Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles

Article abstract of DOI:10.1021/jacs.7b01791

Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this process is the identification of signaling pathways (or receptors) specific to (or overexpressed in) disease cells. In this context, fragment-base

Full text of DOI:10.1021/jacs.7b01791

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Article
Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles
Vinayak Gupta, Jing Yang, Daniel C. Liebler, and Kate S. Carroll
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b01791 • Publication Date (Web): 29 Mar 2017
Downloaded from http://pubs.acs.org on March 31, 2017
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Diverse Redoxome Reactivity Profiles of Carbon Nucleophiles
Vinayak Gupta,‡, [1] Jing Yang,‡, [2] Daniel C. Liebler,[3] and Kate S. Carroll*, [1]
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[1] Department of Chemistry, The Scripps Research Institute, Jupiter, Florida 33458, USA.
[2] State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences, Beijing
102206, China.
[3] Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
ABSTRACT: Targeted covalent inhibitors have emerged as a powerful approach in the drug discovery pipeline. Key to this pro-
cess is the identification of signaling pathways (or receptors) specific to (or over-expressed in) disease cells. In this context, frag-
ment-based ligand discovery (FBLD) has significantly expanded our view of the ligandable proteome and affords tool compounds
for biological inquiry. To date, such covalent ligand discovery has almost exclusively employed cysteine-reactive small-molecule
fragments. However, functional cysteine residues in proteins are often redox-sensitive and can undergo oxidation in cells. Such
reactions are particularly relevant in diseases, like cancer, which are linked to excessive production of reactive oxygen species
(ROS). Once oxidized, the sulfur atom of cysteine is much less reactive toward electrophilic groups used in the traditional FBLD
paradigm. To address this limitation, we recently developed a novel library of diverse carbon-based nucleophile fragments that
react selectively with cysteine sulfenic acid (Cys-SOH) formed in proteins via oxidation or hydrolysis reactions. Here, we report
analysis of sulfenic acid-reactive C-nucleophile fragments screened against a colon cancer cell proteome. Covalent ligands were
identified for >1280 S-sulfenylated cysteines present in ‘druggable’ proteins and orphan targets, revealing disparate reactivity pro-
files and target preferences. Among the unique ligand-protein interactions identified was that of a pyrrolidinedione nucleophile,
PYD that reacted preferentially with protein tyrosine phosphatases (PTPs). Fragment-based covalent ligand discovery with C-
nucleophiles affords an expansive snapshot of the ligandable ‘redoxome’ with significant implications for covalent inhibitor phar-
macology and also affords new chemical tools to investigate redox-regulation of protein function.
ꢀ
INTRODUCTION
also be scavenged by ubiquitous low-molecular-weight nucle-
ophiles such as glutathione (GSH). Moreover, current TCIs
predominantly target protein cysteines and among many nu-
cleophiles present in the cellular system, cysteine thi-
ols/thiolates (Cys-SH/S-) are primary targets for the redox-
based modulation of protein activity. Oxidation of a protein
cysteine thiol (Cys-SH) to sulfenic acid (Cys-SOH) by reac-
tive oxygen species (ROS) (e.g., H2O2) is termed S-
sulfenylation (Fig. 1b). It is a reversible post-translational
modification that plays a crucial role in regulating several
protein functions.4-8 Over the past decade several groups have
reported the role of Cys-SOH in regulation of proteins such as
transcription factors, kinases, phosphatases, ion channels, pe-
roxidases and cysteine proteases, sirtuins, human serum albu-
min, and many others.7,9-18 Aberrant Cys-SOH formations have
been shown to be a biomarker of disease state as well.19,20 The
aforementioned examples and many other reports have estab-
lished protein S-sulfenylation as a global signaling mechanism
and a potential drug target.21-23
In recent years, targeting of disease proteins and pathways
with covalent inhibitors has emerged as a viable option to
overcome drug resistance, mutations and toxicity associated
with non-covalent inhibitors.1,2 Advantages of targeted cova-
lent inhibition (TCI) strategies include extended duration of
action and the ability to target rare residues and shallow bind-
ing sites. The mechanism by which TCIs achieve desired inhi-
bition necessitate two distinct steps – the first involves the
reversible association of a high-affinity ligand with its biologi-
cal target in such a manner that an electrophilic ‘warhead’ on
the ligand is brought into an appropriately proximate position
to form a covalent adduct with nucleophilic residue on the
protein (more often than not, a cysteine). Majority of TCIs are
being developed to target kinases for their role in cancer.3 In-
deed, FDA approval of drugs such as ‘Afatinib’ and ‘Ibrutinib’
validated this strategy of incorporating Michael acceptor (elec-
trophilic in nature) functionality in small-molecule inhibitors
to covalently target cysteine residue in the active sites of dis-
eased proteins (Fig. 1a). While extremely attractive, designing
TCIs is difficult in practice because of the difficulties in strik-
ing the right balance between reactivity and selectivity. In
other words, electrophilic warheads may suffer from chemose-
lectivity issues due to the presence of other nucleophilic spe-
cies (such as lysine, histidine etc.). The electrophiles could
Due to over expression, mutations and constitutive activa-
tion in cancer cells, EGFR is targeted by a major class of co-
valent drugs that are aimed to covalently add at 797Cys-SH.1,3
However, 797Cys present in the catalytic pocket of EGFR is a
target of signal-derived H2O2 oxidation which enhances the
kinase activity of this enzyme.12,14,24 We recently showed that
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probes showed low micromolar inhibitory activity against
phosphatase YopH.30
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Fragment-based ligand discovery (FBLD) and activity-
based protein profiling (ABPP) approaches are powerful tools
for the investigation of protein function and identification of
drug leads for new therapeutics.31,32 Over the years, several
FBLD and ABPP approaches have been reported that profile
the reactivity of carbon electrophiles towards biological nu-
cleophiles (such as Cys-SH) in cellular proteomes.32-34 How-
ever, ligands identified in these studies are incapable of react-
ing with cysteine sulfenic acid (Cys-SOH) under physiological
conditions and cysteine oxidation is expected to impact the
pharmacology of inhibitors based on covalent targeting of
Cys-SH.14 For this reason, we have proposed a complementary
strategy that uses clickable carbon nucleophiles in a chemical
proteomic assay and studied the Cys sulfenylome in RKO
colon adenocarcinoma cells. Based on the results of this work,
the sulfenyl form of therapeutically important proteins could
be exploited to develop a new class of irreversible covalent
inhibitors targeting this unique Cys oxoform.
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Figure 1. Targeting the cysteine thiol (Cys-SH) with covalent
inhibitors. (a) FDA approved Afatinib forms a covalent adduct
with EGFR 797Cys-SH via Michael addition. (b) Oxidation of
Cys-SH results generates cysteine sulfenic acid (Cys-SOH).
(c) Covalent inhibitors with electrophilic warheads (such as
the Michael acceptor highlighted in grey) undergo facile reac-
tion with Cys-SH, but not with Cys-SOH under physiological
conditions.
ꢀ
RESULTS AND DISCUSSION
Current approaches to study protein sulfenylation are based on
-dicarbonyl scaffold that shows moderate reactivity com-
a
β
pared to other biologically relevant reactions (such as disulfide
formation).8,12,35-38 Moreover, due to the lack of scaffold diver-
sity among present Cys-SOH probes, current proteomics-
based approaches (such as FBLD and ABPP) to identify small
molecule ligands targeting sulfenylated proteins could not be
implemented effectively.13,36,39-41 To remedy the moderate
reactivity of current nucleophile probes and to increase the
structure diversity, we recently developed a library of cyclic
and linear C-nucleophiles that showed diverse reactivity pro-
files towards Cys-SOH in a small molecule dipeptide model as
well as in a protein model.42-44 The next logical step is to iden-
tify the biological target preferences of these newly developed
nucleophiles. To do so, we first broadly divided the collection
of ~100 cyclic C-nucleophiles into eight distinct scaffolds -
cyclohexane-1,3-diones (1), (thio)barbituric acids (2), different
chronic oxidation result in an EGFR subpopulation that is
refractory to covalent drugs such as Afatinib (Fig. 1c).14 Inhib-
itors targeting 797Cys-SOH would be better suited for the pur-
pose of preferential reactivity as well as enhanced selectivity.
Another example of enzyme class that is redox regulated is
protein tyrosine phosphatases (PTPs), including PTP1B for
Type II diabetes and obesity, and SHP2 for cancer and others
for rheumatoid arthritis, making them attractive therapeutic
targets.25-29 In the past, we have reported redox-based trifunc-
tional probes consisting of a warhead to covalently target Cys-
SOH of PTPs, a ligand that directs the binding to the target
and a reporter tag used for the identification, purification or
direct visualization of the labeled PTP. These redox-based
sized β-dicarbonyl rings (3), dihydrothiophen-3(2H)-one 1,1-
Figure 2. Novel classes of nucleophile probes to profile cysteine oxidation (a) Structural classes of cyclic C-nucleophiles (1 – 8).
In each structure, the nucleophilic carbon is highlighted in the red circle. Rate constants from ref. 42. (b) From the pool of cyclic C-
nucleophiles, DYn-2 and four new C-nucleophiles were selected based on a range of reaction rate constants and on scaffold diversi-
ty. (c) Energy minimized 3-dimensional representation of the probes.
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Figure 3. Application of new nucleophile probes for studying cellular sulfenylomes. (a) RKO whole cell lysates were prepared
under non-denaturing conditions. Native lysate was divided into equal portions and incubated with one of five nucleophile probes
(or vehicle) in separate reactions. Labeled proteins from each reaction were digested separately and the resulting peptides subjected
to click reaction with UV-cleavable biotin-N3. Enrichment and photo-release afforded different probe-labeled peptides samples for
proteomic analysis.
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dioxides/2-substituted isothiazolidin-4-one 1,1-dioxides (4)
pyrrolidine-2,4-diones (5), 1,3-indandiones (6), piperidine-2,4-
5(6H)-one (7), 1,2-thiazinan-5-one 1,1-dioxides/2H-1,2-
lation showed that BTD adopts a half-boat conformation with
sulfone S out of the plane, similar to TD (Fig. 2c).
thiazin-5(6H)-one
1,1-dioxides/1H-benzo[c][1,2]thiazine-
Application of these nucleophilies to RKO colon adenocar-
cinoma cells under basal conditions, followed by click chemis-
try with a biotin azide reporter tag and visualization of labeled
proteins by SDS-PAGE demonstrated that the panel of nucle-
ophiles exhibited a range of reactivities consistent with their
rate constants (Fig. S2). As expected, the highest reactivity
was observed for the BTD probe, which demonstrated sub-
stantial protein labeling at concentrations as low as 100 ꢀM.
The PRD and PYD probes showed moderate to high levels of
labeling while DYn-2 and TD displayed less extensive label-
ing at low concentrations (Fig. S2). From these results, it was
apparent that if the protein site accessibility is not limiting, the
comparative reactivity of these probes correlates well with
their reaction rate constants towards Cys-SOH.
4(3H)-one 2,2-dioxides (8) (Fig. 2a). Nucleophiles belonging
to these scaffolds showed reaction rates that ranged from 2- to
150-fold higher than 1 (Fig. 2a).42-44 Next, we chose cyclic C-
nucleophiles from the above-described eight scaffolds based
on differences in reactivity rates and structural diversity (Fig.
2b, Fig. S1). An alkyne was incorporated into the framework
of these nucleophiles to provide a click chemistry handle for
gel analysis, enrichment and mass spectrometric analysis.
DYn-2 is an established probe for Cys-SOH detection.12 Ow-
ing to the stability of resultant enolic carbanion (due to reso-
nance stabilization), its reaction rate with Cys-SOH is modest
(10 M-1s-1).42 Minimum energy calculations show that this
resonance stabilization also enables DYn-2 to toggle between
half-chair conformers (Fig. 2c). Thiazolidin-4-one 1,1-dioxide
based TD (kobs = 20 M-1s-1) exists predominantly in a keto
form with an envelope conformation. This is attributed to the
sulfonamide S being out of the plane (due to sterics), thus dis-
torting the tetrahedral geometry around the S atom (Fig. 2c).
In the case of pyrrolidine-2,4-dione based PYD (kobs = 330 M-
1s-1), the keto-enol equilibrium favors the keto form.42 Mini-
mum energy calculations for PYD showed an almost complete
preference for planar geometry (Fig. 2c). Piperidine-2,4-dione
based PRD (kobs = 1155 M-1s-1) is structurally similar to DYn-
2, but exhibits rate enhancement of more than two orders of
magnitude, which is attributed to the instability of the carbani-
on in a keto-enol equilibrium that favors the keto form.42 Min-
imum energy calculation indicated that PRD preferentially
exists in the boat conformation (Fig. 2c). Ben-
zo[c][1,2]thiazine-based BTD (kobs = 1700 M-1s-1) showed the
highest reaction rate towards Cys-SOH, which is attributed to
predominance of the keto conformation and a distorted tetra-
hedron geometry around the S atom.42 Minimum energy calcu-
Subsequently, we further examined the peptide labeling pro-
files for these probes by analyzing their proteome reactivities
using a mass spectrometry platform and a workflow referred
to as SulfenM to site-specifically map the S-sulfenylome in
RKO cells. The selectivity, accuracy and reproducibility of the
workflow have been reported previously.13,39 The workflow
consists of eight steps - lysing the RKO cells under native
conditions (using lysis buffer containing non-ionic detergent
and catalase) in the presence of C-nucelophile (Fig. 3a), re-
ducing the native disulfides with DTT, alkylating the reduced
cysteines, digesting the labeled proteins, conjugation of la-
beled peptides with a photocleavable azide biotin via ‘click
chemistry’, capture of labeled peptides with streptavidin
beads, photorelease by cleavage of the biotin linker with UV
light and MS based proteomic analysis of the probe-labeled
peptides (Fig. S3). A total of 1283 S-sulfenylated sites on 761
proteins were successfully labeled by the five C-nucleophiles
(Fig. 3b-c). Interestingly, only nine proteins (ACLY, EEF2,
GAPDH, HSPA8, PFN1, RPS20, RPS21, RPS27A, RPS3A)
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Figure 4. Chemoproteomic analysis of RKO cellular S-sulfenylome. (a) Crystal structures of GAPDH (PDB: 4WNC) showing
Cys152, Cys156 and Cys247 labeled by different probes. (b) CARM1 (PDB: 5Y4X) showing Cys421 that is labeled by TD. (c)
Comparative sequence analysis of peptides labeled with different probes. (d) Heat map of Z-score normalized peak intensities of S-
sulfenylated sites labeled with various probe. Values in each row direction have been mean centered and scaled. (Probe modified
Cys occurring > 1 indicates different charge states on the corresponding peptide).
were labeled by all five nucleophiles. For example, in
GAPDH, 152Cys-SOH (by DYn-2, PYD, PRD and BTD),
156Cys-SOH (by DYn-2, BTD) and 247Cys-SOH (TD, PYD,
PRD, BTD) were successfully labeled. GAPDH is known as a
major redox-sensitive protein, which has three Cys residues,
one (152Cys) in the exposed active site, and two (156Cys,
247Cys) that are buried (Fig. 4a). While redox-sensitivity of
152Cys is well established, 156Cys and 247Cys were recently
reported to be sensitive to storage-dependent oxidation.45 Even
more interestingly, only two S-sulfenylated sites (17Cys-SOH
on RPS21 and 128Cys-SOH on PFN1) were labeled by all five
nucleophiles (Fig. S4). An interesting target was PARK7 (also
known as DJ-1), which is a highly conserved homodimeric
small protein associated with Parkinson’s disease.46 Studies
have demonstrated that DJ-1 protects cells against oxidative
stress-mediated apoptosis, the mechanism of which is largely
unknown.7 DJ-1 is also known to regulate the activity of phos-
phatase and tensin homolog (PTEN) via transnitrosylation
reaction.47 Structurally, DJ-1 has three potentially redox-active
cysteines (46Cys, 53Cys and 106Cys). However, 106Cys, which is
highly conserved, was shown to be highly sensitive to
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Table 1. Number of distinct probe-modified proteins and sites. PRD modified peptides. Indeed, a pLogo analysis of PRD
1
2
3
4
5
6
7
8
modified peptides show a conserved KxC sequence motif as
well (Fig. S5). Similarly, when sequences of peptides labeled
by TD probe were compared with those modified by PYD
(owing to both having 5-membered ring structure) and BTD
(due to structural analogy), the KxC sequence motif was again
found to be conserved (Fig. 4c, panel 3 - 4), an observation
which also was seen in pLogo sequence analysis of TD (Fig.
S5).
# of distinct labeled # of distinct labeled
Probes
proteins
150
56
sites
183
58
DYn-2
TD
PYD
PRD
BTD
76
87
60
68
715
1186
To further estimate the stoichiometries and selectivity of
nucleophile labeling, we obtained peak intensities of all the
detected S-sulfenylated peptides using MS1 filtering based
label-free quantification as previously described (Fig. S6).13,39
The label-free quantification results from each nucleophile-
labeling experiment were then standardized with Z-score nor-
malization to enable further comparison. The quantitative
comparison of peptide-nucleophile labeling is illustrated in a
heat map (Fig. 4d). These data suggested that the nucleophiles
display different trends of S-sulfenylome reactivity and selec-
tivity in complex proteomes. For example, 442Cys-SOH of
HSPD1 (Hsp60) showed a clear preference for BTD over
DYn-2 (Fig. 4d). In some cases, S-sulfenylated sites on the
same protein showed different probe preferences. DYn-2,
PYD, PRD and BTD labeled 152Cys-SOH of GAPDH with
almost equal efficiencies, but BTD was more efficient in label-
ing surface exposed 247Cys-SOH compared to TD, PYD and
PRD (Fig. 4d). These results could be biased by differences in
ionization efficiency among different peptide-probe adducts.
To evaluate this possibility, we compared the ionization of
Cbz-Cys-Val-OMe peptide labeled with different nucleo-
philes. Each probe was exposed to limiting amount of Cbz-
Cys(SOH)-Val-OMe under standard buffer conditions (Fig.
S7a). Thioether products corresponding to each nucleophile
were HPLC-purified, mixed together in equimolar quantities
and analyzed by LC/MS. The relative ionization intensities of
these adducts differed by one-fold between the lowest and
highest ionizing species with lowest being Cbz-Cys(S-BTD)-
Val-OMe and highest being Cbz-Cys(S-DYn-2)-Val-OMe
indicating limited effect of ionization efficiencies of peptide-
probe adducts (Fig. S7b).
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oxidation and formed a stable Cys-SO2H.7 We showed that
DJ-1 was labeled by DYn-2 and PRD at 106Cys and BTD at
46Cys respectively. The rank order of the number of distinct
probe-modified sites identified by these chemoproteomic
analyses was BTD >> DYn-2 > PYD > PRD > TD (Table 1).
Another interesting target was CARM1, in which 421Cys was
labeled by TD. CARM1 (also known as PRMT4) is a member
of arginine-specific protein methyltransferases (PRMTs).
While most PRMTs methylate Gly- and Arg-rich (GAR) mo-
tifs in their substrates, CARM1 displays unique substrate spec-
ificity by methylating Pro-, Gly-, and Met-rich motifs (PGM
motifs).48
Owing to its superior reaction rate towards Cys-SOH, BTD
exhibited the highest level of S-sulfenylome reactivity. How-
ever, a very restricted reactivity profile was observed for other
probes. Of particular interest was the relatively low number of
sulfenylated proteins labeled by the PYD and PRD probes in
RKO sulfenylome (Table 1), which contrasted sharply with
the robust reactivity shown by these probes in native RKO cell
lysate (Fig. S2). Potential reversibility of thioether adducts
under reducing conditions under the proteomics work-up con-
ditions could be responsible for unexpected labeling profile of
PYD and PRD.44 To further explore structural features that
may define the protein sulfenylation, we examined the flank-
ing sequences of S-sulfenylated Cys residues modified by the
five probes with the pLogo algorithm for the presence of con-
sensus motifs (Fig. S5).49 We found several consensus motifs
for the nucleophile-labeled S-sulfenylome, such as KxC and
KC. Of interest, Lys was significantly overrepresented at al-
most every adjacent position (from -6 to +6) of BTD-labeled
Cys-SOH. This observed enrichment of Lys is intriguing and
may indicate that positive charge in the vicinity of the reactive
cysteine is critical for thiolate generation. However, if this
were strictly the case, then Arg enrichment would also be ob-
served. Another possibility is that due to its elevated reactivi-
ty, BTD was able to capture highly transient sulfenylated pro-
teins. But the abundance of such BTD labeled peptides is low
and probe-tagged peptides containing Lys ionize better, thus
enabling detection of these peptides above the noise. We also
compared the flanking sequences of S-sulfenylated Cys resi-
dues modified by the five probes using Two Sample Logo
(2logP) algorithm (Fig. 4c).50 Structurally, DYn-2 and PRD
differ by just one atom (C4 in DYn-2 vs N in PRD). 2LogP
analyses indicated that, PRD labeled peptides show higher
frequency of Lys (at -2) and Leu (at +4) residues compared to
DYn-2 modified peptides (Fig. 4c, panel 1). On the other
We next investigated whether the S-sulfenylomes labeled by
the five probes had distinct structural features. Based on the
calculations resulting from twenty proteins with structure files
available in Protein Data Bank (PDB) that contained both bur-
ied and exposed Cys residues, we observed that DYn-2 and
Table 2. Surface distribution analysis of probe-labeled pro-
teins. Calculations were based on twenty proteins with 3D
structure files found in the Protein Data Bank (PDB) that con-
tained both buried and exposed cysteine residues. E: exposed
(RSA>25%); B: buried (RSA <10%); UM: Unmodified cyste-
ine residues.
E (%)
35.3
0.0
B (%)
52.9
80.0
71.4
55.6
62.5
58.7
DYn-2
TD
PYD
PRD
BTD
UM
21.4
33.3
25.0
19.6
hand, both PYD and PRD are β-ketolactams, but differ in the
ring size, leading to differences in conformation but which
also resulted in higher frequency of Lys (at -2) and Leu (+4) in
the peptides modified by PRD (Fig. 4c, panel 2). KxC and
CxxxL thus appeared to be the conserved sequence motifs in
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Figure 5. PTPs as privileged targets of PYD. (a) Representative MS/MS spectra of PTPN1 (PTP1B) labeled by PYD probe. (b)
PYD probe preferentially modifies the recombinant oxidized form of PTP1B. (c) Molecular docking (using AutoDock Vina) of
PYD into catalytic pocket of PTPN1 (PDB: 1OET). (d) Abbreviated sequence alignment of PTPs that harbor a Cys-SOH labeled by
PYD probe.
PRD prefer to label S-sulfenylated residues on protein surfac-
es. In contrast, PYD and TD display substantial reactivity to-
wards the buried S-sulfenylated sites (Table 2). Of particular
interest was the distinct reactivity of the PYD probe with the
S-sulfenylated sites within the buried catalytic domains of
several classical protein tyrosine phosphatase (PTPs) such as
PTPN1 (PTP1B), PTPN7, PTPN11 and DUSP23 (Fig. 5a,
Fig. S8). The preferential reactivity of PYD towards PTP1B
was also verified by treatment of recombinant, oxidized phos-
phatase with each nucleophile followed by click chemistry-
mediated conjugation to an biotin-azide reporter tag and visu-
alization by Strepavidin-HRP western blot (Fig. 5b). Molecu-
lar docking (using AutoDock Vina)51 of PYD into the catalytic
pocket of PTP1B showed a good fit with the catalytic pocket,
ostensibly owing to the almost planar conformation of this C-
nucleophile (Fig. 5c). The CX5R sequence is the signature
motif of the PTP superfamily and forms the critical phosphate-
binding loop (also known as the P-loop) in the active site (Fig.
5d). Irrespective of the variations in the X5 segment, confor-
mation of the P-loop is conserved (to keep catalytic Cys and
Arg in close proximity) and can be easily superimposed
among different PTP structures. PTPs are established targets
of ROS because the signature motif CX5R of this family con-
tains an invariant cysteine that has an extremely low pka,
which promotes its function as a nucleophile in catalysis and
renders it highly susceptible to oxidative inactivation.29
future drug design and development. Herein, we utilized a
novel multi-probe approach and a highly efficient chemopro-
teomics platform to globally profile the reactivity of five C-
nucleophile probes, including four novel molecules, in the
RKO S-sulfenylome under normal physiological conditions.
These five C-nucleophiles covalently labeled 1283 redox-
sensitive Cys residues on 761 proteins that are endogenously
S-sulfenylated. We show that distinct structural classes of C-
nucleophiles targeted discrete protein populations and S-
sulfenylated residues with little overlap. Whereas BTD labeled
the greatest numbers of S-sulfenylated sites by far, the smaller
size of the five-membered ring of TD and PYD appear to fa-
cilitate internal site labeling. In addition to physical size, the
conformations of five-membered (envelope for TD and almost
planar for PYD) versus six-membered (chair and boat) probes
also differ, which also would favor buried site labeling by TD
and PYD. Insights from this proteomics study have important
implications in the development of covalent inhibitors that
target cysteines within the thiol ‘redoxome’. For example,
PTPs, including PTP1B for Type II diabetes and obesity, and
SHP2 for cancer and others for rheumatoid arthritis, are attrac-
tive therapeutic targets.25-28 Our study has identified the cata-
lytic Cys in PTP1B (215Cys), PTPN7 (396Cys), PTPN11
(
459Cys) and DUSP23 (95Cys), PTPs as favored targets of pyr-
rolidine-2,4-dione-based nucleophile, PYD. The enrichment of
PYD-labeled PTPs suggests that PYD could be coupled to
binding elements that capture interactions with adjacent pock-
ets surrounding the active site to develop covalent inhibitors
for this class of proteins. This strategy can be expanded and
applied to other attractive targets. Our expanding library of
novel warheads that target S-sulfenylated cysteines will enable
high-throughput profiling to pair distinct C-nucleophilic scaf-
folds with various redox-regulated proteins in a combined
FBLD/ABPP approach to develop effective covalent irreversi-
ble/reversible inhibitors based therapeutics.23,32,52 Finally, these
probes can be used to study how oxidative cysteine modifica-
tions impact both cell signaling and drug pharmacology.
ꢀ
CONCLUSION
In order to develop inhibitors that effectively target redox-
active cysteines in therapeutically important proteins, it is
imperative to complement the structural knowledge of S-
sulfenylated sites with an understanding of warhead reactivity.
With suitable chemical tools, modern chemical proteomics
approaches such as FBLD and ABPP present a way to achieve
this task in a systematic and efficient manner. Not only do
such approaches provide the technology that help identify sites
of covalent modification, but they also differentiate between
selective versus promiscuous covalent modifiers and thus aid
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(16) Alili, L.; Sack, M.; von Montfort, C.; Giri, S.; Das, S.;
Carroll, K. S.; Zanger, K.; Seal, S.; Brenneisen, P. Antioxid Redox
Signal 2013, 19, 765.
ASSOCIATED CONTENT
1
Supporting Information
2
3
4
5
6
7
8
9
(17) Chouchani, E. T.; Kazak, L.; Jedrychowski, M. P.; Lu, G. Z.;
Erickson, B. K.; Szpyt, J.; Pierce, K. A.; Laznik-Bogoslavski, D.;
Vetrivelan, R.; Clish, C. B.; Robinson, A. J.; Gygi, S. P.;
Spiegelman, B. M. Nature 2016, 532, 112.
(18) Hourihan, J. M.; Moronetti Mazzeo, L. E.; Fernandez-
Cardenas, L. P.; Blackwell, T. K. Mol Cell 2016, 63, 553.
(19) Zito, E.; Hansen, H. G.; Yeo, G. S.; Fujii, J.; Ron, D. Mol
Cell 2012, 48, 39.
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental details and protocols including NMR data (PDF).
Proteomics data associated with each probe (XLSX).
AUTHOR INFORMATION
Corresponding Author
(20) Seo, Y. H.; Carroll, K. S. P Natl Acad Sci USA 2009, 106,
16163.
(21) Truong, T. H.; Carroll, K. S. Biochemistry 2012, 51, 9954.
(22) Truong, T. H.; Carroll, K. S. Crit Rev Biochem Mol Biol
2013, 48, 332.
* Correspondence and requests for materials should be addressed
to K.S.C. (kcarroll@scripps.edu)
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13
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22
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24
25
26
27
28
29
30
31
32
33
34
35
36
37
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52
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54
55
56
57
58
59
60
Author Contributions
‡ These authors contributed equally.
(23) Visscher, M.; Arkin, M. R.; Dansen, T. B. Curr Opin Chem
Biol 2016, 30, 61.
Funding Sources
Part of this work is supported by the National Institutes of Health
under awards No. R01 GM102187 and R01 CA174864 to K. S. C.
J.Y. acknowledge the support of the National Key Research and
Development Program (No. 2016YFA0501303), the National
Natural Science Foundation of China (No. 31500666 and No.
81573395), the Beijing Natural Science Foundation (No.
5162009), and Beijing Nova Program.
(24) Heppner, D. E.; Hristova, M.; Dustin, C. M.; Danyal, K.;
Habibovic, A.; van der Vliet, A. J Biol Chem 2016, 291, 23282.
(25) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.;
Collins, S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-
Hagen, J.; Chan, C. C.; Ramachandran, C.; Gresser, M. J.;
Tremblay, M. L.; Kennedy, B. P. Science 1999, 283, 1544.
(26) Saltiel, A. R.; Kahn, C. R. Nature 2001, 414, 799.
(27) He, R. J.; Yu, Z. H.; Zhang, R. Y.; Zhang, Z. Y. Acta
Pharmacol Sin 2014, 35, 1227.
(28) Barr, A. J. Future Med Chem 2010, 2, 1563.
(29) Tonks, N. K. Nat Rev Mol Cell Biol 2006, 7, 833.
(30) Leonard, S. E.; Garcia, F. J.; Goodsell, D. S.; Carroll, K. S.
Angew Chem Int Ed Engl 2011, 50, 4423.
(31) Murray, C. W.; Rees, D. C. Nat Chem 2009, 1, 187.
(32) Backus, K. M.; Correia, B. E.; Lum, K. M.; Forli, S.;
Horning, B. D.; Gonzalez-Paez, G. E.; Chatterjee, S.; Lanning, B.
R.; Teijaro, J. R.; Olson, A. J.; Wolan, D. W.; Cravatt, B. F.
Nature 2016, 534, 570.
(33) Weerapana, E.; Simon, G. M.; Cravatt, B. F. Nat Chem Biol
2008, 4, 405.
(34) Kathman, S. G.; Xu, Z.; Statsyuk, A. V. J Med Chem 2014,
57, 4969.
(35) Reddie, K. G.; Seo, Y. H.; Muse, W. B.; Leonard, S. E.;
Carroll, K. S. Mol Biosyst 2008, 4, 521.
(36) Leonard, S. E.; Reddie, K. G.; Carroll, K. S. ACS Chem Biol
2009, 4, 783.
(37) Qian, J.; Klomsiri, C.; Wright, M. W.; King, S. B.; Tsang, A.
W.; Poole, L. B.; Furdui, C. M. Chem Commun 2011, 47, 9203.
(38) Qian, J.; Wani, R.; Klomsiri, C.; Poole, L. B.; Tsang, A. W.;
Furdui, C. M. Chem Commun 2012, 48, 4091.
(39) Yang, J.; Gupta, V.; Tallman, K. A.; Porter, N. A.; Carroll,
K. S.; Liebler, D. C. Nat Protoc 2015, 10, 1022.
(40) Gould, N. S.; Evans, P.; Martinez-Acedo, P.; Marino, S. M.;
Gladyshev, V. N.; Carroll, K. S.; Ischiropoulos, H. Chem Biol
2015, 22, 965.
(41) Akter, S.; Huang, J.; Bodra, N.; De Smet, B.; Wahni, K.;
Rombaut, D.; Pauwels, J.; Gevaert, K.; Carroll, K.; Van
Breusegem, F.; Messens, J. Mol Cell Proteomics 2015, 14, 1183.
(42) Gupta, V.; Carroll, K. S. Chem Sci 2016, 7, 400.
(43) Gupta, V.; Carroll, K. S. Chem Commun (Camb) 2016, 52,
3414.
(44) Gupta, V.; Paritala, H.; Carroll, K. S. Bioconjug Chem 2016,
27, 1411.
(45) Reisz, J. A.; Wither, M. J.; Dzieciatkowska, M.; Nemkov, T.;
Issaian, A.; Yoshida, T.; Dunham, A. J.; Hill, R. C.; Hansen, K.
C.; D'Alessandro, A. Blood 2016, 128, e32.
ACKNOWLEDGMENT
V.G. thanks Dr. Mauro Lo Conte (Department of Chemistry,
Scripps Florida) for biotin-N3 reagent and Dr. Peng Wu (Depart-
ment of Chemistry, Scripps La Jolla) & Dr. Hua Wang (Depart-
ment of Chemistry, Scripps La Jolla) for providing BTTP ‘click’
ligand used in the ‘click’ chemistry for WB analysis.
REFERENCES
(1) Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. Nat Rev
Drug Discov 2011, 10, 307.
(2) Baillie, T. A. Angew Chem Int Ed Engl 2016, 55, 13408.
(3) Liu, Q.; Sabnis, Y.; Zhao, Z.; Zhang, T.; Buhrlage, S. J.;
Jones, L. H.; Gray, N. S. Chem Biol 2013, 20, 146.
(4) Paulsen, C. E.; Carroll, K. S. Chem Rev 2013, 113, 4633.
(5) Reddie, K. G.; Carroll, K. S. Curr Opin Chem Biol 2008, 12,
746.
(6) Paulsen, C. E.; Carroll, K. S. ACS Chem Biol 2010, 5, 47.
(7) Lo Conte, M.; Carroll, K. S. J Biol Chem 2013, 288, 26480.
(8) Gupta, V.; Carroll, K. S. Biochim Biophys Acta 2014, 1840,
847.
(9) Denu, J. M.; Tanner, K. G. Biochemistry 1998, 37, 5633.
(10) Svoboda, L. K.; Reddie, K. G.; Zhang, L.; Vesely, E. D.;
Williams, E. S.; Schumacher, S. M.; O'Connell, R. P.; Shaw, R.;
Day, S. M.; Anumonwo, J. M.; Carroll, K. S.; Martens, J. R. Circ
Res 2012, 111, 842.
(11) Kulathu, Y.; Garcia, F. J.; Mevissen, T. E.; Busch, M.;
Arnaudo, N.; Carroll, K. S.; Barford, D.; Komander, D. Nat
Commun 2013, 4, 1569.
(12) Paulsen, C. E.; Truong, T. H.; Garcia, F. J.; Homann, A.;
Gupta, V.; Leonard, S. E.; Carroll, K. S. Nat Chem Biol 2011, 8,
57.
(13) Yang, J.; Gupta, V.; Carroll, K. S.; Liebler, D. C. Nat
Commun 2014, 5, 4776.
(14) Truong, T. H.; Ung, P. M.; Palde, P. B.; Paulsen, C. E.;
Schlessinger, A.; Carroll, K. S. Cell Chem Biol 2016, 23, 837.
(15) Barrett, T. J.; Pattison, D. I.; Leonard, S. E.; Carroll, K. S.;
Davies, M. J.; Hawkins, C. L. Free Radic Biol Med 2012, 52,
1075.
(46) Bonifati, V.; Rizzu, P.; van Baren, M. J.; Schaap, O.;
Breedveld, G. J.; Krieger, E.; Dekker, M. C.; Squitieri, F.; Ibanez,
P.; Joosse, M.; van Dongen, J. W.; Vanacore, N.; van Swieten, J.
7
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Page 8 of 9
C.; Brice, A.; Meco, G.; van Duijn, C. M.; Oostra, B. A.; Heutink,
1
2
3
4
5
6
7
8
P. Science 2003, 299, 256.
(47) Choi, M. S.; Nakamura, T.; Cho, S. J.; Han, X. M.; Holland,
E. A.; Qu, J.; Petsko, G. A.; Yates, J. R.; Liddington, R. C.;
Lipton, S. A. J Neurosci 2014, 34, 15123.
(48) Yang, Y.; Bedford, M. T. Nat Rev Cancer 2013, 13, 37.
(49) O'Shea, J. P.; Chou, M. F.; Quader, S. A.; Ryan, J. K.;
Church, G. M.; Schwartz, D. Nat Methods 2013, 10, 1211.
(50) Vacic, V.; Iakoucheva, L. M.; Radivojac, P. Bioinformatics
2006, 22, 1536.
9
(51) Trott, O.; Olson, A. J. J Comput Chem 2010, 31, 455.
(52) Gruner, B. M.; Schulze, C. J.; Yang, D.; Ogasawara, D.; Dix,
M. M.; Rogers, Z. N.; Chuang, C. H.; McFarland, C. D.; Chiou, S.
H.; Brown, J. M.; Cravatt, B. F.; Bogyo, M.; Winslow, M. M. Nat
Methods 2016, 13, 883.
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26
27
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