. Int. Ed. 2014, 53, 12111-
12115.
Funding was provided by National Medical Research Council
20] A. Rutkowska, D. W. Thomson, J. Vappiani, T; Werner, K. M. Mueller, L.
Dittus, J. Krause, M. Muelbaier, G. Bergamini, M. Bantscheff, ACS
Chem. Biol. 2016, 11, 2541-2550.
(
1
CBRG/0038/2013), Singapore, KIST (2E26632/2E26110, CAP-
6-02-KIST) and the Bio & Medical Technology Development
Program of the National Research Foundation (NRF-
016M3A9B6902060) from Ministry of Science, Korea.
[
[
21] L. I. Willems, N. Li, B. I. Florea, M. Ruben, G. A. van der Marel, H. S.
Overkleeft, Angew. Chem. Int. Ed. 2012, 51, 4431-4434.
2
22] H. E. Murrey, J. C. Judkins, C. W. am Ende, T. E. Ballard, Y. Fang, K.
Riccardi, L. Di, E. R. Guilmette, J. W. Schwartz, J. M. Fox, D. S. Johnson,
J. Am. Chem. Soc. 2015, 137, 11461-11475.
Keywords: affinity-based probe • photo-crosslinker •
bioorthogonal chemistry • live-cell imaging • epigenetics
[23] C.-W. Chung, H. Coste, J. H. White, O. Mirguet, J. Wilde, R. L. Gosmini,
C. Delves, S. M. Magny, R. Woodward, S. A. Hughes, E. V. Boursier, H.
Flynn, A. M. Bouillot, P. Bamborough, J.-M. G. Brusq, F. J. Gellibert, E.
J. Jones, A. M. Riou, P. Homes, S. L. Martin, I. J. Uings, J. Toum, C. A.
Clement, A.-B. Boullay, R. L. Grimley, F. M. Blande, R. K. Prinjha, K. Lee,
J. Kirilovsky, E. Nicodeme, J. Med. Chem. 2011, 54, 3827-3838.
[
1]
P. Morgan, P. H. Van der Graaf, J. Arrowsmith, D. E. Feltner, K. S.
Drummond, C. D. Wegner, S. D. A. Street, Drug Discov. Today 2012, 17,
4
19-424.
M. Schurmann, P. Janning, S. Ziegler, H. Waldmann, Cell Chem. Biol.
016, 23, 435-441.
M. E. Bunnage, E. L. P. Chekler, L. H. Jones, Nat. Chem. Biol. 2013, 9,
95-199.
G. M. Simon, M. J. Niphakis, B. F. Cravatt, Nat. Chem. Biol. 2013, 9, 200-
05.
[
[
[
[
2]
3]
4]
5]
[
[
24] S.-E. Ong, Mann, M. Nat. Protocols 2007, 1, 2650-2660.
25] T. Werner, G. Sweetman, M. F. Savitski, T. Mathieson, M. Bantscheff,
M. M. Savitski, Anal. Chem. 2014, 86, 3594-3601.
2
1
[
[
[
26] Z. Li, P. Hao, L. Li, C. Y. J. Tan, X. Cheng, G. Y. J. Chen, S. K. Sze, H.-
M. Shen, S. Q. Yao, Angew. Chem. Int. Ed. 2013, 52, 8551-8556.
27] Z. Li, D. Wang, L. Li, S. Pan, Z. Na, C. Y. J. Tan, S. Q. Yao, J. Am. Chem.
Soc. 2014, 136, 9990-9998.
2
Y. Su, J. Ge, B. Zhu, Y.-G. Zheng, Q. Zhu, S. Q. Yao, Curr. Opin. Chem.
Biol. 2013, 17, 768-775.
28] J. Z. Yao, C. Uttamapinant, A. Poloukhtine, J. M. Baskin, J. A. Codelli, E.
M. Sletten, C. R. Bertozzi, V. V. Popik, A. Y. Ting, J. Am. Chem. Soc.
[
[
[
6]
7]
8]
M. J. Niphakis, B F. Cravatt, Annu. Rev. Biochem. 2014, 83, 341-377.
L. E. Sanman, M. Bogyo, Annu. Rev. Biochem. 2014, 83, 249-273.
E. W. S. Chan, S. Chattopadhaya, R. C. Panicker, X. Huang, S. Q. Yao,
J. Am. Chem. Soc. 2004, 126, 14435-14446.
2012, 134, 3720.
[
[
29] P. Filippakopoulos, S. Knapp, Nat. Rev. Drug Discov. 2014, 13, 339-358.
30] D. Wang, S. Du, A. Cazenave-Gassiot, J. Ge, J.-S. Lee, M. R. Wenk, S.
Q. Yao, Angew. Chem. Int. Ed. 2017, 56, 5829-5833.
[
[
9]
H. Shi, C.-J. Zhang, G. Y. J. Chen, S. Q. Yao, J. Am. Chem. Soc. 2012,
134, 3001-3014.
[
[
31] G. Kaur, R. P. Cholia, A. K. Mantha, R. Kumar, J. Med. Chem. 2014, 57,
10] P. Ranjitkar, B. G. K. Perera, D. L. Swaney, S. B. Hari, E. T. Larson, R.
10241-10256.
Krishnamurty, E. A. Merritt, J. Villen, D. J. Maly, J. Am. Chem. Soc. 2012,
32] J. Zhang, M. Luo, D. Marasco, D. Logsdon, K. A. LaFavers, Q. Chen, A.
134, 19017-19025.
Reed, M. R. Kelley, M. L. Gross, M. M. Georgiadis, Biochemistry 2013,
[
[
[
11] J. J. Hulce, A. B. Cognetta, M. J. Niphakis, S. E. Tully, B. F. Cravatt, Nat.
Methods 2013, 10, 259-264.
5
2, 2955-2966.
33] G. Chen, J. Chen, Z. Yan, Z. Li, M. Yu, W. Guo, W. Tian, Sci. Rep. 2017,
: 40762.
[
12] S. Pan, H. Zhang, C. Wang, S. C. L. Yao, S. Q. Yao, Nat. Prod. Rep.
7
2016, 33, 612-620.
13] M. H. Wright, S. A. Sieber, Nat. Prod. Rep. 2016, 33, 681-708.
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Angewandte Chemie International Edition
10.1002/anie.201706076
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Tagging for More. New “minimalist”
S. Pan, S.-Y. Jang, D. Wang, S. S. Liew,
Z. Li, J.-S. Lee,* Shao Q. Yao*
alkyl
diazirine-containing
with
photo-
crosslinkers
different
bioorthogonal tags were developed,
and used to create a suite of AfBPs
based on a known small molecule
inhibitor of BRD4.
Page No. – Page No.
A Suite of “Minimalist” Photo-
Crosslinkers for Live-Cell Imaging
and Chemical Proteomics: Case
Study with BRD4 Inhibitors
This article is protected by copyright. All rights reserved.