Preparation of small-molecule microarrays by trans-cyclooctene tetrazine ligation and their application in the high-throughput screening of protein-protein interaction inhibitors of bromodomains

Article abstract of DOI:10.1002/anie.201307803

Fast and efficient: A library of trans-cyclooctene (TCO)-modified small molecules were immobilized on tetrazine-functionalized glass slides by using the fastest bioorthogonal reaction known. The resulting small-molecule microarray was screened against a variety of human bromodomains to identify protein-protein interaction inhibitors. Copyright

Full text of DOI:10.1002/anie.201307803

.
Angewandte
Communications
DOI: 10.1002/anie.201307803
Small-Molecule Microarrays
Hot Paper
Preparation of Small-Molecule Microarrays by trans-Cyclooctene
Tetrazine Ligation and Their Application in the High-Throughput
Screening of Protein–Protein Interaction Inhibitors of
Bromodomains**
Chong-Jing Zhang, Chelsea Y. J. Tan, Jingyan Ge, Zhenkun Na, Grace Y. J. Chen,
Mahesh Uttamchandani, Hongyan Sun, and Shao Q. Yao*
The e-N-acetylation of lysine residues (Kac) is one of the most
common posttranslational modifications in proteins that are
associated with epigenetics, and frequently occurs in large
macromolecular complexes that play a role in chromatin
remodelling, DNA damage, and cell-cycle control.[1] In
histones, acetylated lysines reduce electrostatic interactions
with the negatively charged DNA phosphates, thus providing
a more relaxed chromatin structure, which is associated with
transcriptionally active genes. The cellular histone acetylation
levels are strictly controlled by histone acetyltransferases
(HATs; epigenetic “writers”) and histone deacetylases
(HDACs; epigenetic “erasers”).[2,3] Furthermore, Kac affects
gene transcription through interactions with bromodomain
(BRD)-containing proteins (BCPs; epigenetic “readers”).[3,4]
HATs and HDACs have traditionally been the main research
focus in medicinal chemistry and drug discovery for epige-
netic diseases.[3,5] As enzymes, they are considered as
“druggable”. The development of protein–protein interaction
(PPI) inhibitors that target the epigenetic readers, on the
other hand, is much more challenging. It is generally believed
that small molecules do not bind sufficiently tightly to the
much larger protein–protein interface because they contain
only a limited number of functional groups.[6] Such simplistic
views, however, have recently been challenged by two
ground-breaking studies that showed that certain benzodia-
zepine-containing compounds (e.g., JQ1 and I-BET) are
specific nanomolar PPI inhibitors of BET bromodomains
(e.g., BRD4).[7] This has spurred interest in compounds that
might possess similar biological activities against other
BRDs.[8] However, there remains a lack of sensitive assays
for rapid and high-throughput screening (HTS) of other
BRD-binding compounds. Existing assays, including those
based on fluorescence polarization (FP), isothermal titration
calorimetry (ITC), protein stability shift, and surface plasmon
resonance (SPR), are either applicable to only a few well-
known BRDs (e.g., BRD2/3/4), or they suffer from limited
throughput because large amounts of proteins are required.[9]
Furthermore, for most of the 61 human BRDs, their cognate
Kac-binding sequences are not well-understood. Recent
large-scale structural studies and histone–peptide membrane
arrays failed to identify any sub-micromolar-binding acety-
lated peptides that could be suitable for HTS assays.[10] It is
perhaps not surprising that both JQ1 and I-BET were
serendipitously discovered by HTS of random in-house
compound libraries using cell-based reporter assays without
any prior target information.[7] These assays, though useful in
their own right, are not target-oriented, and compounds
identified during these assays may not actually act on their
intended BRDs. Therefore, a prerequisite for drug-develop-
ment efforts that focus on the discovery of new BRD
inhibitors is the development of an HTS platform that is
capable of general, rapid, and systematic large-scale screening
of the interactions between BRDs and small molecules.
We recently developed a powerful small-molecule micro-
array (SMM) that is capable of sensitive, quantitative, and
rapid identification of cell-permeable small-molecule PPI
inhibitors.[11] SMMs are miniaturized assemblies of small
molecules that are immobilized across a planar glass slide, on
which thousands of protein–ligand interactions (strong and
weak) may be measured, with minimal consumption of
proteins and ligands (in the nL to mL range).[12] We reasoned
that BRDs, which are protein-binding domains that normally
bind to cognate acetylated peptides with moderate or weak
affinities, would be well-suited for our SMM approach.
Herein, in a proof-of-concept study, we have successfully
constructed a SMM on which 48 different benzodiazepines
that are based on the core structure of JQ1/I-BET were
immobilized, and screened it against 55 fluorescently labeled
proteins, twelve of which were human BRDs (Figure 1).
Specifically, we have 1) developed the first SMM that is based
on bioorthogonal trans-cyclooctene (TCO) tetrazine ligation
for site-specific covalent immobilization of TCO-modified
benzodiazepines with unprecedented speed; 2) used this
SMM for miniaturized HTS of these compounds against
[*] C.-J. Zhang, C. Y. J. Tan, Dr. J. Ge, Z. Na, G. Y. J. Chen,
Dr. M. Uttamchandani, 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
Homepage: http://staff.science.nus.edu.sg/~syao
Prof. Dr. H. Sun
Department of Biology and Chemistry, City University of Hong Kong
Hong Kong (P.R. China)
[**] Funding was provided by the National Medical Research Council
(CBRG12nov100) and the Ministry of Education (MOE2012-T2-1-
116 and MOE2012-T2-2-051). We also acknowledge a generous
donation of bromodomain plasmids from Dr. James E. Bradner
(Harvard Medical School) and Dr. Stefan Knapp (Oxford).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201307803.
14060
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 14060 –14064

Angewandte
Chemie
Figure 1. a) Synthesis of small molecules with a trans-cyclooctene tag (SM-TCO), followed by immobilization of SM-TCO onto tetrazine-
functionalized slides to generate the corresponding SMMs; these were subsequently screened against fluorescently labeled proteins, including 12
BRDs and 43 other common PPI domains. The newly identified (+)-25T is shown, together with (+)-JQ1 (yellow box) and FITC-TCO, which was
used as a positive spotting control. b) Assessment of the time- and concentration-dependence of the trans-cyclooctene tetrazine method for SMM
immobilization. FITC-TCO (0–100 mm in a 1:1 DMSO/water solution) was used for spotting. Microarray results (left) were quantified and plotted
(right). Fmoc=9-fluorenylmethyloxycarbonyl, NMP=N-methylpyrrolidine, SAR=structure—activity relationship, TFA=trifluoroacetic acid.
a variety of BRD and non-BRD proteins, including SH2 do-
mains, 14-3-3, and other proteins, and established their cross-
reactivity profiles; 3) discovered (+)-25T, a compound that
possesses biological activities comparable to those of (+)-JQ1
in targeting BET bromodomains; and 4) discovered certain
tetrazine-TCO-ligated products, for example, TT-1/3, that are
strong binders of CREBBP and GCN5L2, which are non-
BET BRDs.
A key step of SMM fabrication is the site-specific covalent
immobilization of compound libraries with high efficiency.[12]
This is critical for the study of moderate or weak PPIs, such as
those between BRDs and their ligands, for example, between
acetylated peptides and benzodiazepines. Although many
small-molecule immobilization methods have been developed
over the years,[12,13] none of them employs a covalent, site-
specific, instantaneous, and chemoselective reaction under
aqueous conditions to afford the highest possible concen-
tration of immobilized ligand. Our previously developed
strategy of using the biotin–avidin interaction for compound
immobilization met some of these requirements,[11,14] but
failed to provide sufficiently intense signals for fluorescence
detection (not shown), which is likely due to low compound
loading of the surface. Most conventional bioorthogonal
coupling reactions suffer from slow kinetics,[15] including the
Staudinger ligation (k ꢀ 0.002mÀ1 sÀ1), strain-promoted
alkyne–azide cycloadditions (SPAAC; k ꢀ 1mÀ1 sÀ1), and
even the tetrazine norborene ligation (k ꢀ 1–2mÀ1 sÀ1); when
these transformations are used for microarray immobiliza-
tion, they also require relatively long incubation times (2–
24 h).[13,16] This may lead to solvent evaporation, compound
precipitation, and ultimately a decrease in immobilization
efficiency.[12]
To overcome these problems, we turned to the coupling of
trans-cyclooctene (TCO) and tetrazine.[17] With a second-
order rate constant that exceeds 104 mÀ1 sÀ1, it is the fastest
bioorthogonal reaction known. Although this reaction has
been widely used in bioimaging,[17a,b] material chemistry,[17c]
and activity-based protein profiling,[17d] to the best of our
knowledge, it has not been used for the construction of
microarrays, including SMMs. The most closely related
transformation that has been applied for such a process is
the above-mentioned tetrazine norborene ligation for the
preparation of a carbohydrate microarray, which was de-
scribed by Wittmann et al.,[16] and which took 24 h to reach
complete compound immobilization. In our current study, we
prepared tetrazine-functionalized slides by sequential modi-
fications of plain glass slides with (aminopropyl)triethoxysi-
lane, succinic anhydride, and N-hydroxysuccinimide, followed
by treatment with compound 56 (Supporting Information,
Scheme S1) to give the surface shown in Figure 1a. We next
Angew. Chem. Int. Ed. 2013, 52, 14060 –14064
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 14061

.
Angewandte
Communications
spotted FITC-TCO (a model compound) to assess the
efficiency of the immobilization of TCO-modified small
molecules on these tetrazine slides (Figure 1b); time- and
concentration-dependent results both indicate that the immo-
bilization reaction was extremely rapid and highly efficient,
with near-saturation of the FITC-TCO fluorescence observed
within 1–5 min, even at a low spotting concentration (20 mm).
This unprecedented rate for site-specific covalent immobili-
zation of small molecules in a microarray nearly rivals that of
the non-covalent biotin–avidin method (kon ꢀ 105–
106 mÀ1 sÀ1),[18] but this process affords a significantly higher
immobilized-ligand concentration (data not shown).
Upon successful demonstration of this new immobiliza-
tion method, we next focused on SMM construction. We
synthesized a total of 48 different TCO-modified benzodia-
zepines, with structural variations at three key points of the
core pharmacophore (A, B, C; Figure 1a). Based on the
published X-ray structures of (+)-JQ1 and the BRD4(2)
complex (Figure S1),[7a] the substituents at the C6 position
(red) and on the triazole (purple) and thiophene (blue) rings
in (+)-JQ1 engage in critical interactions with the residues
located in the Kac-binding pocket of BRD4(2). The com-
pounds were synthesized over five steps by known proce-
dures[7] with suitable modifications (Figure 1a; see also the
Supporting Information). In this synthesis, the acid-sensitive
TCO was strategically introduced at the last step to ensure
that its integrity was not compromised during the synthesis,
and to simultaneously obtain TCO-free ligands, such as
(+)-25T, which are needed for post-SMM validation experi-
ments.
A total of 50 samples, including 1–48, TCO-OH (57), and
DMSO, were spotted in duplicate, as 12 identical sub-grids on
single-tetrazine-functionalized glass slides, and simultane-
ously screened against different fluorescently labeled proteins
at uniform concentration (1 mm). In total, 5280 PPI events
with 55 different proteins (including 12 human BRDs, 39 SH2
domains, and 4 human 14-3-3 proteins, with the latter two
classes being common phosphorylation readers) against all 48
benzodiazepines were delineated within hours in a single
experiment by using five slides. The corresponding binding
fingerprints are displayed in a color-coded heat map (Fig-
ure 2a), and representative microarray images are shown in
Figure 2b. In general, weak or no binding between the
immobilized compounds and non-BRD proteins was
observed (Figure 2b; PLCG1(C) and PLCG1(N), from two
SH2 domains). This indicates that the pharmacophore in JQ1
and related benzodiazepines is indeed highly selective
towards BRDs, as was previously observed.[7] To our surprise,
almost all immobilized compounds, including TCO-OH,
showed nearly equal and strong fluorescent-ligand binding
towards two non-BET BRDs, namely GCN5L2 and
CREBBP, in a concentration-dependent manner (Figure 2b;
see also Figure S7). This indicates that these bindings are
likely to be specific. In the meantime, we carried out
against the published Kd of JQ1, which was measured by ITC
experiments,[7a] we found reasonably good correlations. For
example, the app Kd values of the 13/BRD4(1) and 13/
BRD4(2) interactions were shown to be 59.4 Æ 20.4 nm and
131.5 Æ 35.4 nm, respectively, which are similar to the values
reported (49.0 Æ 2.4 nm and 90.1 Æ 4.6 nm). We further
showed that this SMM is highly sensitive, as it is capable of
detecting strong BRD-binding ligands at protein concentra-
tions as low as 10 nm (panel 1, Figure 2c). This indicates that
our platform is indeed suitable for sensitive, rapid, semi-
quantitative, and high-throughput Kd determination of BRD–
ligand interactions. Dual-color ratiometric screening, in which
two proteins, each labeled with a spectrally distinct fluoro-
phore, are mixed in equal concentrations and simultaneously
applied on the same microarray, enables rapid identification
of isoform-specific ligands.[14] Herein, we showed that this
process can be used to discriminate between BET bromodo-
mains (e.g., BRD2-4, BRDT) and other BRDs (e.g., PCAF,
TAF1L, WDR9). BRD4(2)-specific ligands, including 1, 13,
25, and 37, could be visually identified (boxed red spots,
Figure 2e). A selectivity score of log2(Cy5-Brd4(2)/Cy3-
BRDX) was obtained for these compounds against all other
BRDs (right graph; Figure 2e).
Previously, the enantiomerically pure (+)-JQ1 was found
to possess promising antitumor activities in cellular assays and
animal models.[7a] It also down-regulates the transcription of
c-Myc (a well-known oncogene that plays a central role in the
pathogenesis of cancer), which subsequently causes genome-
wide down-regulation of Myc-dependent target genes.[20]
Aside from 13 (TCO-containing JQ1), we have identified
several other potent and specific binders of BET bromodo-
mains, including 1, 25, and 37, by the SMM screening. We
were particularly interested in compound 25, as it was the
most potent in terms of the binding to BRD4(2) (app Kd =
92.38 Æ 31.9 nm; Table S2), and it is a close structural ana-
logue of JQ1 (Figure 1). We therefore prepared its TCO-free
enantiomerically pure versions, (+)-25T and (À)-25T, and
tested their cellular activities (Figure 3a). Both (+)-25T and
(+)-JQ1 potently inhibited the growth of MV4-11 (B-
myelomonocytic leukemia) and THP-1 (acute monocytic
leukemia) cells in a dose-dependent manner, with compara-
ble GI50 values (95 Æ 0.9 nm and 39 Æ 6.8 nm for (+)-25T and
(+)-JQ1, respectively; for the THP-1 results, see Figure S11).
Compound (À)-25T, on the other hand, showed no activity at
all. In a fluorescence-activated cell sorting (FACS) experi-
ment, cells treated with both (+)-25Tand (+)-JQ1 showed an
increase in the percentage of G0/G1 cells (Figure 3b). Finally,
(+)-25T, similar to (+)-JQ1, was able to inhibit c-Myc
expression in a both time- and concentration-dependent
manner (Figure 3c). Taken together, these results indicate
that (+)-25T, a compound identified from our newly devel-
oped SMM, is indeed a potent inhibitor of BET bromodo-
mains and possesses cellular activities comparable to those of
(+)-JQ1.[7a,20]
concentration-dependent
microarray
Kd determination
Earlier, we unexpectedly observed that all of the TCO-
immobilized spots on the SMM, including that of TCO-OH
itself, produced strong and concentration-dependent fluores-
cent-ligand binding that was selective for two of the 12 BRDs
screened, namely GCN5L2 and CREBBP (Figure 2 and
against all 12 BRDs (Figure 2c and Figure S6), and the
results were further extrapolated to obtain the corresponding
app Kd (app Kd = apparent Kd; Figure 2d).[19] By comparing
the app Kd of 13, which is the SMM-immobilized form of JQ1,
14062 www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 14060 –14064

Angewandte
Chemie
Figure 2. SMM screening results with different fluorescently labeled proteins, including 12 human BRDs. a) Color heat map displaying the binding
of the 48 compounds with 55 fluorescently labeled proteins (1 mm), with highlighted rectangles showing the binding profiles of 13 with 12 BRDs
(horizontal), and of all 48 compounds with GCN5L2 and CREBBP (vertical). 13 is the racemic form of TCO-containing JQ1. b) Microarray images
that show the different binding profiles of representative proteins. All compounds were spotted in duplicate (vertical), and in a top-to-bottom and
right-to-left manner (see the Supporting Information, Table S1 and S2). TCO-OH (57) and DMSO were spotted at positions 49 and 50,
respectively. PLCG1(C) & PLCG1(N) are two representative SH2 domains. c) Concentration-dependent SMM-based Kd determination of the 48-
member library against fluorescently labeled BRD4(2) (10–5000 nm). The spots corresponding to 13 are highlighted (box; 1000 nm image).
d) Comparison of SMM-determined app Kd of 13T against selected BRDs (extrapolated from Figure 2c) versus Kd values reported in the
literature.[7a] e) Merged microarray images of dual-color SMM screening results by using an equal mixture of Cy5-labeled BRD4(2) (500 nm;
pseudo-colored in red) and another bromodomain (BRDX) that was labeled with Cy3 (500 nm; pseudo-colored in green). Selected spots (1, 13,
25, and 37) were highlighted (in boxes). On the right side, a bar graph shows the selectivity scores, log2(Cy5-BRD4(2)/Cy3-BRDX), of selected
compounds. A score of “0” indicates no selectivity, and “+” indicates BRD4(2)-preferred ligands. ND=not determined, NA=not available.
Figure S7). A plausible explanation is that these spots have
a common structural motif that does not originate from the
benzodiazepines. We speculate that the conjugate produced
by TCO tetrazine ligation (Figure 3d) might constitute such
a novel binder toward GCN5L2 and CREBBP. Both GCN5
(a histone acetyltransferase) and CREBBP (a protein that
binds to the transcriptional activator CREB) are important
cellular proteins. For the non-BET bromodomains within
these proteins, only few small-molecule binders have been
identified thus far.[8c,21] To test whether compounds such as
TT-1/2/3, in which the R group on one of the aromatic
substituents of the tetrazine was varied, could positively bind
to CREBBP and GCN5L2, ITC experiments were carried out
(Figure S12); the results confirmed that all three compounds
bind to both BRDs with Kd values in the low-micromolar
region. We also synthesized FTT (inset, Figure 3d), a fluo-
rescein derivative of TT-2, and confirmed its binding to
GCN5L2 and CREBBP by fluorescence polarization. These
findings are interesting, as they indicate that TT-1/2/3 are
novel BRD-binding pharmacophores for GCN5 and
CREBBP, and that FTT may serve as a good FP probe for
HTS of potential GCN5/CREBBP inhibitors in the future.[9]
However, as other proteins could also bind to the TCO linker,
we would like to point out that future SMM screenings using
our platform should always be carried out with control spots
that contain only TCO.
In conclusion, the bioorthogonal coupling between trans-
cyclooctene (TCO) and tetrazine was shown to be exceed-
ingly useful for the construction of small-molecule micro-
arrays, and to afford immobilized ligands with unprecedented
speed and surface concentration. By using this new chemo-
selective and covalent immobilization method, we have
constructed the first benzodiazepine SMM suitable for
miniaturized and high-throughput studies of potential PPI
inhibitors against a variety of human bromodomains. We have
identified (+)-25T, a structural analog of (+)-JQ1, as a potent
Angew. Chem. Int. Ed. 2013, 52, 14060 –14064
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 14063

.
Angewandte
Communications
Koehler, Chem. Soc. Rev. 2008, 37, 1385 – 1394; d) C. A. Hassig,
S. L. Schreiber, Curr. Opin. Chem. Biol. 1997, 1, 300 – 308.
[6] a) A. J. Wilson, Chem. Soc. Rev. 2009, 38, 3289 – 3300; b) M. R.
Arkin, J. A. Wells, Nat. Rev. Drug Discovery 2004, 3, 301 – 317.
[7] a) P. Filippakopoulos, J. Qi, S. Picaud, Y. Shen, W. B. Smith, O.
Fedorov, E. M. Morse, T. Keates, T. T. Kickman, I. Felletar, M.
Philpott, S. Munro, M. R. McKeown, Y. Wang, A. L. Christie, N.
West, M. J. Cameron, B. Schwartz, T. D. Heightman, N. L.
Thangue, C. A. French, O. Wiest, A. L. Kung, S. Knapp, J. E.
Brandner, Nature 2010, 468, 1067 – 1073; b) E. Nicodeme, K. L.
Jeffrey, U. Schaefer, S. Beinke, S. Dewell, C.-W. Chung, R.
Chandwani, I. Marazzi, P. Wilson, H. Coste, J. White, J.
Kirilovsky, C. M. Rice, J. M. Lora, R. K. Prinjha, K. Lee, A.
Tarakhovsky, Nature 2010, 468, 1119 – 1123.
[8] For recent examples, see: a) C. W. Chung, A. W. Dean, J. M.
Woolven, P. Bamborough, J. Med. Chem. 2012, 55, 576 – 586;
b) D. S. Hewings, M. Wang, M. Philpott, O. Fedorov, S. Uttarkar,
P. Filippakopoulos, S. Picaud, C. Vuppusetty, B. Marsden, S.
Knapp, S. J. Conway, T. D. Herghtman, J. Med. Chem. 2011, 54,
6761 – 6770; c) D. S. Hewings, O. Fedorov, P. Filippakopoulos, S.
Martin, S. Picaud, A. Tumber, C. Wells, M. M. Olcina, K.
Freeman, A. Gill, A. J. Ritchie, D. W. Sheppard, A. J. Russell,
E. M. Hammond, S. Knapp, P. E. Brennan, S. J. Conway, J. Med.
Chem. 2013, 56, 3217 – 3227.
[9] C.-W. Chung, J. Witherington, J. Biomol. Screening 2012, 16,
1170 – 1185.
[10] P. Filippakopoulos, S. Picaud, M. Mangos, T. Keates, J.-P.
Lambert, D. Barsyte-Lovejoy, I. Felletar, R. Volkmer, S.
Muller, T. Pawson, A.-C. Gingras, C. H. Arrowsmith, S.
Knapp, Cell 2012, 149, 214 – 231.
[11] H. Wu, J. Ge, S. Q. Yao, Angew. Chem. 2010, 122, 6678 – 6682;
Angew. Chem. Int. Ed. 2010, 49, 6528 – 6532.
[12] Y. M. Foong, J. Fu, S. Q. Yao, M. Uttamchandani, Curr. Opin.
Chem. Biol. 2012, 16, 234 – 242.
Figure 3. a) Dose-response growth curves of MV4-11 cells treated with
(+)-JQ1, (+)-25T, or (À)-25T for 72 h. b) FACS results of MV4-11 cells
upon treatment with DMSO, (+)-JQ1, (+)-25T, or (À)-25T (500 nm)
for 24 h. The numbers denote the percentage of cells in the G0/G1
phase. c) Western blot analysis of c-Myc expression in MV4-11 cells
upon treatment with different compounds. MV4-11 cells were treated
with each compound (500 nm) or DMSO for 24 h (Top image).
Concentration- (middle) and time-dependent (bottom) c-Myc expres-
sion in MV4-11 cells that were treated with (+)-25T. d) Structures of
TT-1/2/3, which were obtained by ligation between the three structur-
ally different tetrazines and TCO-OH, and the FP probe FTT (box).
NA=not available.
[13] a) H. Wu, J. Ge, M. Uttamchandani, S. Q. Yao, Chem. Commun.
2011, 47, 5664 – 5670; b) M. Uttamchandani, J. Wang, S. Q. Yao,
Mol. BioSyst. 2006, 2, 58 – 68.
[14] C. H. S. Lu, H. Y. Sun, F. B. Abu Bakar, M. Uttamchandani, W.
Zhou, Y. C. Liou, S. Q. Yao, Angew. Chem. 2008, 120, 7548 –
7551; Angew. Chem. Int. Ed. 2008, 47, 7438 – 7441.
[15] E. M. Sletten, C. R. Bertozzi, Angew. Chem. 2009, 121, 7108 –
7133; Angew. Chem. Int. Ed. 2009, 48, 6974 – 6998.
inhibitor for BET bromodomains, both in vitro and in cell-
based assays. During the course of our study, we unexpectedly
discovered that the ligation products of TCO and tetrazine,
namely TT-1/2/3, relatively strongly and selectively bind to
bromodomains of CREBBP and GCN5. The corresponding
fluorescein-functionalized compound (FTT) may be used for
HTS of potential PPI inhibitors in the future.
[16] H. S. G. Beckmann, A. Niederwieser, M. Wiessler, V. Wittmann,
Chem. Eur. J. 2012, 18, 6548 – 6554.
[17] a) N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand, R.
Weissleder, Angew. Chem. 2009, 121, 7147 – 7150; Angew. Chem.
Int. Ed. 2009, 48, 7013 – 7016; b) N. K. Devaraj, S. Hilderbrand,
R. Upadhyay, R. Mazitschek, R. Weissleder, Angew. Chem.
2010, 122, 2931 – 2934; Angew. Chem. Int. Ed. 2010, 49, 2869 –
2872; c) J. B. Haun, N. K. Devaraj, S. A. Hilderbrand, H. Lee, R.
Weissleder, Nat. Nanotechnol. 2010, 5, 660 – 665; d) K. S. Yang,
G. Budin, C. Tassa, O. Kister, R. Weissleder, Angew. Chem. 2013,
125, 10787 – 10791; Angew. Chem. Int. Ed. 2013, 52, 10593 –
10597.
Received: September 5, 2013
Revised: September 30, 2013
Published online: November 4, 2013
Keywords: bioorthogonal chemistry · epigenetics · inhibitors ·
.
protein–protein interactions · small-molecule microarrays
[18] R. Y. P. Lue, G. Y. J. Chen, Y. Hu, Q. Zhu, S. Q. Yao, J. Am.
Chem. Soc. 2004, 126, 1055 – 1062.
[19] M. Uttamchandani, W. L. Lee, J. Wang, S. Q. Yao, J. Am. Chem.
Soc. 2007, 129, 13110 – 13117.
[1] a) S. L. Berger, Nature 2007, 447, 407 – 412; b) T. Kouzarides,
Cell 2007, 128, 693 – 705.
[2] K. K. Lee, J. L. Workman, Nat. Rev. Mol. Cell Biol. 2007, 8, 284 –
295.
[3] C. H. Arrowsmith, C. Bountra, P. V. Fish, K. Lee, M. Schapira,
Nat. Rev. Drug Discovery 2012, 11, 384 – 400.
[4] A. C. Belkina, G. V. Denis, Nat. Rev. Cancer 2012, 12, 465 – 477.
[5] a) S. Minucci, P. G. Pelicci, Nat. Rev. Cancer 2006, 6, 38 – 51;
b) J. L. Duffner, P. A. Clemons, A. N. Koehler, Curr. Opin.
Chem. Biol. 2007, 11, 74 – 82; c) A. J. Vegas, J. H. Fuller, A. N.
[20] J. E. Delmore, G. C. Issa, M. E. Lemieux, P. B. Rahl, J. Shi, H. M.
Jacobs, E. Kastritis, T. Gilpatrick, R. M. Paranal, J. Qi, M. Chesi,
A. C. Schinzel, M. R. McKeown, T. P. Heffernan, C. R. Vakoc,
P. L. Bergsagel, I. M. Ghobrial, P. G. Richardson, R. A. Young,
W. C. Hahn, K. C. Anderson, A. L. Kung, J. E. Bradner, C. S.
Mitsiades, Cell 2011, 146, 904 – 917.
[21] C. W. Chung, D. F. Tough, Drug Discovery Today Ther. Strategies
2012, 9, e111 – 120.
14064 www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 14060 –14064