Identification of michael acceptor-centric pharmacophores with substituents that yield strong thioredoxin reductase inhibitory character correlated to antiproliferative activity

Article abstract of DOI:10.1089/ars.2012.4909

Aims: The role of thioredoxin reductase (TrxR) in tumorigenesis has made it an attractive anticancer target. A systematic approach for development of novel compounds as TrxR inhibitors is currently lacking. Structurally diversified TrxR inhibitors share in common electrophilic propensities for the sulfhydryl groups, among which include the Michael reaction acceptors containing an α,β-unsaturated carbonyl moiety. We aimed to identify features among structurally diversified Michael acceptor-based compounds that would yield a strong TrxR inhibitory character. Results: Structurally dissimilar Michael acceptor-based natural compounds such as isobutylamides, zerumbone, and shogaols (SGs) were found to possess a poor TrxR inhibitory activity, indicating that a sole Michael acceptor moiety was insufficient to produce TrxR inhibition. The 1,7-diphenyl-hept-3-en-5-one pharmacophore in 3-phenyl-3-SG, a novel SG analog that possessed comparable TrxR inhibitory and antiproliferative potencies as 6-SG, was modified to yield 1,5-diphenyl-pent-1-en-3-one (DPPen) and 1,3-diphenyl-pro-1-en-3-one (DPPro, also known as chalcone) pharmacophores. These Michael acceptor-centric pharmacophores, when substituted with the hydroxyl and fluorine groups, gave rise to analogs displaying a TrxR inhibitory character positively correlated to their antiproliferative potencies. Lead analogs 2,2′-diOH-5,5′-diF-DPPen and 2-OH-5-F-DPPro yielded a half-maximal TrxR inhibitory concentration of 9.1 and 10.5 μM, respectively, after 1-h incubation with recombinant rat TrxR, with the C-terminal selenocysteine residue found to be targeted. Innovation: Identification of Michael acceptor-centric pharmacophores among diversified compounds demonstrates that a systematic approach to discover and develop Michael acceptor-based TrxR inhibitors is feasible. Conclusion: A strong TrxR inhibitory character correlated to the antiproliferative potency is attributed to structural features that include an α,β-unsaturated carbonyl moiety centered in a DPPen or DPPro pharmacophore bearing hydroxyl and fluorine substitutions. Antioxid. Redox Signal. 19, 1149-1165. Mary Ann Liebert, Inc.

Full text of DOI:10.1089/ars.2012.4909

ANTIOXIDANTS & REDOX SIGNALING
Volume 00, Number 00, 2013
ª Mary Ann Liebert, Inc.
DOI: 10.1089/ars.2012.4909
ORIGINAL RESEARCH COMMUNICATION
Identification of Michael Acceptor-Centric Pharmacophores
with Substituents That Yield Strong Thioredoxin Reductase
Inhibitory Character Correlated to Antiproliferative Activity
Fei-Fei Gan, Kamila K. Kaminska, Hong Yang, Chin-Yee Liew, Pay-Chin Leow, Choon-Leng So,
Lan N.L. Tu, Amrita Roy, Chun-Wei Yap, Tse-Siang Kang, Wai-Keung Chui, and Eng-Hui Chew
Abstract
Aims: The role of thioredoxin reductase (TrxR) in tumorigenesis has made it an attractive anticancer target.
A systematic approach for development of novel compounds as TrxR inhibitors is currently lacking. Structurally
diversified TrxR inhibitors share in common electrophilic propensities for the sulfhydryl groups, among which
include the Michael reaction acceptors containing an a,b-unsaturated carbonyl moiety. We aimed to identify
features among structurally diversified Michael acceptor-based compounds that would yield a strong TrxR
inhibitory character. Results: Structurally dissimilar Michael acceptor-based natural compounds such as iso-
butylamides, zerumbone, and shogaols (SGs) were found to possess a poor TrxR inhibitory activity, indicating
that a sole Michael acceptor moiety was insufficient to produce TrxR inhibition. The 1,7-diphenyl-hept-3-en-5-
one pharmacophore in 3-phenyl-3-SG, a novel SG analog that possessed comparable TrxR inhibitory and
antiproliferative potencies as 6-SG, was modified to yield 1,5-diphenyl-pent-1-en-3-one (DPPen) and 1,3-
diphenyl-pro-1-en-3-one (DPPro, also known as chalcone) pharmacophores. These Michael acceptor-centric
pharmacophores, when substituted with the hydroxyl and fluorine groups, gave rise to analogs displaying a
TrxR inhibitory character positively correlated to their antiproliferative potencies. Lead analogs 2,2¢-diOH-5,5¢-
diF-DPPen and 2-OH-5-F-DPPro yielded a half-maximal TrxR inhibitory concentration of 9.1 and 10.5 lM,
respectively, after 1-h incubation with recombinant rat TrxR, with the C-terminal selenocysteine residue found to
be targeted. Innovation: Identification of Michael acceptor-centric pharmacophores among diversified com-
pounds demonstrates that a systematic approach to discover and develop Michael acceptor-based TrxR inhib-
itors is feasible. Conclusion: A strong TrxR inhibitory character correlated to the antiproliferative potency is
attributed to structural features that include an a,b-unsaturated carbonyl moiety centered in a DPPen or DPPro
pharmacophore bearing hydroxyl and fluorine substitutions. Antioxid. Redox Signal. 00, 000–000.
Introduction
The Trx system consists of Trx, thioredoxin reductase
(TrxR), and NADPH. TrxR catalyzes the NADPH-dependent
edox homeostasis is maintained by cellular antioxi- reduction of a dithiol/disulfide active site (in human Trx:
R
dant systems such as the thioredoxin (Trx) and glutathi- -Trp-Cys32-Gly-Pro-Cys35-Lys-) in oxidized Trx. Through the
one (GSH) system. These thiol systems act as double-edged thiol–disulfide exchange, Trx reduces protein disulfides,
swords; they protect cells from oxidative stress, but in cancer, which leads to protection of cellular proteins against an oxi-
they contribute to tumor growth and progression. Perturba- dative insult. As an electron donor to ribonucleotide reduc-
tion of the redox state-sustaining systems in malignant cells tase and peroxide scavengers peroxiredoxins, Trx also
can thereby form an important basis for chemotherapeutic participates in DNA biosynthesis/repair and redox homeo-
strategies.
stasis, respectively. The redox protein is broadly involved in
Department of Pharmacy, National University of Singapore, Singapore, Republic of Singapore.
1

2
GAN ET AL.
moiety centered in a 1,5-diphenyl-pent-1-en-3-one (DPPen) or
1,3-diphenyl-pro-1-en-3-one (DPPro; also known as chalcone)
pharmacophore bearing hydroxyl- and fluorine substitutions.
Of note, the good correlation between the TrxR inhibitory and
antiproliferative potencies among the DPPen and DPPro
compounds suggests TrxR inhibition as the underlying
mechanism of their antitumor effects. Taken together, the
findings in this study have offered opportunities not only for
possibly a more predictive identification and characterization
of novel compounds as potential TrxR inhibitors, but also to
be potentially applied for future design and development of
TrxR inhibitors.
Innovation
The structural diversity of Michael acceptor-based
thioredoxin reductase (TrxR) inhibitors hinders carrying
out of large-scale structure–activity relationship studies.
Those possessing a weak TrxR inhibitory activity are also
little known. In this present study, initial screening of a
number of Michael acceptor-based natural compounds
had not only demonstrated that a sole Michael acceptor
moiety was insufficient to produce TrxR inhibition, but
also led to the synthesis of compounds bearing 1,5-
diphenyl-pent-1-en-3-one and 1,3-diphenyl-pro-1-en-3-
one pharmacophores that displayed well-correlated TrxR
inhibitory and antiproliferative activities. The findings
have suggested the feasibility of a systematic approach
to discover and develop Michael acceptor-based TrxR
inhibitors.
Results
Evaluation of naturally occurring a,b-unsaturated
amides and unconjugated a,b-unsaturated ketones
for in vitro TrxR inhibitory and antiproliferative activities
In an attempt to identify the structural features in the Mi-
cell signaling by exerting redox control over the activity of chael acceptors that were required for TrxR inhibition, a
several proteins, including activator protein-1, nuclear factor number of structurally diversified natural products that bear
jB, and apoptosis signal-regulating kinase 1 (ASK1) (1, 19, a common a,b-unsaturated carbonyl moiety were selected for
40). Homodimeric mammalian TrxR has each of the 55-kDa evaluation in the DTNB reduction assay. These compounds
subunit containing an N-terminal–Cys59-Val-Asn-Val-Gly- included a,b-unsaturated amides such as hexa-2,4-dienoic
Cys64- dithiol/disulfide active site and a C-terminal–Gly- acid isobutylamide (Hexa-IBA) and dodeca-2(E),4(E)-dienoic
Cys497-Sec498-Gly- active site. The C-terminal selenocysteine acid isobutylamide (Dodeca-IBA) that are found in the species
(Sec)-containing active site has a range of substrates, in- of Echinacea, as well as unconjugated a,b-unsaturated ke-
cluding Trx, 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB) and tones such as zerumbone isolated from rhizomes of Zingiber
protein disulfide isomerase (19, 33). In recent years, discov- zerumbet Smith and shogaols (SGs), active constituents iso-
ery and development of anticancer agents targeting the Trx lated from rhizomes of Zingiber officinale Roscoe. As of yet, no
system are gaining momentum, partly attributed to the studies have reported the possible effects of these compounds
evidence demonstrating that Trx and TrxR are over- on the TrxR activity. The IBAs and zerumbone had failed to
expressed in aggressive tumors and such overexpression are inhibit the TrxR activity even at a high concentration of
related to cancer drug resistance and poor patient prognosis 100 lM. Among the SGs, 4-SG and 6-SG containing shorter
(5, 23, 24, 37, 43). Further, in mouse xenografts, TrxR is found carbon-chain lengths displayed weak TrxR inhibition with a
to be essential for tumorigenesis (51), and in cancer cells, respective IC50 value of 64.5 and 88.0 lM at 1-h incubation,
knockdown of TrxR retards self-sufficient growth and DNA whereas 8-SG and 10-SG with longer chain lengths exhibited
replication (52). Indeed, mammalian TrxR is an attractive negligible TrxR inhibition (Table 1). We further synthesized a
anticancer target of many electrophilic compounds due to its novel SG analog 3-phenyl-3-SG (3-Ph-3-SG; structure as
two unique structural properties (30, 45). First, being located shown in Table 1) and found that it possessed a comparable
at the flexible C-terminal tail, the Sec-containing active site TrxR inhibitory activity as 6-SG (IC50 at 1-h incubation:
is highly accessible for attack by inhibitors. Secondly, the 87.1 lM).
low pKa of the free selenol group (-SeH) in Sec enables
The effects of these compounds on the HCT 116 and
ionization to nucleophilic selenolate (Se- ) at physiological MCF-7 cell viabilities were evaluated. The IBAs were found
pH. A number of chemotherapeutic agents used clinically to possess negligible antiproliferative activities that were
or currently in clinical trials such as nitrosoureas (49), cy- correlated to their poor TrxR inhibitory activities. On the
clophosphamide (47), gold complexes such as auranofin other hand, in agreement with the studies reporting its
(8, 18), platinum complexes such as cisplatin (4), arsenic antitumor effects (32, 35), zerumbone had displayed potent
trioxide (29), and motexafin gadolinium (20) have been rec- cytotoxic effects. For the series of SGs with increasing side-
ognized to inhibit TrxR.
chain lengths, we had observed previously for HCT 116
Currently, there is no documentation of a systematic ap- cells (17) and in this present study for MCF-7 cells that the
proach that allows a rational design and development of antiproliferative efficacies decreased as the chain length
compounds as selective TrxR inhibitors. Structurally diversi- increased. This trend of diminishing antiproliferative activ-
fied small molecules acting on TrxR share in common elec- ities was similar to the observed trend of decreasing TrxR
trophilic propensities for sulfhydryl groups, among which inhibition. Similar to its TrxR inhibitory activity, the syn-
include metal complexes with reactive valencies and Michael thetic SG analog 3-Ph-3-SG possessed comparable anti-
reaction acceptors containing an a,b-unsaturated carbonyl proliferative potencies as 6-SG. Taken together, zerumbone
moiety (7, 30, 45). Our laboratory has interest in discovering and SGs had exhibited potent antiproliferative effects, but a
the Michael acceptor-based TrxR inhibitors. In this study, we lack of or weak TrxR inhibition, suggesting that TrxR tar-
report that a strong TrxR inhibitory character is attributed to geting is unlikely the mechanism underlying their antitu-
structural features that include an a,b-unsaturated carbonyl mor action.

MICHAEL ACCEPTORS INHIBIT THIOREDOXIN REDUCTASE
3
Table 1. Mammalian TrxR Inhibitory and Antiproliferative Activities of Classes of Compounds
Containing an a,b-Unsaturated Carbonyl Moiety
Antiproliferative activity
TrxR inhibitory activity
HCT116
MCF-7
Mean Mean
IC50 (30min) IC50 (60min)
Mean
Mean
Mean
Mean
Compounds
Source
(mM)
(mM)
GI50 (mM) LC50 (mM) GI50 (mM) LC50 (mM)
a,b-unsaturated amides
Hexa-IBA
Dodeca-IBA
Echinacea purpurea
Echinacea purpurea
Zanthoxylum piperitum
ND
ND
ND
ND
ND
ND
ND
84.8 – 7.0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
b-sanshool
Unconjugated a,b-unsaturated ketone
Zerumbone
Shogaol (SG): 4-SG
6-SG
8-SG
10-SG
Zingiber zerumbet Smith
Zingiber officinale Roscoe 87.5 – 6.8
Zingiber officinale Roscoe
Zingiber officinale Roscoe
Zingiber officinale Roscoe
Synthetic
ND
ND
64.5 – 5.2
88.0 – 9.6
ND
ND
87.1 – 8.4
10.9 – 0.8 28.2 – 1.0 15.9 – 2.8 93.5 – 7.0
1.3 – 0.5* 21.5 – 5.4*
4.3 – 0.3* 27.0 – 1.5*
3.8 – 1.5 26.2 – 1.4
8.5 – 0.7 28.0 – 1.7
ND
ND
ND
ND
13.7 – 1.8* 84.0 – 7.6* 34.8 – 7.2 95.8 – 8.3
16.1 – 3.7* 84.6 – 5.3* 32.8 – 9.4 92.1 – 7.6
3-Ph-3-SG
7.2 – 1.9 27.3 – 1.2
8.3 – 1.3 26.4 – 1.8
1,5-diphenyl-pent-1-en-3-one (DPPen)
Unsub-DPPen
2-OH-5-F-DPPen
2¢-OH-5¢-F-DPPen
2,2¢-diOH-5,5¢-diF-DPPen Synthetic
Conjugated a,b-unsaturated ketone
1,3-diphenyl-propen-1-one (DPPro/Chalcone)
Synthetic
Synthetic
Synthetic
ND
ND
54.1 – 2.8 92.5 – 4.1 42.7 – 3.0 92.1 – 7.3
40.4 – 7.0
66.0 – 7.9
10.9 – 3.2
28.0 – 3.7 20.1 – 2.4 85.3 – 7.0 24.6 – 3.3 83.9 – 6.1
40.5 – 2.9 41.2 – 7.1 82.7 – 4.3 26.3 – 1.6 91.6 – 2.8
9.1 – 1.1 11.8 – 1.5 27.3 – 2.3 13.1 – 1.4 28.0 – 0.9
Unsub-DPPro
Synthetic
Synthetic
Synthetic
69.3 – 1.3
11.8 – 3.2
36.8 – 1.5
23.0 – 0.7
37.3 – 2.2 17.5 – 0.5 27.9 – 1.1 14.9 – 7.5 74.2 – 3.5
2-OH-5-F-DPPro
2¢-OH-5¢-F-DPPro
2,2¢-diOH-5,5¢-diF-DPPro Synthetic
10.5 – 2.4
4.1 – 0.2
9.3 – 0.2
3.8 – 0.6 24.3 – 2.7
27.8 – 3.3 16.0 – 2.7 29.5 – 0.8 14.3 – 1.6 85.4 – 2.9
16.7 – 2.6 6.5 – 1.0 10.0 – 0.4 6.1 – 1.0 26.0 – 2.4
Values for 50% TrxR inhibition concentration (IC50), 50% growth inhibition concentration (GI50), and 50% lethal concentration (LC50) are
presented as means – SD from three independent experiments. *Results as obtained in ref. 17.
TrxR, thioredoxin reductase; Hexa-IBA, hexa-2,4-dienoic acid isobutylamide; Dodeca-IBA, dodeca-2(E),4(E)-dienoic acid isobutylamide.

4
GAN ET AL.
Evaluation of analogs bearing DPPen or DPPro
pharmacophore for in vitro TrxR inhibitory
and antiproliferative activities
2-OH-5-F-DPPro had caused a dose-dependent decrease in
the TrxR activity, where lethal doses of 40 and 50 lM brought
about a significant decline in the enzyme activity. The Wes-
tern blot analyses showed that the protein levels of TrxR had
remained relatively constant across the concentrations (Fig.
4E), which ruled out the possibility that reduction in the TrxR
activity was attributed to a reduced enzyme expression. The Trx,
GR, and GPx activities in the lysate samples were found to re-
main generally unsuppressed by the lead DPPen/DPPro ana-
logs (Fig. 4B–D). The results obtained therefore indicate that the
DPPen and DPPro analogs displayed selective TrxR inhibition.
We wanted to synthesize the structural derivatives of either
6-SG or 3-Ph-3-SG that possessed comparable and weak TrxR
inhibitory activities. 3-Ph-3-SG was selected as the chemical
scaffold based on which derivatives were synthesized, be-
cause the two phenyl rings allowed feasible structural modi-
fications via chemical synthesis. To investigate whether the
distance between the two phenyl rings in 3-Ph-3-SG would
affect TrxR inhibition, compounds containing shortened
phenyl–phenyl distances, namely, those bearing a DPPen or
DPPro pharmacophore, were synthesized. Analogs bearing Mechanistic characterization of cytotoxic activities
the 2-hydroxy- and 5-fluorine substitutions on the phenyl ring of DPPen and DPPro compounds
were also synthesized (structures as shown in Table 1), and the
HCT 116 cells were selected for the examination of the cy-
TrxR inhibitory activities were evaluated by the in vitro DTNB
totoxicities elicited by the DPPen and DPPro analogs. First,
reduction assay. For the series of DPPen-based compounds,
the Western blot analyses showed that the lead analogs 2,2¢-
the unsubstituted analog (unsub-DPPen) lacked the TrxR in-
diOH-5,5¢-diF-DPPen and 2-OH-5-F-DPPro caused apoptosis
hibitory activity. In contrast, analogs containing the hydroxyl
dose-dependently, as indicated by cleaved forms of caspase 3
and fluorine substituents on the phenyl rings displayed an
and poly (ADP-ribose) polymerase (PARP) (Fig. 5A). The
elevated TrxR inhibitory activity with concentration- and
compounds at a lethal concentration (40 lM) caused a time-
time-dependent potencies stronger than the SGs (Fig. 1A, B
dependent shift of reduced Trx to the oxidized form (Fig. 5B).
and Table 1). It was found that the hydroxyl and fluorine
One of the mechanisms by which Trx exerts antiapoptotic
substitutions at both Ring A and B of the DPPen pharmaco-
effects involves reduced Trx inhibiting ASK1 through a direct
phore produced the analog 2,2¢-diOH-5,5¢-diF-DPPen that
protein–protein interaction (40). ASK1 is a mitogen-activated
displayed the strongest TrxR inhibitory effect (IC50 at 1-h in-
protein kinase kinase kinase (MAPKKK) that lies upstream of
cubation: 9.1lM). Compounds containing a DPPro pharma-
the MAPK signaling pathway. Its activation by phosphory-
cophore, also commonly known as chalcones, were found to
lation leads to activation (also by phosphorylation) of two
exert a TrxR inhibition. Unlike the inactive unsub-DPPen, the
downstream MAPK signaling cascades, the c-Jun N-terminal
unsubstituted DPPro analog (unsub-DPPro) possessed a
kinase (JNK) and p38 pathway, which eventually leads to
greater TrxR inhibitory efficacy than the SGs, with hydroxyl
apoptosis (22). The drug effects on the Trx-ASK1 interaction
and fluorine substitutions enhancing the inhibitory effects
were examined by Trx immunoprecipitation reactions. As the
(Fig. 1A, C and Table 1). Among the DPPro-based analogs,
endogenous ASK1 levels were nondetectable, the cells were
2-OH-5-F-DPPro produced the strongest dose- and time-
transfected with pCMV-SPORT6-ASK1 to attain detectable
dependent enzyme inhibition that was comparable to the
ASK1 levels. As shown in Figure 5C, a decrease in the amount
inhibitory activity of the most active DPPen analog 2,2¢-diOH-
of coimmunoprecipitated ASK1 in the lysates of cells treated
5,5¢-diF-DPPen.
with 40 lM 2,2¢-diOH-5,5¢-diF-DPPen and 2-OH-5-F-DPPro
Evaluation of the effects of DPPen and DPPro compounds
was observed. This was accompanied by an increase in the
on the cell viability had revealed a positive correlation
phosphorylated forms of JNK and p38. Taken together, the
between the antiproliferative and TrxR inhibitory potencies
results had suggested that cytotoxicity elicited by DPPen/
(Fig. 2). The linear correlation coefficient r was calculated as
DPPro-mediated TrxR inhibition was at least partly a result of
0.81. In contrast, these properties of SGs were poorly corre-
dissociation of oxidized Trx from ASK1, leading to ASK1-
lated. Expectedly, DPPen and DPPro analogs that displayed
dependent apoptosis induction.
a greatest TrxR inhibition were also the most cytotoxic
analogs. Of note, the DPPro-based compounds generally
elicited greater antiproliferative activities than their DPPen Characterization of TrxR inhibition
counterparts.
by DPPen and DPPro compounds
TrxR inhibition by the DPPen and DPPro compounds was
NADPH dependent, with the activity of oxidized TrxR re-
maining relatively unaffected while the NADPH-reduced
form was inhibited (Fig. 6A). To investigate whether the
Determination of effects of DPPen and DPPro analogs
on TrxR, Trx, GSH reductase, and GSH peroxidase
cellular activities
In vitro GSH reductase (GR) and GSH peroxidase (GPx) compounds inhibited the enzyme reversibly or irreversibly,
activity assays revealed that the lead DPPen/DPPro analogs recombinant rat TrxR that had activity completely inhibited
2,2¢-diOH-5,5¢-diF-DPPen and 2-OH-5-F-DPPro failed to in- by 20 lM of 2,2¢-diOH-5,5¢-diF-DPPen or 2-OH-5-F-DPPro
hibit both enzymes (Fig. 3A, B). The selectivity of their in- was passed through an NAP-5-desalting column (GE Health-
hibitory effects against Trx- and GSH-related enzymes in HCT care) to remove the excess drug. The enzyme activity failed to
116 and MCF-7 cells were next determined. As shown in recover when aliquots of the eluted enzyme were assayed for
Figure 4A, 6-SG and 3-Ph-3-SG were found to produce a activity (in the presence of NADPH) at various timepoints
marginal decline in the cellular TrxR activity. The cell-based (Fig. 6B), indicating that TrxR was irreversibly inhibited by
results corroborated with the SGs’ weak in vitro TrxR inhibi- DPPen and DPPro compounds. We also found that the lead
tory activity. Conversely, 2,2¢-diOH-5,5¢-diF-DPPen and DPPen and DPPro analogs did not induce increased NADPH

MICHAEL ACCEPTORS INHIBIT THIOREDOXIN REDUCTASE
5
FIG. 1. In vitro inhibitory activity of a,b-unsaturated carbonyl (Michael acceptor) moiety-bearing compounds on
mammalian thioredoxin reductase (TrxR). Dose-dependent (left panels) and time-dependent (right panels) TrxR inhibitory
activities of (A) Michael acceptor-based natural compounds, (B) analogs bearing a 1,5-diphenyl-pent-1-en-3-one (DPPen)
pharmacophore, and (C) analogs bearing a 1,3-diphenyl-pro-1-en-3-one (DPPro) (also known as chalcone) pharmacophore.
Enzyme activities were evaluated by the 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB) assay, upon 1-h incubation of the
compounds at indicated concentrations with 110 nM recombinant rat TrxR (for dose-dependence assay conditions) or in-
cubation of 25 lM of the compounds with 110 nM recombinant rat TrxR for different times (for time-dependence assay
conditions). All data points represent means – SD of at least two independent experiments.
oxidase activity (data not shown), suggesting that DPPen and 30-min incubation of TrxR protein with 2,2¢-diOH-5,5¢-diF-
DPPro analogs are unlikely substrates of TrxR.
DPPen or 2-OH-5-F-DPPro, the enzyme showed a decline in
Whether the lead DPPen and DPPro compounds targeted the activity over time (top panel of Fig. 6C). The results were
the C-terminal Sec in mammalian TrxR was determined by consistent with the observed progressive reduction in BIAM
the N-(Biotinoyl)-N¢-(Iodoacetyl) Ethylenediamine (BIAM) labeling at pH 6.5 (middle panel of Fig. 6C). In contrast, the
labeling assay. In view that Sec and Cys possess a different same labeling experiment conducted at pH 8.5 showed no
pKa value of 5.2 and around 8, respectively, only Sec is significant reduction in the BIAM labeling along time (bottom
available for BIAM alkylation at pH 6.5, whereas at pH 8.5, panel of Fig. 6C), suggesting that it was the C-terminal Sec,
both Cys and Sec are available for BIAM labeling. During a but not the Cys residues that was targeted by the

6
GAN ET AL.
is targeted by the enone-containing compounds bearing the
DPPen and DPPro pharmacophores.
Molecular simulation of the interaction
between the C-terminal active site in TrxR with the SG,
DPPen, and DPPro analogs
To study the plausible drug–protein interactions, molecu-
lar docking exercises were performed for 6-SG, 3-Ph-3-SG,
2,2¢-diOH-5,5¢-diF-DPPen and 2-OH-5-F-DPPro. In our
docking studies, we hypothesized that the candidate inhibi-
tors would interact with the charged Cys498 (i.e., U498C) and
also block other residues within the TrxR active site that might
be important for facilitating and/or stabilizing Trx binding.
As observed in the first crystal structure of the human TrxR-
Trx complex (15), besides the disulfide bond between C32 in
Trx and U498C in TrxR, several amino acid interactions, such
as those involving the Trx residues 58 to 74 and TrxR helical
residues Glu103 to Glu122, could further stabilize the TrxR-
Trx complex. Therefore, the smaller site at the protein surface
that contained residues within the stretch of amino acids
Glu103 to Glu122 was selected. Although the selected site
differed from previous studies that had explored the docking
of curcuminoids (42) and ethaselen (46) into the larger
solvent-exposed 193-amino-acid site, it might be useful in
the context of our studies to gain some insights into the in-
FIG. 2. Correlation of the TrxR inhibitory and anti-
proliferative potencies of Michael acceptor moiety-bearing
compounds. The in vitro 50% TrxR inhibition concentration
(IC50) values of 4-, 6-, and 3-Ph-3-SG, as well as the DPPen
and DPPro analogs were plotted against their corresponding
50% growth inhibition concentration (GI50) values obtained
in HCT 116 cells. The DPPen and DPPro analogs, but not the
shogaols (SGs), showed a positive correlation with the linear
correlation coefficient r = 0.81.
compounds. In addition, the trypsin-digested peptide mas-
ses of TrxR protein treated with 2,2¢-diOH-5,5¢-diF-DPPen terferences (if any) posed by potential TrxR inhibitors on the
were analyzed by matrix-assisted laser desorption/ioniza- TrxR-Trx interaction. Analysis of the top five poses showed
tion (MALDI) mass spectrometry. When the mass spectra
that the interactions of U498C in TrxR with 6-SG and 3-Ph-3-
obtained for untreated protein and drug-treated protein SG were often with the hydroxyl group in the vanilloid moi-
were compared, a mass peak of 1449.7075 had appeared in ety. The remaining poses involving the SGs did not identify
the mass spectrum of the latter sample (Fig. 7A, B). This
any other points of interaction with U498C. Notably, the a,b-
peak corresponded to a calculated mass of 1448.7 of a pep- unsaturated carbonyl group was found to be orientated away
tide fragment containing the C-terminal Sec residue from U498C in all poses (Fig. 9A, B). Docking results obtained
(SGGDILQSGCUG, mass peak of 1144.4–1 H) plus one for 2,2¢-diOH-5,5¢-diF-DPPen revealed that the hydroxyl-
molecule of 2,2¢-diOH-5,5¢-diF-DPPen, and plus 1 H
group in Ring B and the b-carbon atom in the enone group
(1143.4 + 304.3 + 1). The proposed mechanism of alkylation could interact with U498C, whereas for 2-OH-5-F-DPPro, the
by 2,2¢-diOH-5,5¢-diF-DPPen at the Sec residue to form a hydroxy-group in Ring A and the b-carbon atom interacted
Michael acceptor adduct is as illustrated in Figure 8. The
with U498C (Fig. 9C, D). Additionally, a number of findings
results obtained from the BIAM labeling assay and MALDI not observed for the SGs were noted for the DPPen and DPPro
mass spectrometry suggest that the penultimate Sec residue lead analogs. First, most of the poses showed that the enone
FIG. 3. In vitro effects of the DPPen and DPPro lead analogs on glutathione reductase (GR) and glutathione peroxidase
(GPx) activities. The activity of yeast GR (20 nM) incubated with 200 lM NADPH and indicated compounds at various
concentrations (1 to 100 lM) for (A) 30 min and (B) 1 h was determined by following the absorbance at 340 nm after adding
glutathione disulfide and NADPH. The activity of bovine GPx (100 nM) incubated with 20 nM yeast GR, 200 lM NADPH,
1 mM glutathione, and indicated compounds at various concentrations for (A) 30 min and (B) 1 h was determined by after the
absorbance at 340 nm after adding H2O2. All data points are means – SD of two independent experiments.

MICHAEL ACCEPTORS INHIBIT THIOREDOXIN REDUCTASE
7
FIG. 4. Effects of the
DPPen and DPPro lead ana-
logs on enzyme activities in
HCT 116 and MCF-7 cells.
Lysates of HCT 116 and
MCF-7 cells treated with in-
dicated concentrations of SG,
DPPen, and DPPro analogs
for *12 h were collected for
enzyme activity and protein
expression
determination.
TrxR (A), Trx (B), GR (C), and
GPx (D) activities were ex-
pressed as a percentage of
dimethyl sulphoxide control.
All data points are means –
SE of two to four indepen-
dent experiments. *Statisti-
cally significant difference
( p < 0.05) in enzyme activity
compared to control. (E) The
cell lysates with equal protein
were analyzed by Western
blotting with indicated anti-
bodies for TrxR and Trx ex-
pression.
Western
blot
images are representative of
at least two independent ex-
periments.

8
GAN ET AL.
FIG. 5. Effects of the DPPen
and DPPro lead analogs
on the Trx-apoptosis signal-
regulating kinase 1 (ASK1)
interaction and apoptosis in-
duction in HCT 116 cells. (A)
Dose-dependent induction of
apoptosis, indicated by pres-
ence of cleaved caspase 3 (17/
19kDa) and poly (ADP-ribose)
polymerase (PARP) (89 kDa),
after 12-h treatment with
2,2¢-diOH-5,5¢-diF-DPPen or
2-OH-5-F-DPPro. (B) Time-
dependent changes in the
redox state of Trx after treat-
ment with 2,2¢-diOH-5,5¢-diF-
DPPen or 2-OH-5-F-DPPro at a
lethal concentration of 40 lM
at indicated timepoints. (C)
The collected lysates of ASK1-
transfected cells treated or
untreated for 8 h with 40 lM
2,2¢-diOH-5,5¢-diF-DPPen or
2-OH-5-F-DPPro were sub-
jected with immunoprecipita-
tion using an anti-human Trx
antibody. Immunoprecipitates
and aliquots of the cell lysates
were analyzed by Western
blotting with the indicated
antibodies. The images shown
are representative Western
blot results of three indepen-
dent experiments.
group was proximal to and orientated toward U498C. Sec- drug–protein adduct. The docking results had suggested that
ondly, the DPPen and DPPro lead analogs established more 2,2¢-diOH-5,5¢-diF-DPPen and 2-OH-5-F-DPPro were docked
points of interaction with residues within the docking site. into the TrxR C-terminal active site more favorably than the
Besides the interactions between the hydroxyl group and SGs, with an electrophilic b-carbon atom establishing the in-
b-carbon atom with U498C, the top pose for 2,2¢-diOH-5,5¢- teraction with the Sec residue.
diF-DPPen revealed possible hydrogen bonding between the
compound and TrxR’s Ser111 (Fig. 9C), an amino acid resid-
Discussion
ing within the stretch of TrxR helical residues (Glu103 to
Glu122) identified to interact with the residues in Trx to sta-
TrxRs belong to the flavoprotein family of the pyridine
bilize the TrxR-Trx complex (15). Such interaction suggests nucleotide–disulfide oxidoreductases that include GR. The
that docking of the DPPen or DPPro analogs into TrxR’s active types of TrxRs found in mammals include cytosolic TrxR1
site may result in (1) the Sec residue being targeted, and (2) a (53), mitochondrial TrxR2 (28), and testis-specific Trx GR (44).
blocking effect to prevent formation of or to disrupt stabili- All types of TrxR and GR have a common domain structure
zation of the TrxR-Trx complex. Thirdly, the hydroxyl- and comprising a binding domain for flavin adenine dinucleotide
fluorine substituents on the lead DPPen and DPPro analogs and NADPH, as well as an interface domain. In addition,
showed a large solvent-accessible area, as indicated by the mammalian TrxR and GR possess a similar N-terminal -Cys-
dark blue regions surrounding each atom on the compound in XXXX-Cys- active site and therefore share a similar reaction
the ligand interaction map. Conversely, a large part of the mechanism with regard to the reductive half-reaction (3, 41).
respective nonpolar aliphatic and 3-phenyl (Ring A) side The C-terminal Sec-containing active site, being absent in GR,
chain of 6-SG and 3-Ph-3-SG was much exposed to the sol- is unique of mammalian TrxR. Upon reduction by accepting
vent. Given that the active site was largely exposed to the the reducing equivalents from the N-terminal dithiol active
solvent, the presence of the relatively small and hydrophilic site, this C-terminal tail moves such that the selenothiol motif
substituents might favor the exposure of the compounds at becomes solvent exposed and transfers electrons over to
the solvent surface, which in turn could help stabilize the substrates (6, 10, 16, 41). Such unique properties have made

FIG. 6. Irreversibility of TrxR inhibition and selenocysteine (Sec) residue targeting by the DPPen and DPPro lead
analogs. (A) Oxidized recombinant rat TrxR (110 nM) in the presence or absence of 200 lM NADPH was incubated with
different concentrations of 2,2¢-diOH-5,5¢-diF-DPPen or 2-OH-5-F-DPPro for 20 min, and the TrxR activity was determined by
the DTNB assay. All data points are means of two independent experiments. (B) Recombinant rat TrxR (0.9 lM) was
incubated with 20 lM of 2,2¢-diOH-5,5¢-diF-DPPen or 2-OH-5-F-DPPro for 20 min in a 50 mM Tris-HCl, 1 mN EDTA, pH 7.5
buffer containing 200 lM NADPH. The excess drug was removed by passing the protein through a NAP-5-desalting column,
followed by determination of the activity of the eluted protein at indicated times using the DTNB assay. (C) Recombinant rat
TrxR (0.9 lM) was incubated with 200 lM NADPH and 20 lM of 2,2¢-diOH-5,5¢-diF-DPPen or 2-OH-5-F-DPPro. At different
timepoints, an aliquot of enzyme mixture was withdrawn for TrxR activity measurement by the DTNB assay and N-
(Biotinoyl)-N¢-(Iodoacetyl) Ethylenediamine (BIAM) labeling of Sec at pH 6.5 and 8.5. Top panel: time course of TrxR enzyme
activity; middle panel: horseradish peroxidase (HRP)-conjugated streptavidin detection of BIAM labeling of free selenol at pH
6.5 at various incubation times; bottom panel: HRP-conjugated streptavidin detection of BIAM labeling of free selenol and
sulfhydryls at pH 8.5 at various incubation times. Results shown are representative of three independent experiments.
9

10
GAN ET AL.
FIG. 7. Peptide mass analysis
of TrxR-2,2¢-diOH-5,5¢-diF-DPPen
protein adduct by matrix-assisted
laser desorption/ionization (MAL-
DI) mass spectrometry. NADPH-
reduced recombinant rat TrxR
(3 lM) was incubated at 37ꢀC with
or without 50 lM 2,2¢-diOH-5,5¢-
diF-DPPen for 30 min, desalted to
remove excess drug, denatured in
urea, and trypsin-digested. Finally,
MALDI mass spectrometry was
performed to obtain the peptide
mass spectrum of the (A) untreated
and (B) 2,2¢-diOH-5,5¢-diF-DPPen-
treated protein sample. (To see this
illustration in color, the reader is
referred to the web version of this
article at www.liebertpub.com/ars.)
TrxR susceptible to inhibition by electrophilic compounds (12). We therefore screened a number of commonly known
that can react with the accessible and nucleophilic selenoate Michael acceptor moiety-bearing natural compounds that as
at the C-terminal active site. Another related enzyme GPx is of yet have not been reported as TrxR inhibitors. These natural
also a selenoprotein, except that the Sec residue in the active products, which serve as potential scaffolds for diversity-
site is not as solvent exposed as that in mammalian TrxR oriented synthesis, belong to distinct structural families:
(27, 38). Efforts to discover and develop TrxR inhibitors are a,b-unsaturated amides and unconjugated a,b-unsaturated
underway. For instance, high-throughput screening of the ketones. The a,b-unsaturated IBAs exhibited poor anti-
1280-member LOPAC1280 collection of compounds has been proliferative activities correlated to their lack of TrxR inhibi-
conducted to identify the novel inhibitors and substrates tory activities. Conjugation of electrons on the amide nitrogen
of TrxR (36).
alleviated the electronegative effect of the carbonyl group,
TrxR inhibitors are classified based on their mechanisms of thus reducing the electrophilic character of the b carbon atom
enzyme inhibition and structural types (7, 30, 45). Among and in turn their ineffectiveness for TrxR inhibition. Un-
them are compounds containing a,b-unsaturated carbonyl conjugated a,b-unsaturated ketones zerumbone and SGs
groups, which allow them to undergo a Michael addition displayed no or weak TrxR inhibitory activity in contrast to
reaction between the b-carbon atom and the Se- of Sec in their potent antiproliferative effects. In these compounds, an
TrxR. Such electrophilic compounds, also called the Michael electron-donating alkyl group linked to the Michael acceptor
acceptors, are structurally diversified, which hinder the car- group had reduced electrophilicity of the b-carbon atoms,
rying out of large-scale structure–activity relationship studies. which explained the poor TrxR inhibition. The poor correla-
To add to the difficulty for the rational design of Michael tion between zerumbone’s and SGs’ inhibitory effects on cell
acceptor-based TrxR inhibitors, Michael acceptors that lack proliferation and TrxR activity had indicated that TrxR tar-
the TrxR inhibitory effects are not clearly known. We hy- geting was unlikely the mechanism underlying their antitu-
pothesized that despite the structural diversity, an a,b- mor action. The results agreed with findings by published
unsaturated carbonyl moiety and other structural features studies, which have reported the apoptotic activities of zer-
formed a TrxR-inhibiting pharmacophore. Nature is a source umbone and SGs in association with other signaling path-
of bioactive compounds that has led to discovery of Michael ways (17, 21, 26, 32, 35). Molecular docking results obtained
acceptor-based TrxR inhibitors such as palmarumycin (34, for the SGs also revealed plausible reasons for their poor TrxR
48), curcumin (14), flavonoids (31), and trans-cinnamaldehyde inhibitory properties. First, the a,b-unsaturated carbonyl

MICHAEL ACCEPTORS INHIBIT THIOREDOXIN REDUCTASE
11
FIG. 8. Proposed mechanism of
inhibition of mammalian TrxR
by 2,2¢-diOH-5,5¢-diF-DPPen. The
electrophilic b-carbon atom in 2,2¢-
diOH-5,5¢-diF-DPPen undergoes a
Michael addition reaction with the
Sec residue in NADPH-reduced
TrxR to form a Michael acceptor
adduct.
group was orientated away from the Sec residue, which re- were synthesized. In the DPPen and DPPro series, both un-
duced the probability of the interaction between the electro- substituted analogs registered weakest TrxR inhibition,
philic b-carbon atom with Sec. Secondly, the high solvent whereas 2,2¢-diOH-5,5¢-diF-DPPen and 2-OH-5-F-DPPro
exposure of the nonpolar aliphatic chain might discourage a were the respective analogs recording the strongest TrxR in-
stable drug interaction at the enzyme’s active site. Collec- hibitory effect. Clearly, 2-hydroxy and 5-fluorine substitu-
tively, the findings had suggested that having a Michael ac- tions at the phenyl ring adjacent to the a,b-unsaturated bond
ceptor group could not unequivocally indicate the TrxR (Ring A) yielded a greater TrxR inhibitory activity than hav-
inhibitory activity; other structural features might be required ing similar substitutions at the phenyl ring closer to the car-
for enzyme inhibition.
bonyl group (Ring B). This could be attributed to the stronger
3-Ph-3-SG, a novel SG analog that possessed a comparable influence exerted by an electron-withdrawing fluorine sub-
antiproliferative and TrxR inhibitory activity as 6-SG was stituent at Ring A on the b-carbon atom, making it more
selected as the compound on which structural derivatives electrophilic for reaction with Se- . Molecular docking work
were synthesized. Working along our hypothesis that other had revealed high solvent exposure of the hydrophilic
structural features were required for enzyme inhibition, we hydroxyl and fluorine substituents, which might contribute to
investigated whether a shortened distance between the two the TrxR inhibition by favoring the stability of the ligand–
phenyl rings (Ring A and B) would affect the TrxR inhibitory protein adduct formed at the enzyme’s active site. The
activity. Compounds carrying the DPPen or DPPro pharma- hydroxyl and fluorine substitutions at Ring B, being distant
cophores were therefore synthesized. Based on the knowl- from the b-carbon atom, would supposedly exert a dimin-
edge from published studies that Michael acceptor-based ished electron-withdrawing effect. Nonetheless, a favorable
TrxR inhibitors such as quinones such as juglone (9), engagement of the substituents with solvent might have sta-
quinols such as PMX464 (11), 4-hydroxynonenal (13), pal- bilized the compound’s interaction with the active site, which
marumycin CP1 (48), and flavonoids such as myricetin and possibly explained the greater inhibitory potencies of these
quercetin (31) contain a hydroxyl group proximal to the a,b- analogs (2¢-OH-5¢-F-DPPen and 2¢-OH-5¢-F-DPPro) over their
unsaturated carbonyl group, and our recent findings that unsubstituted counterparts. On the combined bases of the
2-hydroxy and 5-fluorine substitutions on the phenyl ring favorable electronic and hydrophilic character, we would
adjacent to the a,b-unsaturated carbonyl group led to an en- expect analogs bearing the 2-hydroxy and 5-fluorine substit-
hanced TrxR inhibitory activity (12), the DPPen and DPPro uents in both Ring A and B to display the strongest TrxR
analogs carrying the hydroxyl and fluorine substitutions inhibitory activity. This was the case for the DPPen series of

12
GAN ET AL.
FIG. 9. Molecular simulation of the interaction between C-terminal active site in TrxR with SGs, DPPen, and DPPro
compounds. Molecular docking exercises were conducted for (A) 6-SG, (B) 3-Ph-3-SG, (C) 2,2¢-diOH-5,5¢-diF-DPPen, and (D) 2-
OH-5-F-DPPro, with the ligand interaction maps of the top five poses obtained for each ligand presented in descending order.
analogs, with 2,2¢-diOH-5,5¢-diF-DPPen found most potent. DPPen and DPPro compounds caused a selective dose-de-
Unexpectedly, for the DPPro series of analogs, 2-OH-5-F- pendent reduction in the TrxR activity with Trx, GR, and GPx
DPPro was found most potent instead, suggesting that the activity minimally affected. Michael acceptor-based com-
hydroxyl and fluorine substituents at Ring B might have im- pounds tend to target multiple cellular pathways and are often
peding effects on TrxR inhibition. One proposed explanation multifunctional. Components of these signaling pathways
was being adjacent to each other; 2¢-hydroxy on Ring B and usually contain soft nucleophiles such as thiols that are integral
the carbonyl oxygen in 2,2¢-diOH-5,5¢-diF-DPPro were en- to protein functions. Selenothiol in Sec is also a soft nucleophile
gaged in intramolecular hydrogen bonding, reducing elec- that the Michael acceptor moiety reacts with in preference over
tronegativity of the latter atom and in turn reduced hard nucleophiles such as amino- and hydroxyl groups. In-
electrophilicity on the b-carbon atom.
deed, the BIAM labeling assay and MALDI mass spectrometry
TrxR inhibitory efficacies of the DPPen and DPPro analogs provided evidence that suggests that the C-terminal Sec resi-
were positively correlated to their antiproliferative potencies, due in TrxR is the site of target by compounds that contain
and TrxR inhibition was found to be NADPH dependent and the DPPen or DPPro pharmacophores. Our study has there-
irreversible. The compounds’ selectivity for TrxR was demon- fore concluded that structural features that include an a,b-
strated in cell-based enzyme inhibition studies, where the lead unsaturated carbonyl-centric core contained in a DPPen or

MICHAEL ACCEPTORS INHIBIT THIOREDOXIN REDUCTASE
13
DPPro pharmacophore, together with hydroxyl and fluorine dehydrated by refluxing in benzene in the presence of toluene
substitutions, contribute toward a strong TrxR inhibitory char- sulfonic acid to produce 3-Ph-3-SG. The compound was
acter. TrxR inhibition caused Trx oxidation and dissociation of characterized by proton and carbon nuclear magnetic reso-
the Trx-ASK1 complex, resulting in ASK1-dependent activation nance (NMR), and its purity as determined by reverse-phase
of the JNK and p38 pathways that culminated in apoptosis in- high-performance liquid chromatography (HPLC) was found
duction. Nonetheless, the possibility that compound-induced to be 95%.
TrxR inhibition mediates apoptosis induction independent of
ASK1 signaling is not ruled out. As such, while the C-terminal
2-OH-5-F-DPPen and 2,2¢-diOH-5,5¢-diF-DPPen. To a
active site of TrxR is known to broadly accommodate a mixture of benzylacetone or 2-hydroxy-5-fluoro-benzylacetone
structurally diversifying range of Michael acceptor-based (2 mmol) and benzaldehyde or 2-hydroxy-5-fluoro-benzaldehyde
compounds, our findings have suggested that a systematic (2 mmol) dissolved in ethanol (1.5 ml), two drops of piperidine
approach for discovery of Michael acceptor-based TrxR in- were added. The mixture was stirred at room temperature for
hibitors is feasible, and can be potentially applied for future 2 weeks, with one drop of piperidine added every 3 days. The
design of novel TrxR inhibitors as anticancer agents.
viscous reaction mixture was diluted with alcohol and made
acidic with HCl. The pure product was isolated from silica-gel
column chromatography with hexane/ethyl acetate as the mobile
phase. Compounds were characterized by proton and carbon
NMR, and their purities of at least 95% were determined by
Materials and Methods
Chemicals, materials, and cell culture
Dodeca-IBA and Hexa-IBA were from Chromadex. reverse-phase HPLC.
Synthesis of 4-, 6-, 8-, and 10-SG has been described previ-
ously (17). Dimethyl sulphoxide (DMSO) drug stocks of
Unsub-DPPen and 2¢-OH-5¢-F-DPPen. A solution of
50 mM were made and stored at - 20ꢀC. Recombinant rat benzylacetone (5 mmol) and benzaldehyde or 2-hydroxy-5-
TrxR was prepared as previously described (2). Yeast GR, fluoro-benzaldehyde (5 mmol) was added to a solution of
bovine GPx, insulin, GSH disulfide (GSSG), and trypsin of 85 mM KOH and stirred at 55ꢀC for 4–6 h. The reaction mixture
proteomics grade were from Sigma. Recombinant human Trx was then transferred to an ice bath with pH being adjusted to
and anti-human Trx antibody were obtained from IMCO 2–3 by adding HCl. The mixture was extracted by ethyl ace-
Corporation. BIAM and horseradish peroxidase-conjugated tate. The silica-gel column chromatography was performed to
streptavidin were from Molecular Probes. Antibodies against yield the desired DPPen compounds, whose structures were
caspase 3, cleaved caspase 3, PARP, JNK, phospho-JNK verified by proton and carbon NMR, and the purities of at least
(Thr183/Tyr185), p38, and phospho-p38 (Thr180/Tyr182) 95% were determined by reverse-phase HPLC.
were from Cell Signaling Technology, while those specific for
human TrxR, ASK1, and b-actin were from Santa Cruz Bio-
DPPro compounds (also known as chalcones).
technology. Polyclonal antibodies against rat TrxR1 were Benzaldehyde (2 mmol) and acetophenone (2 mmol) were
raised in rabbits and purified as described previously (39). condensed in 20 ml of 3% w/v NaOH in methanol at room
Human-derived colon HCT 116 and MCF-7 breast carcinoma temperature for 4 h. The reaction mixture was next sub-
cells were maintained in a Rose Park Memorial Institute 1640 merged in ice bath, and HCl was added to adjust pH to 2–3.
medium supplemented with 10% fetal bovine serum, 100 The mixture was stirred overnight, and the crude product was
units/ml penicillin, and 100 lg/ml streptomycin and incu- extracted by ethyl acetate. Further purification was done by
bated at 37ꢀC in an humidified atmosphere of 95% air and 5% recrystallization in ethanol. All DPPro analogs were charac-
CO2.
terized by proton and carbon NMR, and the purities as de-
termined by reverse-phase HPLC were at least 95%.
Extraction and purification of zerumbone
Cell viability assay
Zerumbone was extracted using a published method (25)
with modifications. Slices of fresh rhizomes of Zingiber zer-
umbet Smith (1.1 kg) harvested in Chiang Mai, Thailand,
were subjected to steam distillation. The distillate was kept
for 1 or 2 days at room temperature to give a crystalline solid
of an essential oil. The crystals were collected, and the re-
maining water–oil mixture was subjected to extraction by
hexane. The pooled hexane layers were concentrated and
used for recrystallization. Pure zerumbone was collected
(yield: 0.19%).
The viabilities of HCT 116 and MCF-7 cells treated with
various concentrations of indicated compounds were deter-
mined by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide as described previously (12).
Determination of TrxR activity by in vitro DTNB
reduction assay
TrxR activity was determined as previously described (11).
Chemical synthesis
Preparation of cell lysates and Western blot analysis
3-phenyl-3-shogaol. Vanillin was condensed with ace-
tone in NaOH at room temperature for 12 h, and the product
was hydrogenated to zingerone. The phenolic group in zin-
gerone was protected by tetrahydropyran (THP). At - 78ꢀC,
THP-protected zingerone was activated by lithium diisopro-
pylamide in anhydrous THP, followed by an aldol conden-
sation reaction with benzaldehyde. The formed gingerol was
The cell lysate was prepared, and the Western blot analysis
was performed, as previously described (12).
Determination of TrxR and Trx activity in cell lysates
The TrxR and Trx activity in the cell lysates was determined
by an endpoint insulin assay as previously described (12).

14
GAN ET AL.
GR and GPx activity assays
recombinant rat TrxR was performed using the procedures as
described previously (12).
The GR activity was determined in vitro as described
previously (11). For determination of the cellular GR activity,
25 lg of cell lysate was mixed with a solution of GSSG and
NADPH in 0.1 M sodium phosphate/2 mM EDTA, pH 7.5, to
a final volume of 100 ll (final GSSG and NADPH concen-
MALDI mass spectrometric analysis of drug-treated
recombinant rat TrxR protein
Recombinant rat TrxR (3lM) in a 50 mM Tris-HCl, 1 mM
tration: 1 mM and 200 lM respectively). The enzyme activity EDTA, pH 7.5 buffer containing 200lM NADPH was incu-
was determined by measuring the decrease in absorbance at bated at 37ꢀC with 50lM 2,2¢-diOH-5,5¢-diF-DPPen for 30 min.
340 nm during the initial 5 min and expressed as a percent- An aliquot of the protein was tested in the DTNB reduction
age of the enzyme activity of that of the DMSO-treated assay to verify that enzyme activity was abolished. Excess drug
sample.
and NADPH were removed using a Sephadex G-25-containing
For in vitro determination of GPx activity, to a mixture NAP-5-desalting column (GE Healthcare). An aliquot of the
containing 100 nM GPx, 20 nM GR, 1 mM GSH, and 200 lM desalted enzyme was tested in the DTNB reduction assay to
NADPH in 0.1 M sodium phosphate/2 mM EDTA, pH 7.5, confirm the null activity. The enzyme was denatured in 8 M
H2O2 was added to a final concentration of 1.5 mM to initiate urea at 60ꢀC for 1 h, diluted 10-fold in 50 mM Tris–HCl/1 mM
the reaction. The GPx activity was determined by measuring CaCl2, pH 7.6, buffer, and concentrated using an Amicon Ultra
the decrease in absorbance at 340 nm during the initial 6 min 30K NMWL filter (Merck Millipore) to a final concentration
and expressed as a percentage of enzyme activity of that of the range of 0.05–0.1 lg/ll. The protein was digested by trypsin
DMSO-treated sample. The cellular GPx activity was deter- (trypsin-to-protein ratio 1:10) at 37ꢀC for 6 h, followed by de-
mined similarly except to replace bovine GPx with 25 lg of salting using C18 ZipTips (Merck Millipore) with the peptides
cell lysates.
eluted in 80% acetonitrile/0.1% trifluoroacetic acid (TFA). The
desalted sample (0.5ll) was spotted on a 384-well target plate
and crystallized with 0.5 ll of a-cyano-4-hydroxycinnamic acid
matrix solution in 50% acetonitrile/0.1% TFA. The sample was
analyzed on the MALDI-Tof-Tof MS: 4800 MALDI TOF/TOF
Analyzer (Applied Biosystems). The mass-over-charge ratio
(m/z) data of the sample were acquired in the reflector mode
using the reflectron method (accelerating voltage: 20000V; laser
intensity: 3300–3600; acquisition mass range: 1100 to 1500). The
peptide mass fingerprints were analyzed using the peptide-
Cutter online tool (www.web.expasy.org).
Preparation of lysates and determination of Trx redox
state in cells
The Trx redox state in cells was determined as described
previously (50) with slight modifications. Upon reaching
70%–80% confluency, HCT 116 seeded in 60-mm plates were
treated with 40 lM 2,2¢-diOH-5,5¢-diF-DPPen, 2-OH-5-F-
DPPro, or DMSO for 6 or 12 h. The lysates were collected in a
lysis buffer comprised of 50 mM Tris–HCl (pH 8.3), 2 mM
EDTA, 6 M guanidine HCl, 0.5% Triton-X, and 50 mM
iodoacetamide. The lysates were incubated at room temper-
ature in the dark for 30 min, followed by brief sonication and Molecular modeling
passing through a MicroSpin G-25 column (GE Healthcare).
The mammalian TrxR1 structure PDB ID 3QFA (resolution
The desalted samples were subjected to native polyacryl-
amide gel electrophoresis (PAGE), followed by transfer of
separated proteins onto nitrocellulose membranes for im-
munoblotting with an anti-human Trx antibody.
˚
of 2.20 A) carrying double mutations C497S and U498C was
obtained from the Protein Data Bank. Docking simulations
were performed using Molecular Operating Environment
(MOE), version 2011.10. The 3QFA structure was processed
using the recommended Structure Preparation application in
MOE, which was used to diagnose and correct structural is-
Immunoprecipitation
At 40% confluency, HCT 116 cells seeded in 60-mm plates sues such as incorrect number of hydrogens and missing at-
were transfected with 1.5 lg of pCMV-SPORT6-ASK1 per oms. The S-atom in Cys498 was added a negative charge to
plate. The transfected cells were allowed to grow to 70%–80% mimic the nucleophilic selenoate ion for docking simulations.
confluency, followed by treatment with 40 lM 2,2¢-diOH-5,5¢- Visual inspection of the TrxR-Trx complex based on the crystal
diF-DPPen, 2-OH-5-F-DPPro, or DMSO for 8 h, and the lysates structure elucidated by Fritz-Wolf et al. (15) and the Site Finder
were collected. For each lysate sample, a volume containing results of MOE revealed two potential sites for molecular
equal amount of protein (1–1.2 mg protein) was mixed with docking involving the charged residue. The first was a large
1.5 lg of Trx antibody coupled to 20 ll of protein G-sepharose site that had 193 residues accessible to solvent. The other po-
(GE Healthcare) and tumbled at 4ꢀC for 2 h. The sepharose tential site, being smaller, was located nearer at the protein
beads were washed three times in a cold washing buffer con- surface and contained residues such as Asn104, Asn107, and
taining 20 mM Tris (pH 7.5), 0.5 M NaCl, and 1 mM ethylene Ser111. The latter site was chosen for docking with S- in
glycol-bis (b-amino ethyl ether)-N,N,N¢N¢-tetraacetic acid and Cys498 identified as an essential binding atom. During the
once in buffer containing 50 mM Tris (pH 7.5). The im- automated docking process, each ligand was docked into the
munoprecipitated proteins were denatured in a sodium dodecyl active site in different ways, creating different poses each with a
sulfate (SDS) sample buffer containing 5% b-mercaptoethanol docking score. The top five poses of each ligand were obtained
and subjected to SDS-PAGE and Western blotting.
and analyzed for their interactions with TrxR.
BIAM labeling assay
Acknowledgments
Biotin labeling through BIAM alkylation of the free selenol
We thank Dr. Elias Arner and Dr. Qing Cheng (Division of
at pH 6.5, or the free selenol and sulfhydryls at pH 8.5, in Biochemistry, Karolinksa Institutet) for providing recombinant

MICHAEL ACCEPTORS INHIBIT THIOREDOXIN REDUCTASE
15
rat TrxR, and Dr. Thilo Hagen (Department of Biochemistry,
Yong Loo Lin School of Medicine, National University of Sin-
gapore) for providing pCMV-SPORT6-ASK1. This work was
supported by a National Medical Council Grant NMRC/NIG/
0050/2009 and a National University of Singapore Academic
Research Fund Tier 1 R-148-116-112.
maldehydes inhibit thioredoxin reductase and induce Nrf2:
potential candidates for cancer therapy and chemopreven-
tion. Free Radic Biol Med 48: 98–111, 2010.
13. Fang J and Holmgren A. Inhibition of thioredoxin and
thioredoxin reductase by 4-hydroxy-2-nonenal in vitro and in
vivo. J Am Chem Soc 128: 1879–1885, 2006.
14. Fang J, Lu J, and Holmgren A. Thioredoxin reductase is
irreversibly modified by curcumin:
mechanism for its anticancer activity. J Biol Chem 280: 25284–
25290, 2005.
a novel molecular
Author Disclosure Statement
No competing financial interests exist.
15. Fritz-Wolf K, Kehr S, Stumpf M, Rahlfs S, and Becker K.
Crystal structure of the human thioredoxin reductase-
thioredoxin complex. Nat Commun 2: 383, 2011.
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Address correspondence to:
Dr. Eng-Hui Chew
Department of Pharmacy
National University of Singapore
Science Drive 4
Singapore 117543
Republic of Singapore
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E-mail: phaceh@nus.edu.sg
Date of first submission to ARS Central, August 21, 2012; date
of final revised submission, December 19, 2012; date of ac-
43. Soini Y, Kahlos K, Napankangas U, Kaarteenaho-Wiik R,
Saily M, Koistinen P, Paaakko P, Holmgren A, and Kinnula ceptance, January 1, 2013.

MICHAEL ACCEPTORS INHIBIT THIOREDOXIN REDUCTASE
Abbreviations Used
17
GSH ¼ glutathione
Hexa-IBA ¼ hexa-2,4-dienoic acid isobutylamide
JNK ¼ c-Jun N-terminal kinase
MALDI ¼ matrix-assisted laser desorption/ionization
MAPK ¼ mitogen-activated protein kinase
MOE ¼ molecular operating environment
Sec ¼ selenocysteine
AP-1 ¼ activator protein-1
ASK1 ¼ apoptosis signal-regulating kinase 1
Dodeca-IBA ¼ dodeca-2(E),4(E)-dienoic acid
isobutylamide
DPHep ¼ 1,7-diphenyl-hept-3-en-5-one
DPPen ¼ 1,5-diphenyl-pent-1-en-3-one
DPPro ¼ 1,3-diphenyl-pro-1-en-3-one
DTNB ¼ 5,5¢-dithiobis-(2-nitrobenzoic acid)
GPx ¼ glutathione peroxidase
SG ¼ shogaol
TFA ¼ trifluoroacetic acid
THP ¼ tetrahydropyran
Trx ¼ thioredoxin
GR ¼ glutathione reductase
TrxR ¼ thioredoxin reductase