Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents

Article abstract of DOI:10.1002/anie.201505641

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bea

Full text of DOI:10.1002/anie.201505641

.
Angewandte
Communications
International Edition: DOI: 10.1002/anie.201505641
German Edition: DOI: 10.1002/ange.201505641
Protein Profiling
Proteome-Wide Profiling of Targets of Cysteine reactive Small
Molecules by Using Ethynyl Benziodoxolone Reagents
Daniel Abegg, Reto Frei, Luca Cerato, Durga Prasad Hari, Chao Wang, Jerome Waser, and
Alexander Adibekian*
Abstract: In this study, we present a highly efficient method for
proteomic profiling of cysteine residues in complex proteomes
and in living cells. Our method is based on alkynylation of
cysteines in complex proteomes using a “clickable” alkynyl
benziodoxolone bearing an azide group. This reaction pro-
ceeds fast, under mild physiological conditions, and with a very
high degree of chemoselectivity. The formed azide-capped
alkynyl–cysteine adducts are readily detectable by LC-MS/MS,
and can be further functionalized with TAMRA or biotin
alkyne via CuAAC. We demonstrate the utility of alkynyl
benziodoxolones for chemical proteomics applications by
identifying the proteomic targets of curcumin, a diarylhepta-
noid natural product that was and still is part of multiple
human clinical trials as anticancer agent. Our results demon-
strate that curcumin covalently modifies several key players of
cellular signaling and metabolism, most notably the enzyme
casein kinase I gamma. We anticipate that this new method for
cysteine profiling will find broad application in chemical
proteomics and drug discovery.
lent results on peptides and recombinant proteins, only very
few of them have been shown to be also effective in labeling
cysteines in cells or cellular lysates and therefore suitable for
proteomics applications. Iodoacetamide (IAA) is widely
accepted as gold standard among MS-compatible cysteine-
reactive chemical probes,[6] but it displays rather low chemical
stability in aqueous buffers and it only reacts with a fraction of
functional cysteines.[7] Moreover, iodoacetamide also modi-
fies lysines and the formed artifacts lead to misassignment of
lysine ubiquitination sites by mass spectrometry.[8] Thus, there
is a clear need for new electrophilic probes complementary to
IAA that would enable labeling of so far “inaccessible”
subsets of proteomic cysteines, which would then allow more
comprehensive target profiling. Herein, we present discovery
and an in-depth evaluation of a “clickable” alkynyl benz-
iodoxolone as a highly efficient and chemoselective cysteine-
reactive probe with an iodoacetamide-complementary pro-
teomic profile.
Small-molecule thiols are readily alkynylated by alkynyl
benziodoxolones (EBX reagents).[9] This class of reagents is
selective for thiols in presence of other nucleophilic func-
tional groups such as aromatic rings, alcohols, amines or
carboxylates. While these transformations were fast, high-
yielding and air-tolerant, they were performed on dipeptides
in water/organic solvent mixtures and required stoichiometric
amounts of base. Thus, reaction of the alkynyl benziod-
oxolones with cysteines directly on proteins and under native
physiological conditions would constitute a formidable chal-
lenge. We chose the azide-functionalized alkynyl benziod-
oxolone JW-RF-010 for our studies, as the azide group can be
used for further functionalization via the copper(I)-catalyzed
alkyne–azide cycloaddition (CuAAC), whereas the obtained
internal thioalkynes are usually inert in copper-catalyzed
cycloadditions (Figure 1A). Compared to fluorescent dye-
and biotin-conjugated ABPP probes that have poor cell
membrane permeability and are too bulky to enable efficient
binding to some of the proteins, such two-step labeling
approach is advantageous for applications in cells[3b] and
animals[10] and is now widely used.
D
espite their relatively low abundance in proteins, cysteines
are vital for cellular biochemistry. For example, cysteines
form disulfide bridges, are actively involved in enzyme
catalysis as nucleophiles and can be posttranslationally
oxidized or modified through palmitoylation, prenylation or
nitrosylation.[1] Many of the so-called functional cysteines
display pronounced hyperreactivity and these reactive hot
spots can be precisely identified in complex proteomes with
broad-range electrophilic probes.[2] Such probes represent
particularly useful tools for competitive activity-based protein
profiling[3] (ABPP) of targets of covalently binding cysteine-
reactive drugs and metabolites, as impressively demonstrated
in recent publications.[4] Over the past few years, a broad
variety of novel methods for broad-spectrum, yet chemo-
selective targeting of cysteine residues have been reported.[5]
However, while most of these transformations deliver excel-
[*] D. Abegg, L. Cerato, C. Wang, Prof. Dr. A. Adibekian
School of Chemistry and Biochemistry
NCCR Chemical Biology, University of Geneva
30 quai Ernest-Ansermet, Geneva (Switzerland)
E-mail: alexander.adibekian@unige.ch
Before starting with the labeling experiments, it was
important to test the aqueous stability of JW-RF-010 by
obtaining 1H NMR spectra of this reagent in D2O over
prolonged period of time. Even after 14 days in D2O, only
traces (< 3%) of decomposition were detectable (Figure S1 in
the Supporting Information). Next, we compared the gluta-
thione (GSH)-directed reactivity between the azide-function-
alized alkynyl benziodoxolone JW-RF-010 and iodoaceta-
mide through a colorimetric GSH-binding assay (Figure S2).
While both probes showed rather moderate activity in this
Dr. R. Frei, Dr. D. Prasad Hari, Prof. Dr. J. Waser
Laboratory of Catalysis and Organic Synthesis
Ecole Polytechnique FØdØrale de Lausanne
EPFL SB ISIC LCSO, BCH 4306, 1015 Lausanne (Switzerland)
Supporting information for this article (synthesis of compounds, cell
culture, bioassays, labeling and sample preparation for proteomics,
and mass spectrometry) is available on the WWW under http://dx.
doi.org/10.1002/anie.201505641.
10852
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 10852 –10857

Angewandte
Chemie
Figure 1. Evaluating the proteomic reactivity of JW-RF-010 by SDS-PAGE. A) A general reaction scheme showing alkynylation of cysteine-containing
proteins by JW-RF-010. B) Gel-based fluorescence image of HeLa cell lysates labeled with various concentrations of JW-RF-010 or iodoacetamide
alkyne (left) and quantification of the fluorescence labeling (right). C) Gel-based fluorescence image after labeling of living HeLa cells with various
concentrations of JW-RF-010.
experiment, we observed a twofold higher activity for JW-RF-
010 (RC50 = 75 mm) compared to IAA (RC50 = 145 mm).
Interestingly, when monitoring this transformation by mass
spectrometry we did not detect increased disulfide bond
formation or any other cysteine oxidation as possible side
reactions (Figure S3). We then compared the efficiency of
labeling of purified protein catalase by an EBX reagent versus
iodoacetamide using a gel-based competitive assay (Fig-
ure S4). Here, catalase was labeled with 5 mm 2-iodo-N-(prop-
2-yn-1-yl)acetamide (iodoacetamide alkyne), then reacted
with carboxymethylrhodamine (TAMRA) azide through
CuAAC, and visualized by in-gel fluorescence scanning
after SDS-PAGE. In a competitive labeling with the methyl
alkyne-substituted EBX reagent JW-RF-001 a ca. 3 to 5-fold
higher efficiency compared to iodoacetamide was observed
(Figure S4). To assure that the alkynyl cysteine adduct is
stable under physiological conditions even after longer period
of time, we labeled catalase with 1 mm of JW-RF-010, then
removed the excess of JW-RF-010 by membrane filtration
and incubated the reacted protein for various amounts of time
prior to “clicking” with TAMRA alkyne and visualization by
SDS-PAGE (Figure S5). Even after 18 h of incubation, no
noticeable loss of fluorescence was detectable, implying that
the formed alkynyl cysteine adduct is indeed stable under the
tested conditions.
lysates. We started by incubating HeLa cell lysates in
phosphate-buffered saline with different concentrations of
JW-RF-010 for 1 h. Multiple fluorescent bands were detected
even at submicromolar concentrations of JW-RF-010 and
clear concentration-dependent increase in labeling intensity
was observed (Figure 1B, S6). With iodoacetamide alkyne as
probe, an about fivefold lower overall fluorescence intensity
compared to JW-RF-010-treated proteomes was observed.
Moreover, the higher proteome reactivity of EBX reagents
compared to iodoacetamide (IAA) was also clearly observ-
able in cross-competition experiments (Figure S7). Here,
pretreatment with high concentrations of JW-RF-001 com-
pletely abolished proteome labeling by iodoacetamide
alkyne, but not vice versa. Interestingly, while the overall
labeling pattern of both probes showed significant degree of
similarity, several fluorescence bands appeared to be specif-
ically present in JW-RF-010-labeled proteomes.
We next examined whether JW-RF-010 could also be used
for labeling proteomes directly in living cells. Because broad
range cysteine-reactive electrophilic probes are intrinsically
cytotoxic,[11] we compared the cytotoxicities of JW-RF-010
and IAA in HeLa cells using WST-1 assay (Figure S8).
Whereas significant cytotoxicity was observed after 20 h in
cells treated with 1 mm IAA, JW-RF-010 did not show any
cellular toxicity at concentrations up to 1 mm, while at 3 mm
significant cell death was observed. We then tested whether
JW-RF-010 could permeate the cellular membranes and react
with the intracellular proteome. HeLa cells were treated with
Having demonstrated the efficiency of the alkynyl benz-
iodoxole probes on both peptides and proteins, the stage was
now ready for the much more challenging labeling in total cell
Angew. Chem. Int. Ed. 2015, 54, 10852 –10857
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 10853

.
Angewandte
Communications
1 mm JW-RF-010 for 4 h, fixed and TAMRA alkyne and
CuAAC reagents were added. The obtained confocal micros-
copy images showed homogeneous intracellular TAMRA
labeling in JW-RF-010 treated cells, while no significant
fluorescence was detected in cells treated with DMSO as
control (Figure S9). To visualize the proteomic targets by gel-
based ABPP, HeLa cells were again treated with JW-RF-010
for 4 h, lysed, and the probe-labeled proteins were reacted
with TAMRA alkyne and separated by SDS-PAGE. The gel
scan documented strong fluorescence labeling at the concen-
tration as low as 100 nm (Figure 1C, S6), thus well below the
concentration toxic to cells.
Having successfully established proteome labeling in vitro
and in situ, we continued our studies by comparing the exact
modification sites of JW-RF-010 and IAA alkyne in complex
proteomes using a capture-and-release strategy coupled with
LC-MS/MS analysis that allowed us specific enrichment and
detection of probe-labeled peptides (Figure 2A). In total,
2257 cysteine-containing peptides were identified as enriched
in proteomes labeled by 10 mm JW-RF-010 and 2184 peptides
were enriched by 10 mm IAA alkyne. While 1391 peptides
were commonly found in both datasets, 866 peptides (38%)
were only enriched by JW-RF-010 (Figure 2B and Table S1.
Thus, the proteomic profile of JW-RF-010 shows significant
complementarity to IAA alkyne to allow more comprehen-
sive profiling of proteomic cysteines. To exclude the possi-
bility of labeling of amino acids other than cysteines as
possible reason for the large amount of unique peptides
observed, the resulting MS data were searched for mass
adducts on all nucleophilic amino acids (Cys, Asp, Glu, His,
Lys, Ser, Thr, Tyr) using the Sequest HT algorithm[12] and the
percentage of unique peptides carrying the covalent modifi-
cation on each amino acid was determined (Figure 2C and
Tables S1, S2). Gratifyingly, formed alkynyl adducts were
predominantly found on cysteines (97%). For comparison, we
observed significantly lower cysteine selectivity with 10 mm
IAA alkyne as probe (91.4%). This is an important advantage
of the EBX probes, as they will limit the presence of “false
positives” in proteomics experiments.[8]
Finally, to demonstrate the utility of our method for drug
target discovery applications, we used JW-RF-010 to identify
the proteomic targets of curcumin (Figure 3A). Curcumin,
a diarylheptanoid natural product isolated from turmeric
(Curcumin longa), has been extensively studied over the last
few decades as an anticancer and an anti-inflammatory
agent.[13] Due to these intriguing biomedical properties and
also the fact that even multigram high dosages of curcumin
are well tolerated by humans, curcumin has been part of many
human clinical trials including multiple myeloma, colon and
pancreatic cancer.[14] While countless studies have been
performed with the goal of iden-
tification of entire biochemical
and signaling pathways modu-
lated by curcumin,[15] surprisingly
little is known about the direct
molecular targets of this natural
product.[16] For instance, several
studies have demonstrated that
curcumin treatment results in
decrease of phosphorylation of
S473 in Akt,[17] a modification
that is crucial for the full activa-
tion of this potent oncogene. Akt
is a suppressor of apoptosis and
a major target for anticancer drug
discovery, but the molecular link
between curcumin and Akt is not
understood. Examining the chem-
ical structure of curcumin made
us speculate whether possible
covalent bond formation between
the a,b-unsaturated ketone group
of this natural product and the
proteomic cysteines could poten-
tially explain the connection to
Akt signaling and also many
other intriguing bioactivities of
curcumin.
We first tested and confirmed
Figure 2. Comparative evaluation of JW-RF-010 versus iodoacetamide alkyne using chemical enrich-
ment and LC-MS/MS analysis. A) A capture-and-release strategy using a photocleavable biotin alkyne
linker for detection of exact probe modification sites in complex proteomes. B) A Venn diagram of
cysteine-containing peptides enriched by 10 mm JW-RF-010 versus 10 mm iodoacetamide alkyne (n=4).
C) Chemoselectivities observed in HeLa lysates using 10 mm JW-RF-001 versus 10 mm IAA alkyne
(n=4). Error bars represent standard deviation (SD).
the cytotoxic effect of curcumin
on HeLa cells using the WST-
1 assay (Figure 3B, S10). Using
the caspase-directed fluorescent
probe FITC-VAD-FMK and con-
10854 www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 10852 –10857

Angewandte
Chemie
Figure 3. Effect of curcumin treatment on HeLa cells. A) Chemical structure of curcumin. B) HeLa cell viability curve after treatment with various
concentrations of curcumin (n=3). C) Detection of caspase activation and apoptosis in HeLa cells after treatment with 1 mm staurosporine
(positive control) or 10 mm curcumin using FITC-VAD-FMK fluorescent marker (confocal microscopy images are shown). D) Gel-based competitive
profiling of curcumin targets after treatment of HeLa cell lysates with 30 mm curcumin and using 10 mm JW-RF-010 or 10 mm iodoacetamide alkyne
as cysteine-reactive probes. The contrast of the IAA alkyne gel was adjusted for better comparison between both probes.
focal microscopy, we determined that the cell death is caused
by caspase activation and subsequent apoptosis (Figure 3C).
A gel-based competition experiment in HeLa cells employing
10 mm IAA alkyne or 10 mm JW-RF-010 as probes evidenced
disappearance of few protein bands upon pretreatment with
30 mm curcumin, suggesting that curcumin may indeed have
reacted with proteomic cysteines (Figure 3D).
Based on this observation, we then performed a compet-
itive LC-MS/MS experiment using both cysteine-reactive
probes at 10 mm concentration and employing the capture-
and-release strategy described above coupled with the
method of stable isotope labeling of amino acids in culture
(SILAC).[18] A detailed experimental workflow is shown in
Figure 4A. In total, 2689 different cysteine-modified peptides
were enriched from HeLa cells by both probes and quantified
(Table S3), but labeling of only 57 of 2689 peptides,
corresponding to 57 different proteins, was ꢀ 75% competed
by 30 mm curcumin (SILAC ratio ꢁ 0.25). To further confirm
the identified targets we synthesized an alkyne-tagged
derivative of curcumin and confirmed this probeꢀs bioactivity
in HeLa cells using the WST-1 assay (Figure S11). We then
treated HeLa cells with 30 mm curcumin alkyne and identified
the “click”-enriched proteins by LC-MS/MS (Table S4).
Although enrichment with curcumin alkyne allowed identify-
ing and confirming target proteins, in contrast to our
comparative profiling approach, it did not provide the exact
modification sites due to complex fragmentation pattern of
cysteine–curcumin adducts and some high abundance pro-
teins, known as contaminants in proteomic pulldown experi-
ments, were also enriched by this probe. In total, 42 of 57
(74%) initially identified candidate curcumin targets were
also enriched by curcumin alkyne (Figure 4B and Table S5).
To the best of our knowledge, only 1 of 42 identified proteins
has been reported as direct biological target of curcumin
before.[16a] Remarkably, among the 42 targets, 19 proteins
were only enriched by iodoacetamide alkyne and 16 proteins
were exclusively enriched by JW-RF-010, further underscor-
ing the importance of using multiple cysteine-reactive probes
for more comprehensive proteome-wide target profiling. For
instance, MAP2K4 and MAP2K6, both members of the same
family of mitogen-activated protein kinase kinases, were
enriched exclusively by IAA alkyne or JW-RF-010, respec-
tively (Figure 4B).
Angew. Chem. Int. Ed. 2015, 54, 10852 –10857
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 10855

.
Angewandte
Communications
Figure 4. Identification of molecular targets of curcumin in HeLa cells using cysteine-reactive probes JW-RF-010 (10 mm) and IAA alkyne (10 mm)
and SILAC proteomics. A) Experimental workflow for the competitive proteomic profiling of curcumin targets in SILAC-labeled HeLa cells.
B) Selected proteomic targets of curcumin in HeLa cells. The corresponding enrichment probes and the “light”/“heavy” SILAC ratiosÆSD are
indicated (n=3). C) Protein structure of H. sapiens CSNK1G3 (PDB: 2IZS) with the P loop displayed in blue and Cys51 displayed in red. The image
was created using PyMOL (V1.7.2.1). D) Recombinant human CSNK1G3 is labeled by JW-RF-010, but not by IAA alkyne (both 10 mm; top).
CSNK1G3 is labeled by 10 mm curcumin alkyne and the labeling is competed by curcumin in concentration-dependent manner (middle). The
labeling of CSNK1G3 by 30 mm curcumin alkyne is also competed by ATP in concentration-dependent manner (bottom).
Most intriguingly, one of the identified targets, casein
kinase I gamma (CSNK1G, all three isoforms), may provide
a direct mechanistic link to Akt phosphorylation. The protein
kinase CSNK1G isoform 3 (CSNK1G3, EC:2.7.11.1) was
shown to directly participate in Akt signaling and siRNA
knockdown of CSNK1G3 reduced the phosphorylation on
S473 of Akt.[19] CSNK1G3 knockdown in combination with an
Akt inhibitor treatment also resulted in improved killing of
renal carcinoma cells.[19] In our LC-MS/MS-based proteomic
profiling experiments CSNK1G was labeled exclusively by
JW-RF-010. The modification was identified on Cys51 that is
located in the glycine-rich loop (P loop) in the immediate
proximity to the ATP-binding pocket of this kinase (Fig-
ure 4C, S12, S13) and was ꢀ 90% competed by curcumin
pretreatment. Cysteine residues in P loops of other protein
kinases are well known as binding sites for some ATP
competitive, covalent kinase inhibitors, for example, pan-
FGFR inhibitors.[20] As expected, purified human CSNK1G3
was labeled by 10 mm JW-RF-010, but not by 10 mm iodoacet-
amide alkyne (Figure 4D, top and S14). We confirmed
CSNK1G3 as direct molecular target of curcumin by labeling
the protein with curcumin alkyne and competing this labeling
with curcumin (Figure 4D, middle and S14). Labeling by
curcumin alkyne was also competed by ATP in dose-
dependent manner (Figure 4D, bottom and S14), confirming
that curcumin does indeed bind in the close proximity to the
ATP-binding pocket of CSNK1G3. Hence, treatment with
30 mm curcumin also inhibited ATP hydrolysis by recombi-
10856 www.angewandte.org
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 10852 –10857

Angewandte
Chemie
[5] a) G. J. Bernardes, J. M. Chalker, J. C. Errey, B. G. Davis, J. Am.
nant CSNK1G3 (Figure S15), indicating that curcumin not
only binds, but also inhibits the catalytic activity of
CSNK1G3. More detailed biochemical follow-up studies are
currently in progress in our laboratories and these results will
be reported in due course.
Chem. Soc. 2008, 130, 5052 – 5053; b) M. E. Smith, F. F. Schu-
macher, C. P. Ryan, L. M. Tedaldi, D. Papaioannou, G. Waks-
man, S. Caddick, J. R. Baker, J. Am. Chem. Soc. 2010, 132, 1960 –
1965; c) F. Li, A. Allahverdi, R. Yang, G. B. Lua, X. Zhang, Y.
Cao, N. Korolev, L. Nordenskiold, C. F. Liu, Angew. Chem. Int.
Ed. 2011, 50, 9611 – 9614; Angew. Chem. 2011, 123, 9785 – 9788;
d) N. Toda, S. Asano, C. F. Barbas 3rd, Angew. Chem. Int. Ed.
2013, 52, 12592 – 12596; Angew. Chem. 2013, 125, 12824 – 12828;
e) A. M. Spokoyny, Y. Zou, J. J. Ling, H. Yu, Y. S. Lin, B. L.
Pentelute, J. Am. Chem. Soc. 2013, 135, 5946 – 5949; f) A. Abbas,
B. Xing, T. P. Loh, Angew. Chem. Int. Ed. 2014, 53, 7491 – 7494;
Angew. Chem. 2014, 126, 7621 – 7624; g) P. M. Cal, G. J. Ber-
nardes, P. M. Gois, Angew. Chem. Int. Ed. 2014, 53, 10585 –
10587; Angew. Chem. 2014, 126, 10758 – 10760.
In summary, we have presented the discovery and detailed
proteomic evaluation of EBX reagents as biocompatible
cysteine-reactive chemical probes. A range of advantageous
properties render them desirable probes for many life science
applications. EBX reagents are stable in water and cysteine
alkynylation can be performed under physiological conditions
and with high chemoselectivity. Moreover, the described
modification can be performed directly in living cells with up
to micromolar probe concentrations. Finally, our method-
ology is especially well suited for chemical proteomics
applications, because the formed thioalkyne adducts are
readily detected in routine LC-MS/MS experiments and,
when the probe contains an azide group, can be further
functionalized via CuAAC. We demonstrated the utility of
alkynyl benziodoxolones as useful, iodoacetamide-comple-
mentary probes for chemical proteomics by successfully
identifying the biological targets of curcumin in HeLa cells.
These results will hopefully provoke new drug discovery
endeavors towards identification of pharmacologically
improved analogs of curcumin as promising anticancer drugs.
[6] D. A. Shannon, E. Weerapana, Curr. Opin. Chem. Biol. 2015,
24C, 18 – 26.
[7] H. L. Wong, D. C. Liebler, Chem. Res. Toxicol. 2008, 21, 796 –
804.
[8] M. L. Nielsen, M. Vermeulen, T. Bonaldi, J. Cox, L. Moroder, M.
Mann, Nat. Methods 2008, 5, 459 – 460.
[9] R. Frei, M. D. Wodrich, D. P. Hari, P. A. Borin, C. Chauvier, J.
Waser, J. Am. Chem. Soc. 2014, 136, 16563 – 16573.
[10] A. Adibekian, B. R. Martin, J. W. Chang, K. L. Hsu, K. Tsuboi,
D. A. Bachovchin, A. E. Speers, S. J. Brown, T. Spicer, V.
Fernandez-Vega, J. Ferguson, P. S. Hodder, H. Rosen, B. F.
Cravatt, J. Am. Chem. Soc. 2012, 134, 10345 – 10348.
[11] M. J. Plewa, E. D. Wagner, S. D. Richardson, A. D. Thruston,
Y. T. Woo, A. B. McKague, Environ. Sci. Technol. 2004, 38,
4713 – 4722.
[12] J. K. Eng, A. L. Mccormack, J. R. Yates, J. Am. Soc. Mass
Spectrom. 1994, 5, 976 – 989.
[13] a) A. Goel, A. B. Kunnumakkara, B. B. Aggarwal, Biochem.
Pharmacol. 2008, 75, 787 – 809; b) B. B. Aggarwal, A. Kumar,
A. C. Bharti, Anticancer Res. 2003, 23, 363 – 398.
[14] C. H. Hsu, A. L. Cheng, Molecular Targets and Therapeutic Uses
of Curcumin in Health and Disease, Vol. 595, Springer, Heidel-
berg, 2007, pp. 471 – 480.
[15] M. K. Shanmugam, G. Rane, M. M. Kanchi, F. Arfuso, A.
Chinnathambi, M. E. Zayed, S. A. Alharbi, B. K. H. Tan, A. P.
Kumar, G. Sethi, Molecules 2015, 20, 2728 – 2769.
[16] a) L. S. Angelo, D. S. Maxwell, J. Y. Wu, D. L. Sun, D. H. Hawke,
I. E. McCutcheon, J. M. Slopis, Z. H. Peng, W. G. Bornmann, R.
Kurzrock, Bioorg. Med. Chem. 2013, 21, 932 – 939; b) Z. Firouzi,
P. Lari, M. Rashedinia, M. Ramezani, M. Iranshahi, K. Abnous,
Life Sci. 2014, 98, 12 – 17.
[17] a) C. S. Beevers, F. J. Li, L. Liu, S. L. Huang, Int. J. Cancer 2006,
119, 757 – 764; b) C. Wang, X. Zhang, Z. P. Teng, T. Zhang, Y. Li,
Eur. J. Pharmacol. 2014, 740, 312 – 320.
Acknowledgements
University of Geneva, EPFL, Swiss National Science Foun-
dation and the NCCR Chemical Biology are acknowledged
for financial support. The work of R.F. was further supported
by a Marie Curie International Incoming Fellowship (Grant
Number 331631). Dr. Fides Benfatti and Marie-Madeleine
Stempien from Syngenta Crop Protection Münchwilen AG
are kindly acknowledged for providing DSC measurements.
Keywords: activity-based protein profiling · curcumin ·
mass spectrometry · proteomics · target identification
How to cite: Angew. Chem. Int. Ed. 2015, 54, 10852–10857
Angew. Chem. 2015, 127, 11002–11007
[1] S. M. Marino, V. N. Gladyshev, J. Mol. Biol. 2010, 404, 902 – 916.
[2] E. Weerapana, C. Wang, G. M. Simon, F. Richter, S. Khare, M. B.
Dillon, D. A. Bachovchin, K. Mowen, D. Baker, B. F. Cravatt,
Nature 2010, 468, 790 – 795.
[3] a) M. Uttamchandani, C. H. S. Lu, S. Q. Yao, Acc. Chem. Res.
2009, 42, 1183 – 1192; b) L. I. Willems, W. A. Van der Linden, N.
Li, K. Y. Li, N. Liu, S. Hoogendoorn, G. A. Van der Marel, B. I.
Florea, H. S. Overkleeft, Acc. Chem. Res. 2011, 44, 718 – 729;
c) G. C. Rudolf, W. Heydenreuter, S. A. Sieber, Curr. Opin.
Chem. Biol. 2013, 17, 110 – 117.
[18] S. E. Ong, B. Blagoev, I. Kratchmarova, D. B. Kristensen, H.
Steen, A. Pandey, M. Mann, Mol. Cell. Proteomics 2002, 1, 376 –
386.
[19] S. Morgan-Lappe, K. W. Woods, Q. Li, M. G. Anderson, M. E.
Schurdak, Y. Luo, V. L. Giranda, S. W. Fesik, J. D. Leverson,
Oncogene 2006, 25, 1340 – 1348.
[20] Q. S. Liu, Y. Sabnis, Z. Zhao, T. H. Zhang, S. J. Buhrlage, L. H.
Jones, N. S. Gray, Chem. Biol. 2013, 20, 146 – 159.
[4] a) E. Weerapana, G. M. Simon, B. F. Cravatt, Nat. Chem. Biol.
2008, 4, 405 – 407; b) C. Wang, E. Weerapana, M. M. Blewett,
B. F. Cravatt, Nat. Methods 2014, 11, 79 – 85.
Received: June 18, 2015
Revised: July 7, 2015
Published online: July 24, 2015
Angew. Chem. Int. Ed. 2015, 54, 10852 –10857
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 10857