Synthesis and molecular recognition studies on small-molecule inhibitors for thioredoxin reductase

Article abstract of DOI:10.1021/jm5012098

Thioredoxin reductase (TrxR), which is overexpressed in many aggressive cancers, plays a crucial role in redox balance and antioxidant function, including defense of oxidative stress, control of cell proliferation, and regulation of cell apoptosis. Deactivation of TrxR can destroy the homeostasis of the cancer cells, inducing elevation of reactive oxygen species (ROS) levels and the oxidation of enzymatic substrates. Here, we synthesized and identified a new gold(I) small molecule (D9) that possesses two strong electron-donating moieties, i.e., 4-methylphenyl alkynyl and thionyldiphenyl phosphine, exhibiting an enhanced p-π conjunction effect. The resulting compound shows the increased soft Lewis acids and the stability of gold(I). And we demonstrated that D9 could efficiently and specifically inhibit the activity of TrxR in vitro and in vivo, and it could effectively avoid the ligand exchange with albumin that was one of the most abundant proteins in blood. We believe that these comprehensive studies on the relationship between the structure and performance will provide inspiring information on the precise synthesis and design of new compounds for targeting TrxR.

Full text of DOI:10.1021/jm5012098

Article
pubs.acs.org/jmc
Synthesis and Molecular Recognition Studies on Small-Molecule
Inhibitors for Thioredoxin Reductase
Di Zhang,† Zhonghe Xu,‡ Jia Yuan,§ Ying-Xi Zhao,† Zeng-Ying Qiao,† Yu-Juan Gao,† Guang-Ao Yu,*,§
Jingyuan Li,*,‡ and Hao Wang*,†
†CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology
(NCNST), No. 11 Beiyitiao, Zhongguancun, Haidian District, Beijing, 100190, China
‡CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, 19 B, Yuquan Road,
Beijing, China
§Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Central China Normal University, Wuhan, 430079, China
S
* Supporting Information
ABSTRACT: Thioredoxin reductase (TrxR), which is overex-
pressed in many aggressive cancers, plays a crucial role in redox
balance and antioxidant function, including defense of oxidative
stress, control of cell proliferation, and regulation of cell
apoptosis. Deactivation of TrxR can destroy the homeostasis of
the cancer cells, inducing elevation of reactive oxygen species
(ROS) levels and the oxidation of enzymatic substrates. Here, we
synthesized and identified a new gold(I) small molecule (D9)
that possesses two strong electron-donating moieties, i.e., 4-
methylphenyl alkynyl and thionyldiphenyl phosphine, exhibiting
an enhanced p−π conjunction effect. The resulting compound
shows the increased soft Lewis acids and the stability of gold(I).
And we demonstrated that D9 could efficiently and specifically
inhibit the activity of TrxR in vitro and in vivo, and it could effectively avoid the ligand exchange with albumin that was one of the
most abundant proteins in blood. We believe that these comprehensive studies on the relationship between the structure and
performance will provide inspiring information on the precise synthesis and design of new compounds for targeting TrxR.
micromolar concentration in vitro.29,30 Despite their in vitro
efficiency, the chemical stabilities are poor and systematic
INTRODUCTION
■
TrxR is the electron donor for the enzyme of ribonucleotide
toxicities are significant for gold(III) compounds in in vivo
reductase which plays critical roles in DNA synthesis,
applications.4 Furthermore, gold(I) compounds are effective
replication, and repair.1,2 Overexpressed TrxR in cancer cells3
inhibitors of TrxR as well. Gold(I) compounds, e.g.,
is potentially related to the imbalanced deoxynucletide pools
and may accelerate the development of the malignant
phenotype by gene amplification, genetic rearrangements, and
Auranofin,19,20 triphenyl phosphine gold chloride,22 gold
sodium thiomalate,31 sodium aurothiosulfate,31 and gold N-
heterocyclic carbene complexes,26 have been investigated for
their inhibition of the TrxR activity, with half maximal enzyme
inhibition concentration (EC50) values ranging from 4 to 4000
nM.4 Notably, alkynyl phosphine gold(I) compounds exhibited
high activity of TrxR inhibition in vitro.23 Many gold(I)
compounds are practically ineffective in vivo, due to their
potentiality of exchanging ligand with biomolecules, which
usually leads to the reduction or even loss of their activity.4
Herein, we systematically design and synthesize 55 gold(I)
compounds with varying molecular features for targeted
inhibition of TrxR. The screened D9 compound exhibits high
efficiency and specificity for TrxR in vitro. Moreover, we
comprehensively evaluate the inhibition effect of D9 compound
even therapy resistance.4 Recent studies demonstrated that
overexpressed TrxR is necessary for growth of cancer cells and
development of tumors, such as sustained progression of cell
cycle, the evasion of growth-inhibitory signals, and the
resistance to necrosis or apoptosis.3,5−7 On the contrary,
deactivation of TrxR can destroy the redox homeostasis of
cells3,7−9 through the elevation of cellular reactive oxygen
species (ROS) levels10−12 and further lead to the inhibition of
proliferation, and even induction of necrosis or apoptosis of
cells.3,13 Accordingly, numerous TrxR-targeting compounds,
such as platinum,14,15 arsenic,16,17 and gold18−20-related
chemoprevention agents,4,21−26 have been developed as
potential TrxR inhibitors for anticancer treatment. Recent
studies suggested gold(III) compounds could be TrxR-targeted
drug candidates.27,28 Preliminary results showed that apoptosis
of cancer cells was induced by gold(III) compounds with
Received: July 15, 2014
© XXXX American Chemical Society
A
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Figure 1. (a) Structures of a series of elaborately designed gold (I) compounds with host and R radical. (b) Screening of the highly efficient host
structures from A, B, C, D, and E with phenyl as R radical. (c) Screening of the highly efficient R radical (1−11). (d) Investigating the highly efficient
R radical combined with different host structures to screen the highest efficacy TrxR inhibitors. (e) Screening of the potential inhibitor (dark dash
ellipse) of TrxR through designed concentrations (10, 20, and 50 nM) from 15 gold compounds. Error bars in (b), (c), (d), and (e) represent the
SD of experimental duplicates.
Table 1. EC50 Values of 15 Gold Compounds to TrxR and the Homologous Enzyme of GR
EC50 (nM)
EC50 (nM)
Gold compds
TrxR
GR
GR/TrxR
Gold compds
TrxR
GR
GR/TrxR
>10
A9
A1
B9
C2
C10
C4
C8
C9
∼10
∼10
∼10
∼100
∼100
∼100
∼100
∼100
>1000
>1000
>1000
>1000
∼ 50
>1000
>1000
>1000
>100
>100
>100
>10
C1
C5
D7
D8
D9
D10
E9
∼100
>50
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>20
>10
∼100
∼20
<10
>50
∼ 0.5
>100
>10
>10
>10
>10
∼100
∼100
>10
at enzyme, cells, and animal levels. The merits of D9 are (i)
high activity of in vitro TrxR inhibition with nanomolar EC50
value (2.8 nM), which is comparable with the clinically
practiced gold(I) compound Auranofin (EC50 = 4 nM);4 (ii)
good selectivity and high toxicity toward cancer cells and
limited side effects toward normal cells; and (iii) high
stabilityit can prohibit ligand exchange with albuminand
high antitumor efficiency in vivo.
small energy gap between s, p, and d states, which results in
proficient s/d or s/p hybridizations.32 The host structures
include the following: phenyl, 4-methylphenyl, 1-hydroxycyclo-
hexane, 4-methoxyphenyl, and 4-fluorophenyl, denoted A, B, C,
D, and E, respectively (Figure 1a). These host structures with
different electron-donating moieties enhanced the stability of
positive gold atom. To further improve the stability of gold
atom, we designed a series of phosphines with different degrees
of electron-donating as R radical (shown in Figure 1a, R 1−11).
Therefore, 55 kinds of gold(I) compounds were synthesized
through orthogonal combination of host and radical R
RESULTS AND DISCUSSION
■
To realize the specific inhibition of TrxR activity both in vitro
and in vivo, we elaborately designed a series of gold(I)
compounds with ligands of alkynyl and phosphine. The host
structure of gold acetylide compound was combined with
alkynyl ligand with the linear geometry which is attributed to a
1
structures. All these structures were confirmed by H, 13C,
and 31P NMR and by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF)
(see Supporting Information). In order to identify possible
B
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Figure 2. (a) Curves of TrxR activity after incubation with different concentrations of D9. The activity was evaluated through the color changes of
TNB monitored through UV/vis absorption. (b) Kinetic curve of TrxR inhibition by D9; (inset) linear fitting of the reaction ratio curve. (c) Specific
inhibition of TrxR by D9 was verified through comparison with the homologous enzyme of GR. (d) The stability of D9 was investigated through
incubation with BSA at different times. MALDI-TOF was used to monitor the MW changes of BSA. The MW (66.3 kDa) of BSA without gold
compounds incubation was set as control. References I, II, and III were [2-(4-methoxyphenyl)ethyn-1-yl] (triphenylphosphine) gold(I); [2-
phenylethyn-1-yl] (triphenylphosphine) gold(I); and triphenylphosphine gold chloride, and their structures are shown in Supporting Information,
Figure S5.
TrxR inhibitors from these gold compounds, the inhibition
efficacy of gold compounds was first investigated at two
concentrations (50 and 100 nM). The results of the
compounds with host structure (D) and radical R (9) are
shown in Figure 1b, c, d, and Figure S1. And 15 gold
compounds were identified (structures shown in the Support-
ing Information, Figure S2) with relatively high activity. All
these gold compounds similarly bear strong electron-donating
moieties, which may result in the delocalization of the metal
active center.
In addition, the remaining 15 gold compounds were further
screened with detailed concentrations (10, 20, 50 nM). D9
compound (dashed rectangle in Figure 1e) with 4-methyl-
phenyl alkynyl gold (host D) and phosphine ligand of
thienyldiphenylphosphine (R 9) was found to be the most
highly efficient TrxR inhibitor with low inhibition concen-
tration (<10 nM). Meanwhile, the specific inhibition of these
15 gold compounds was roughly investigated using homolo-
gous redox enzyme, i.e. glutathione reductase (GR) with three
gold compound concentrations (20, 500, and 1000 nM; Figure
S3). The EC50 values of these 15 compounds to TrxR and GR
were summarized in Table 1. D9 compound had a minimum
value of EC50 (<10 nM) for TrxR and EC50 > 1000 nM for GR.
The ratio of EC50 for TrxR/GR was higher than 100, suggesting
that D9 compound may have capability to inhibit TrxR with
high efficiency and remarkable specificity.
Next, we further investigated the inhibition efficiency and
specific targeting capabilities of D9 compound in detail. The
concentration-dependent (0−20 nM) TrxR inhibition is shown
in Figure 2a. D9 almost completely inhibited the activity of
enzyme with the concentration as low as 6 nM. The kinetic
studies were also carried out with various concentrations
(Figure 2b).
On the basis of linear dependence of the reaction rate (R2 =
0.9960), the EC50 value of D9 (Figure 2b inset) was 2.8 nM,
which was comparable with the EC50 value of the gold
compound (auranofin). The EC50 value of auranofin toward
TrxR was approximately 4 nM (see Supporting Information,
Figure S4), which was consistent with the previous report.4 And
the detailed inhibition specificity of D9 was also evaluated
through comparing the inhibition of the activities of TrxR and
GR. TrxR activity was reduced by approximately 60% with 2
nM D9 treatment and 80% with 4 nM D9 treatments.
Furthermore, the catalytic activity of GR was not significantly
inhibited even when the concentration of D9 compound
increased to more than 1000 nM. The EC50 value of GR was at
least 337-fold higher than that of TrxR (Figure 2c, inset).
Therefore, the results of these detailed studies confirmed that
D9 had an efficient and specific profile for targeted inhibition of
TrxR activity. Previous studies suggested that auranofin or
many other gold compounds that quickly exchange the ligands
with albumin existed abundantly in blood serum, leading to no
significant effect on TrxR inhibition.31 Therefore, the stability
C
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Figure 3. (a) Inhibition efficacy of host structures (A, B, C, D, and E) combined with phenyl as R. (b) Efficient inhibition of R radical (1−11). (c)
Efficient inhibition of R radical combined with different host structures to screen the highest efficacy agents for anticancer cells. (d) D9 was screened
out (dark dash ellipse) from 15 gold compounds under different concentrations (1, 5, and 10 μM). (e) Curves of general capability of anticancer
cells (MDA MB-231 cells (MD-231 shown in e and f), KB cells, HT-29 cells, A549 cells, HeLa cells, and MCF-7 cells) by D9 under different
concentrations (0.05−1.40 μM). (f) Cytotoxicity of D9 (0.6 μM) to cancer cells and normal cells (293T cell lines, LO2 cell lines). Error bars in the
figure represent the SD of experimental duplicates.
of D9 should be crucial to its anticancer activity in vivo. For this
purpose, the kinetics of ligand exchange with albumin were
studied and compared with previously reported gold com-
pounds (Figure 2d). D9 (0.4 μM) was incubated with albumin
(0.1 mg/mL, 66.3 kDa) in PBS at room temperature. The
change of molecular weight (MW) of albumin was examined by
MALDI-TOF with different incubation times. D9 can stably
exist in the albumin solution almost without ligand exchange:
the MW of albumin did not considerably increase even after 3 h
of incubation. However, the MW of albumin incubation with
other compounds (References I, II, and III in Figure 2d are [2-
(4-methoxyphenyl)ethyn-1-yl] (triphenylphosphine) gold(I);
[2-phenylethyn-1-yl] (triphenylphosphine) gold(I); and tri-
phenyl phosphine gold chloride, and their structures are shown
in Supporting Information, Figure S5) rapidly increased (ΔMW
≈ 800−1400) after 3 h of incubation. Taken together, D9
compound should be stable enough to be utilized for anticancer
in vivo.
Then we investigated the inhibition of cell proliferation by
gold compounds due to the reduction of TrxR activity. Similar
to the effects of the inhibition of enzyme activity, the impacts of
all 55 gold compounds were investigated with different
concentrations separately (Figure 3a−c and Supporting
Information, Figures S6 and S7). The results showed that 15
gold compound was also identified with relatively high
efficiency (Figure 3d). The half inhibition concentration
(IC50) values of 15 gold compounds for MCF-7 and HT-29
cells are summarized in Table 2. Almost 70% MCF-7 cells and
Table 2. IC50 Values of 15 Gold Compounds to MCF-7 and
HT-29 Cells
IC50 (μM)
IC50 (μM)
MCF-7 HT-29
Gold compds
MCF-7
HT-29
Gold compds
A9
A1
B9
C2
C10
C4
C8
C9
1.5
1.25
2.2
3
>6
5.1
>6
>6
>6
>6
>6
>6
C1
C5
D7
D8
D9
D10
E9
4.75
1.25
0.55
0.55
0.03
0.51
0.62
>6
>6
0.75
0.56
0.10
0.62
0.6
2.6
2
1.65
1.25
50% HT-29 cells were killed by D9 with the concentration as
low as 0.1 μM (Figure 3d and Supporting Information, Figure
S8. D9 had the highest efficacy to inhibit the cell proliferation
with minimum values of IC50 for MCF-7 cells (0.03 μM) and
HT-29 cells (0.10 μM). As indicated by these results, the
inhibition of TrxR can further result in the reduction of cell
viability. Hence, we further investigated potential capacities of
D9 for anticancer cells in general. The activities of different
kinds of cancer cells, including mouth epidermal carcinoma
cells (KB), colorectal adenocarcinoma (HT-29), breast cancer
cells (MDA MB-231 and MCF-7), human lung cancer cells
(A549), and human cervical cancer cells (HeLa), were analyzed
after the incubation of D9 with different concentrations (0−1.4
μM).
D
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Figure 4. (a) Schematic illustration of the treatment protocol of the mice by D9. D9 (200 μL, 5 mg/kg) was intravescularly injected into BALB/c
nude mice bearing a MCF-7 tumor. The mice were treated every 2 days (d = 1, 3, 5, 7, 9, and 11). (b) In vivo antitumor efficacy studies of D9 (5 mg/
kg) with PBS solution as controls. Values were expressed as means SD (N = 5). (c) Representative photos of the tumor-bearing mice of two
groups after days 1 and 15 of treatment. (d) Body weight changes of the mice during the course of treatments. Values were expressed as means SD
(N = 5).
The cell viability was obtained after 72 h of incubation with
D9 compound in complete cell culture medium (with 10%
FBS). The proliferation of all cancer cells was completely
inhibited by D9 (0.60 μM), and the IC50 values of all cancer
cells could be as low as 0.55 μM (Figure S9). PI/Hoechst was
used to evaluate the necrosis/apoptosis induced by D9. In this
assay, we incubated MCF-7 cells with D9 (0.80 μM) for 4 and
8 h separately, and the necrosis/apoptosis of cells was examined
by adding the solution of PI/Hoechst and maintaining the
incubation for 15 min. Confocal microscopy was then used for
the fluorescent imaging of cells. After 4 h of treatment, there
was more than 50% necrosis/apoptosis of cells compared to
control (without treatments). And all cells had complete
necrosis/apoptosis after 8 h of incubation (see Supporting
Information, Figure S10). These results suggested that the
efficient anticancer activity of D9 compound may be attributed
to its impact of inducing cell necrosis/apoptosis.
We further investigated the selectivity of its anticancer cells
activity by examining the potential cytotoxicity of D9
compound to normal cells as well as tumor cells. Normal
cells (human embryonic kidney cell (293T) and immortal
hepatic cell (LO2)) and tumor cells were separately incubated
with D9 (0.6 μM). The cell viability was measured through
CCK-8 assay after 4 h of incubation in complete medium with
10%-FBS. The cytotoxicity is shown in Figure 3f. D9 has much
higher toxicity to tumor cells than normal cells. The tumor cells
activity decreased to below 40%, and even 80% tumor cells
were completely killed by D9, while the cell viability of normal
cells was not significantly affected. These results suggested that
D9 can be a potential anticancer drug which can selectively kill
cancer cells but has a limited side effect on normal cells.
Auranofin and other gold compounds can similarly inhibit the
activity of TrxR, while it is still very difficult to achieve an
efficient antitumor effect in vivo.31 Therefore, it is very
important or crucial to investigate the tumor inhibition of D9
in vivo. In vivo tumor suppression of D9 was carried out using a
MCF-7 cells xenografted BALB/c nude mice model. MCF-7
cells (5 × 106) in 100 μL of PBS solution were subcutaneously
injected into the right flank of BALB/c nude mice. The mice
were randomly divided into two groups (N = 5). The designs of
the in vivo antitumor protocol are shown in Figure 4a. When
the tumors developed to approximately 80 mm3, D9 was
intravenously injected into mice (200 μL, 5 mg/kg) at days 1,
3, 5, 7, 9, and 11 (Figure 4a). The same solutions (PBS with 1%
DMSO) without D9 were intravenously injected into mice as
control. The injections were performed every 2 days, and
treatment was maintain to 15 days. During the process of the
treatment, the tumor volumes and body weight were measured
every day. The time-dependent tumor volumes of mice treated
by D9 were compared with the control group (Figure 4b). It is
clear that D9 compound can effectively inhibit the growth of
tumors. And the efficacy of inhibition was significantly different
after 10 days of treatment. After 15 days of treatment, the
tumor inhibition ratio was calculated from the equation as
follow:
Inhibition ratio (%) = (V − V) × 100%/V
c
t
c
where Vc is the average tumor volume of the control group after
treatment with intravascular injections of PBS for 15 days, and
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Figure 5. (a) The schematic illustration of the TrxR electron transfer processes; verified the inhibition site of TrxR by D9 through the substrates
inhibition approaches: the activity of N-terminal (b) and C-terminal sites (c) was investigated through the catalysis of Juglone and 9,10-
phenanthrenequinone, respectively, after 75 min incubation of TrxR and D9. (d) The irreversible inhibition of TrxR by D9 was carried out through
verifying the catalytic activity of the system of TrxR-Trx: the substrates of insulin was added into after 75 min incubation of TrxR and D9. The
activity of TrxR in (b), (c), and (d) was evaluated through the NADPH reduction at 340 nm absorption.
Vt is the average tumor volume of treatments with D9. From
the results, we can see that D9 compound has effective tumor
inhibition in in vivo treatments with high tumor inhibition ratio
(IR ≈ 91.5%, see Supporting Information, Figure S11). The
representative images of tumor-bearing mice before and after
administration are shown in Figure 4c. The biodistribution of
D9 compound in major organs was studied by inductively
coupled plasma−atomic emission spectroscopy (ICP-AES)
analysis of ex vivo tissues. The gold contents in different
organs, i.e., tumor, head, lung, liver, kidney, spleen, and heart,
were estimated. The results revealed that D9 mainly distributed
in spleen (106.5 μg/g), kidney (52.4 μg/g), and liver (41.8 μg/
g), and the concentration of D9 in tumor was 25.3 μg/g after 4
h of treatment (see Supporting Information, Figure S12), which
was sufficient to inhibit the proliferation and induce the
necrosis/apoptosis of tumor cells. In the control group, the
gold concentration was negligible and close to the baseline.
Meanwhile, no obvious body weight loss was observed in all
groups (Figure 4d), suggesting no acute toxicity of D9 after in
vivo treatments. In addition, the toxicity of D9 was evaluated
through blood chemistry and the activity of transaminase in
liver (ALT and AST) of the mice model (see Supporting
Information, Table S1). There was no significant damage after
in vivo treatment with D9. Therefore, we successfully
demonstrated that D9 has the capability to inhibit tumor
proliferation both in vitro and in vivo.
Moreover, we tried to investigate the corresponding
mechanism about the TrxR inhibition of D9. TrxR is a
selenoprotein with two redox active sites, i.e., between Cys-57
and Cys-64 (N-terminal) and between Cys-497 and CSe-498
(C-terminal); the latter is considered as the binding site of the
protein substrate, e.g. Trx. The electron transfer pathway of
TrxR was depicted in Figure 5a. Briefly, the electrons are first
transferred from NADPH to FAD and subsequently transferred
to the disulfide of N-terminal active site. Finally, this dithiol of
the N-terminal reduces the C-terminal selenenylsulfide of the
enzyme. The selenolthiol has high activity to reduce Trx or
other substrates. The substrates inhibition approaches were
utilized to investigate the inhibition mechanism of D9
compound. 5-Hydroxy-1,4-naphthoquinone (Juglone) serves
as the substrate of the N-terminal redox active motif, and 9,10-
phenanthrenequinone can receive electrons from redox-active
selenolthiol of the C-terminal motif.33−35 D9 compound and
TrxR were incubated at room temperature for 75 min in the
environment of inert gas, and mixture solution preprepared was
added into the solution; finally Juglone and 9,10-phenanthre-
nequinone (50 μM) were respectively added into the solution.
The reduction of NADPH (absorption at 340 nm) was
immediately detected. The activity of enzyme was estimated
according to the reduction of NADPH. The activity of TrxR to
catalyze 9,10-phenanthrenequinone was completely inhibited,
suggesting the redox-active selenenylsulfide motif was com-
F
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was added into the solution of enzyme and then incubated in the
environment of inert gas for 75 min at room temperature. The mixture
solution (175 μL, 100 mM potassium phosphate, pH = 7.0, 10 mM
EDTA, 20 mg/mL BSA, and 0.045 mg/mL NADPH) was added into
96-well plates. The rate of NADPH oxidation was immediately
monitored after addition of 9,10-phenanthrene quinone (50 μM) and
5-hydroxy-1,4-naphtoquinone (Juglone, 50 μM). The control experi-
ments were done with the same processes without the addition of gold
compounds. To investigate the irreversible inhibition of TrxR, the
mixture solutions containing potassium phosphate (50 mM, pH 7.0),
EDTA (1 mM), insulin (0.5 mg/mL), NADPH (0.2 mM), and Trx (3
pM) were added into the culture solutions of TrxR and gold
compounds. The absorption of NADPH at 340 nm was monitored by
microplate reader.
Antiproliferation Assay by Gold Compounds. MCF-7 cells
were cultured in DMEM supplemented with 10% fetal bovine serum
(FBS) at 37 °C in a humified atmosphere containing 5% CO2. And
then the cells were seeded in 96-well plates at a density of 6 × 104 cells
per well. After 15 h adhesion, different concentrations of gold
compounds (50, 100, and 500 μM for initial screening; 0.1, 0.5, and 1
μM for detailed screening of 15 gold compounds; 0.02−1.4 μM for
investigating the IC50 values of different cells) were added into the
culture mediums with 72 h of incubation. And then the cells were
washed 3 times with phosphate buffer solution (PBS). Finally the cell
viability of different treatments was monitored with the cell counting
kit-8 assay (CCK-8, see Supporting Information). To investigate the
cytotoxicity of D9 compound to tumor cells and normal cells, D9 (0.6
μM) was added into the medium of cells with 4 h of incubation, and
then the medium was replaced with the fresh medium without D9
compound and cultured for another 24 h, and the cell viability was
evaluated through the kit of CCK-8.
pletely inhibited by D9. However, the catalytic activity of TrxR
to reduce Juglone was moderately inhibited. The redox-activity
of the N-terminal motif was not severely inhibited by D9. In
the control system, TrxR effectively catalyzed the compounds
of Juglone and 9,10-phenanthrenequinone without the
incubation of D9 compound (Figure 5b and c). Meanwhile,
Trx reduction is important to maintain the homeostasis of cells.
Insulin is one of the TrxR dependent substrates of Trx, and we
used insulin to characterize the catalytic activity of the TrxR-
Trx system. The results are shown in Figure 5d, and the
catalytic activity of the TrxR-Trx system was completely
inhibited by D9 compound. These results showed that D9 had
the capability to efficiently inhibit the redox-active selenenyl-
sulfide motif of TrxR, and the inhibition was irreversible.
CONCLUSIONS
■
In summary, we designed a novel compound D9 by screening
55 gold compounds. D9 was found to have specific and efficient
inhibition of TrxR activity in vitro and in vivo. As indicated by
this work, D9 compound exhibits great potential in the
application of biomedicine.
EXPERIMENTAL SECTION
■
Elaborately Designed and Synthesized Gold Compounds.
The host structures of gold(I) compounds were elaborately designed
with 5 different alkynyl ligands, denoted as A−E: phenyl (A), 4-
methylphenyl (B), 1-hydroxycyclohexane (C), 4-methoxyphenyl (D),
and 4-fluorophenyl (E), which represent classical electron donors and
acceptors. Meanwhile, we also synthesized 11 ligands of phosphine,
denoted as R radical, with different electron donating degrees.
Through orthogonal combination of all these host structures and R
radicals, we designed 55 kinds of gold compounds and tested their
capabilities for the inhibition of the TrxR activity in vitro and in vivo.
The synthetic processes of these gold compounds were as follows:
The mixture solution of acetone and water (1:1, v/v, 14 mL) was
added to the aqueous solution (4 mL) of chlorauric acid (1.94 mmol)
and potassium bromide (1.12 g), and aerated with sulfur dioxide in the
ice−water bath. Then sodium acetate and the acetone solution of
alkyne were added and reacted for 1 h at room temperature. The
yellow precipitates were isolated by filtration and washed with acetone
and ether. The obtained compounds of alkyne-gold (0.1 mmol),
phosphine ligand (0.1 mmol), and dichloromethane (2 mL) were
added into the tube and reacted for 2 h at room temperature. The gold
compounds were obtained after recrystallization with n-hexane twice.
Screening of High Efficient Inhibitors to TrxR. The activity of
TrxR was measured by NADPH-dependent reduction of 5,5′-dithiobis
2-nitrobenzoic acid (DTNB) at room temperature according to the
assay of ref 23. Briefly, the samples of gold compounds were dissolved
into the solution of dimethyl sulfoxide (DMSO) and then diluted with
respective buffers. In the assays of TrxR inhibition, 25 μL-TrxR (0.75
U) and two different concentrations (50 and 100 nM) of gold
compounds (25 μL) were added into the 96-well plates and cultured
for 75 min at room temperature. Finally, the 175 μL mixture solution
(potassium phosphate: 100 mM, pH = 7.0; ethlenediamine tetraacetic
acid: EDTA, 10 mM; bovine serum albumin: BSA, 20 mg/mL; and
NADPH: 0.045 mg/mL) was added into 96-well plates, and
incubation was continued for 0.5 min. DTNB (28 mM) was added
into the solution, and the absorption of color changes was immediately
measured at 412 nm, monitoring the formation of 5-thio-2-
nitrobenzoic acid (5-TNB). The control experiments were examined
through the activity of TrxR without addition of gold compounds. All
assays were carried out in triplicate.
In Vivo Antitumor of D9. All animal experiments were performed
complying with the NIH guidelines for the care and use of laboratory
animals and according to the protocol approved by the Institutional
Animal Care. The initial body weight of mice was about 17−18 g.
ICP-AES Analysis. The gold distribution in different organs of
treated mice was evaluated through the concentrations of gold
detected by ICP-AES analysis (ELAN 6100, PerkinElmer SCIEX).
Organs were dissected and dissolved into aqua regia. The resulting
solution was diluted with 2% HNO3 to constant volumes.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental materials and methods, Figures S1−S12, Table
1
S1, the molecular formula strings, and H, 13C, 31P NMR and
MS of gold compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: wanghao@nanoctr.cn (H.W.).
*E-mail: lijingyuan@ihep.ac.cn (J.L.).
*E-mail: yuguang@mail.ccnu.edu.cn (G.A.Y.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Basic Research Program
of China (973 Program, 2013CB932701, 2013CB933704) and
the 100-Talent Program of the Chinese Academy of Science
(Y2462911ZX), the National Natural Science Foundation
(21374026, 21304023, 21072071, and 51303036), and the
Beijing Natural Science Foundation (2132053).
Demonstration of the Inhibition Site of TrxR. To determine
the inhibition site of TrxR, the assays were conducted according to the
protocol of reference.34 Briefly, 25 μL- aliquots of the enzyme solution
(5 U/mL) were added into 96-well plates; and 25-μL of potassium
phosphate buffer (pH 7.0) containing the gold compounds (50 nM)
G
dx.doi.org/10.1021/jm5012098 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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