Design, synthesis, and biological evaluation of a novel indoleamine 2,3-dioxigenase 1 (IDO1) and thioredoxin reductase (TrxR) dual inhibitor

Article abstract of DOI:10.1016/j.bioorg.2020.104401

Targeting the Trp-Kyn pathway is an attractive approach for cancer immunotherapy. Thioredoxin reductase (TrxR) enzymes are reactive oxygen species (ROS) modulators that are involved in the tumor cell growth and survival processes. The 4-phenylimidazole scaffold is well-established as useful for indoleamine 2,3-dioxygenase 1 (IDO1) inhibition, while piperlongumine (PL) and its derivatives have been reported to be inhibitors of TrxR. To take advantage of both immunotherapy and TrxR inhibition, we designed a first-generation dual IDO1 and TrxR inhibitor (ZC0101) using the structural combination of 4-phenylimidazole and PL scaffolds. ZC0101 exhibited better dual inhibition against IDO1 and TrxR in vitro and in cell enzyme assays than the uncombined forms of 4-phenylimidazole and PL. It also showed antiproliferative activity in various cancer cell lines, and a selective killing effect between normal and cancer cells. Furthermore, ZC0101 effectively induced apoptosis and ROS accumulation in cancer cells. Knockdown of TrxR1 and IDO1 expression induced cellular enzyme inhibition and ROS accumulation effects during ZC0101 treatment, but only reduced TrxR1 expression was able to improve ZC0101′s antiproliferation effect. This proof-of-concept study provides a novel strategy for cancer treatment. ZC0101 represents a promising lead compound for the development of novel antitumor agents that can also be used as a valuable probe to clarify the relationships and mechanisms of cancer immunotherapy and ROS modulators.

Full text of DOI:10.1016/j.bioorg.2020.104401

Bioorganic Chemistry 105 (2020) 104401
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, and biological evaluation of a novel indoleamine
2
,3-dioxigenase 1 (IDO1) and thioredoxin reductase (TrxR) dual inhibitor
a
,
1
b
,
1
a
a
a
Qing-Zhu Fan , Ji Zhou , Yi-Bao Zhu , Lian-Jun He , Dong-Dong Miao ,
a
a,
*
a,*
Sheng-Peng Zhang , Xiao-Ping Liu , Chao Zhang
a
Center of Drug Screening and Evaluation, Wannan Medical College, Wuhu, Anhui 241000, PR China
b
Center for Reproductive Medicine, The First Affiliated Hospital of Wannan Medical College, Wuhu, Anhui 241000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
IDO1
Targeting the Trp-Kyn pathway is an attractive approach for cancer immunotherapy. Thioredoxin reductase
(
TrxR) enzymes are reactive oxygen species (ROS) modulators that are involved in the tumor cell growth and
survival processes. The 4-phenylimidazole scaffold is well-established as useful for indoleamine 2,3-dioxygenase
(IDO1) inhibition, while piperlongumine (PL) and its derivatives have been reported to be inhibitors of TrxR.
TrxR
Dual inhibitor
Antitumor
1
To take advantage of both immunotherapy and TrxR inhibition, we designed a first-generation dual IDO1 and
TrxR inhibitor (ZC0101) using the structural combination of 4-phenylimidazole and PL scaffolds. ZC0101
exhibited better dual inhibition against IDO1 and TrxR in vitro and in cell enzyme assays than the uncombined
forms of 4-phenylimidazole and PL. It also showed antiproliferative activity in various cancer cell lines, and a
selective killing effect between normal and cancer cells. Furthermore, ZC0101 effectively induced apoptosis and
ROS accumulation in cancer cells. Knockdown of TrxR1 and IDO1 expression induced cellular enzyme inhibition
and ROS accumulation effects during ZC0101 treatment, but only reduced TrxR1 expression was able to improve
′
ZC0101 s antiproliferation effect. This proof-of-concept study provides a novel strategy for cancer treatment.
ZC0101 represents a promising lead compound for the development of novel antitumor agents that can also be
used as a valuable probe to clarify the relationships and mechanisms of cancer immunotherapy and ROS
modulators.
1
. Introduction
Combination drugs targeting multiple molecules are commonly used
L-tryptophan (L-Trp), which is the least abundant essential amino
acid, can be metabolized via four distinct mechanisms: decarboxylation
to tryptamine, protein synthesis, the serotonergic pathway, and the
kynurenine (Kyn) pathway. Kyn pathway (KP) metabolism accounts for
approximately 95% of all mammalian dietary Trp [3]. Indoleamine 2,3-
dioxygenase (IDO1) is a heme enzyme that catalyzes the oxygenation of
the indole ring of Trp to produce N-formylkynurenine (NFK) in the first
rate-limiting step of the KP [4,5]. NFK is then metabolized to Kyn and its
subsequent bioactive metabolites [6]. Tryptophan depletion results in
inhibiting the proliferation of T lymphocytes, which are sensitive to low
Trp levels. The production of KP metabolites can enhance immune
tolerance by activating the aryl hydrocarbon receptor. Both contribute
in cancer treatment to improve efficacy, decrease toxicity, and prevent
the development of drug resistance [1]. A promising strategy that is
gaining interest in drug discovery is the development of a single com-
pound containing a combination of pharmacophores that are capable of
modulating multiple targets simultaneously. This strategy of using a
single multitargeted agent, called a “designed multiple ligand,” avoids
the problems of complicated pharmacokinetic and pharmacodynamic
relationships and drug–drug interactions associated with combination
therapy [2].
Abbreviations: TLC, thin layer chromatography; THF, tetrahydrofuran; EA, ethyl acetate; DCM, dichloromethane; DMAP, 4-dimethylaminopyridine; LDA, lithium
diisopropylamide; Boc, t-butyloxy carbonyl; DMF, dimethyl formamide; PE, petroleum ether; TFA, trifluoroacetic acid; IDO1, indoleamine 2,3-dioxigenase 1; TrxR,
thioredoxin reductase; ROS, reactive oxygen species; PL, piperlongumine; L-Trp, L-tryptophan; Kyn, kynurenine; KP, kynurenine pathway; Trx, thioredoxin; NFK, N-
′
′
formylkynurenine; PIM, 4-phenylimidazole; PI, propidium iodide; DCFH-DA, 2 -,7 -dichlorofluorescein diacetate; DMSO, dimethyl sulfoxide.
*
Corresponding authors.
E-mail addresses: liuxiaoping@wnmc.edu.cn (X.-P. Liu), zhangchao@wnmc.edu.cn (C. Zhang).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.bioorg.2020.104401
Received 13 March 2020; Received in revised form 12 October 2020; Accepted 17 October 2020
Available online 21 October 2020
0
045-2068/© 2020 Elsevier Inc. All rights reserved.

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
to the immunosuppressed state of the tumor microenvironment [7,8]. In
addition, evidence indicates that elevated levels of IDO1 expression in
both tumor cells and antigen-presenting cells are correlated with a poor
prognosis and reduced survival [9,10]. Given its important role in tumor
immune escape, IDO1 represents a valuable therapeutic target in cancer
immunotherapy.
inhibition of IDO1 and TrxR (Fig. 1). To our knowledge, there have been
no other studies combining the key moieties to create a dual inhibitor for
IDO1 and TrxR. Therefore, a novel phenylimidazole dihydropyridine
derivative co-targeting IDO1 and TrxR was designed and synthesized to
develop an effective anticancer agent exerting synergistic effects
through an ROS-based mechanism and simultaneous activation of the
antitumor immune response via IDO1 blockage.
More recently, thioredoxin reductase (TrxR) has been recognized as
an attractive target for anticancer drug development [11]. Together
with its substrate thioredoxin (Trx), TrxR maintains redox homeostasis
in cells, preventing oxidative damage and mutations [12–14]. Both Trx
and TrxR have been reported to be overexpressed in numerous cancer
cells, and they have been observed to be associated with a poor prog-
nosis and chemotherapy resistance [15–18]. The validity of TrxR as a
target for anticancer treatment has been demonstrated by experiments
showing that knockdown of TrxR in cancer cells brings about a reversal
of tumor phenotype, as well as inhibition of DNA replication and cancer
cell growth [19–21]. The Trx/TrxR system is a key component for
maintenance of intracellular pathways involving redox homeostasis in
mammalian cells. TrxR is upregulated in cancerous cells to combat
reactive oxygen species (ROS) overproduction [22]. TrxR inhibition
could modulate antioxidant levels and enhance intracellular ROS, and
then disturb the cellular oxidative environment and induce cell death,
thereby serving as a novel therapeutic agent. Notably, it has been shown
that knocking down IDO1 using shRNA or IDO1 inhibitors heightens
ROS levels, which in turn significantly inhibits cancer cell growth [23].
In the current work, we developed a novel small molecule that targets
both IDO1 and TrxR.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, compound ZC0101 was synthesized by using
tert-butyl 4-(2-oxoethyl) piperidine-1-carboxylate as a starting material.
The key intermediate 5 was obtained based on a previous reference [33].
Further reaction with bromoacetyl chloride yielded compound 6.
Treatment of 6 with PPh in THF resulted in the quaternary phosphate,
3
7. Compound 10 was synthesized via a coupling reaction using 2-formyl-
phenylboronic acid and 4-iodo-1H-imidazole as the starting material.
Subsequently, treatment of compound 10 with Boc O using DMAP as a
2
base yielded another key intermediate, 11. Finally, the target compound
ZC0101 was obtained through a coupling reaction of compounds 7 and
11, followed by deprotection of Boc. The structure of the target com-
pound was confirmed by 1H NMR and electrospray ionization mass
spectrometry (ESI-MS). It was purified by silica gel column chroma-
tography, and HPLC was used to determine its purity (>95%).
4
-phenylimidazole (PIM) has been reported to bind to a deep pocket
2
.2. In vitro IDO1 and TrxR inhibitory activity evaluation
of IDO1 with its phenyl ring oriented toward the lipophilic cavity, and
its N-1 atom interacting with the heme iron [24,25]. Based on this
finding, some derivatives have been reported that have improved ac-
tivity at the micromolar level [26]. Piperlongumine (PL) is a natural
product obtained from the fruit of the long pepper. PL is a form of
traditional Chinese medicine that exhibits anticancer activity through a
number of mechanisms, including inhibiting the phosphorylation of
serine/threonine kinase Akt (Akt)[27] and blockade of both nuclear
factor κB (NF-κB) and JAK-STAT3 signaling pathways [28,29]. PL has
recently been reported to be a selective, irreversible inhibitor of TrxR
that demonstrates antiproliferative activity against selected human
cancer cell lines, with the Michael acceptor of PL postulated to react
with a selenocysteine residue of the TrxR enzyme via a Michael addition
reaction to form covalent adducts [30].
Initially, ZC0101 was evaluated for IDO1 inhibitory activity using
recombinant human IDO1 as well as PIM (Aladdin, no. P135879, purity:
8%) and Epacadostat (MCE, no. HY-15689, purity: 99.95%) as refer-
9
ence drugs. As shown in Fig. 2A, ZC0101 exhibited potent IDO1 inhib-
itory activity with an IC50 value in the nanomolar range, which was
comparable to Epacadostat, but far superior to PIM.
The Docking results showed that the nitrogen of the imidazole in
ZC0101 was directly bound to the heme iron which is similar to PIM and
that the phenyl group was located at the expanded pocket A which is
formed by Tyr126, Val30 and Gly262. Furthermore, it is predicted that
the side chain dihydropyridinone occupied the pocket B which is
composed of residues Arg231, Phe262, and Leu234 and the dihy-
dropyridinone moiety took a position adjacent to Arg231 (Fig. 3). These
docking results supported that the bulky hydrophobic group to fill
pocket B of IDO1 is necessary for improving the IDO1 inhibitory activity
Since IDO1 inhibitors are generally developed as combination ther-
apies with cytotoxic antitumor agents, radiotherapy, therapeutic
vaccination, and other targeted therapies [9,31,32]. Our laboratory has
focused on exploiting the combination of PIM and PL pharmacophoric
scaffolds to produce novel single molecules that can improve anti-tumor
effects both in vitro and in vivo, at least partly through simultaneous
[
25]. Then, ZC0101 was evaluated for in vitro inhibitory activity against
rat TrxR, with PL and Auranofin (MCE, no. HY-N2329 and HY-B1123;
purity: 99.19% and 98%) as reference drugs. As shown in Fig. 2B,
ZC0101 exhibited potent TrxR inhibitory activity, with an IC50 value of
7
.98 ± 0.02
anofin showed more potent activity with an IC50 value of 0.085 ± 0.002
M. These observations demonstrate that ZC0101 acts as a dual enzy-
matic inhibitor in vitro.
μ
M, superior to that of PL at 21.9 ± 0.3
μ
M, while Aur-
μ
2
.3. Cellular cytotoxicity screening
We next performed cytotoxicity screening, which is summarized in
Table 1. The cytotoxicity of ZC0101 was primarily investigated in non-
small cell lung and colon cancer cells, with PL used as a reference drug,
which was further compared to BEAS-2B (normal lung epithelial cells)
and FHC (normal colon epithelial cells) in terms of IC50 values. ZC0101
showed more cytotoxicity than PL in all screened cells, and especially in
HCT-116 cells. In addition, both ZC0101 and PL could selectively kill
cancer cells, although this selectivity was more obvious among FHC and
tested colon cancer cells. Thus, follow-up cellular TrxR activity studies
were mainly performed using HCT-116 cells.
Fig. 1. Design of a novel inhibitor targeting thioredoxin reductase (TrxR) and
indoleamine 2,3-dioxygenase (IDO1) by merging the TrxR inhibitor piperlon-
gumine (PL) and the IDO1 inhibitor 4-phenylimidazole (PIM).
2

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
◦
◦
Scheme 1. Reagents and conditions: (i) LDA, THF, ꢀ 78 C, PhSSPh, 50%; (ii) m-CPBA, DCM, NaHCO
2%; (v) Bromoacetyl chloride, THF, rt., 20%; (vi) PPh , THF, rt., 70%; (vii) DMF, Pd(PPh , Na CO
CHCl , rt., 50%; (x) CF COOH, rt., 72%.
3
; (iii) Toluene, reflux, 80 C, two steps , 76%; (iv) DCM, TFA,
7
3
3
)
4
2
3 2 3
, 40%; (viii) Boc O, DMAP, Et N, Toluene, rt., 65%; (ix) DMAP,
3
3
2
.4. Cellular IDO1 and TrxR inhibitory activity evaluation
expression in HCT-116 and HeLa cells in a concentration-dependent
manner, while cells treated with ZC0101 had lower levels of TrxR
than those treated with the same concentration of PL (Fig. 4A). Taken
together, these results indicate that ZC0101 showed better cellular TrxR
inhibitory activity than PL.
To evaluate the IDO1 inhibitory activity of ZC0101 in a cellular
environment, a HeLa cell-based assay measuring Kyn was performed to
calculate the EC50 of ZC0101 as well as that of PIM and Epacadostat
[
34]. As shown in Fig. 2C, ZC0101 had an EC50 of 1.41 ± 0.01
shows greater closeness to the EC50 of Epacadostat (0.040 ± 0.002
than the closeness between PIM (>20 M) and Epacadostat. Addition-
ally, ZC0101 had an LC50 of 13.49 ± 0.38 M to HeLa cells as measured
μ
M, which
μM)
2.5. Apoptosis analysis in cancer cells
μ
μ
To investigate whether the cell growth inhibition was associated
with apoptosis, HCT-116 cells were treated with either DMSO or various
concentrations of ZC0101 and PL for 24 h. The cells were stained with
Annexin-V and propidium iodide (PI), and the apoptotic ratio was
determined by flow cytometry. As shown in Fig. 5, the percentages of
by cytotoxicity tests (Table 1), with a LC50/EC50 ratio of 9.6. This in-
dicates that ZC0101 effectively inhibited IDO1 activity in HeLa cells,
and that the cell-based IDO1 activity was not caused by cytotoxicity. In
addition, ZC0101 treatment had almost no effect on IDO1 protein
expression in HCT-116 and HeLa cells at low concentrations, while a
high dose of ZC0101 treatment reduced IDO1 protein expression in a
concentration-dependent manner (Fig. 4B). Taken together, these find-
ings show that ZC0101 may inhibit cellular IDO1in terms of both
enzyme activity and protein expression.
apoptotic cells for ZC0101 were 16.93% (5
μ
M), 30.34% (10
μ
M), and
46.46% (20 M), respectively. The ability of ZC0101 to induce apoptosis
μ
was stronger than that of PL (8.22%, 12.94%, and 27.16% at the same PL
concentrations). Thus, we presumed that ZC0101 exhibited better can-
cer cell growth inhibition than PL due to the combination of its better
cell apoptosis-inducing activity and its superior cellular TrxR inhibitory
activity.
To investigate whether ZC0101 could effectively suppress TrxR ac-
tivity in cancer cells, we first evaluated its cellular TrxR inhibition using
an endpoint insulin reduction assay in HCT-116 cell lysates [35].
Treating HCT-116 cells with ZC0101 led to a remarkable inhibition of
2
.6. RNA interference analysis in HCT-116 and HeLa cells
cellular TrxR activity, with an IC50 of around 4.22
fold lower than that of PL (10.5 M, Fig. 2D). However, both ZC0101
and PL exhibited weaker inhibitory activity than Auranofin (IC50: 0.70
0.07 M, Fig. 2D). We also synthesized a TrxR probe, TRFS-green,
μ
M, which was 2.5-
μ
As we fully demonstrated that ZC0101 inhibited both TrxR1 and
IDO1 enzyme activity in both the in vitro assay and the cellular assay, we
next questioned the physiological significance of TrxR1 and IDO1 in-
hibition on the cellular actions of ZC0101. We reduced TrxR1 or IDO1
expression in HCT-116 and HeLa cells by transfecting a siRNA specif-
ically targeting TrxR1 or IDO1. The knockdown efficiency was validated
in Fig. 6A and B. The suppression of both TrxR1 and IDO1 expression
improved the effect of ZC0101 treatment, as indicated by cellular
±
μ
developed by Fang et al [36]. By measuring and quantifying the fluo-
rescence signal of this probe, we confirmed a greater decrease in TrxR
activity in living HCT-116 and HeLa cells treated with ZC0101
compared with cells receiving PL treatment (Fig. 4C-E). In addition,
both ZC0101 and PL treatment significantly suppressed TrxR protein
3

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
Fig. 2. ZC0101 inhibited both IDO1 and TrxR1 in vitro. (A, B) Concentration-effect curves for IDO1 and TrxR1 enzyme inhibitory activity of ZC0101. ZC0101 was
more potent than PIM and PL in initial activity assays. (C) ZC0101 more effectively inhibited Kyn production than PIM in IFN-γ-treated human HeLa cells. (D)
ZC0101 more effectively inhibited TrxR1 activity than PL in human HCT-116 cells. Dimethyl sulfoxide (DMSO) was used as the negative control. The results are
expressed as the mean ± standard deviation of three independent experiments.
Fig. 3. (A) Binding mode of PIM in the IDO1 active site (PDB: 2D0T). (B) Proposed binding mode of compound ZC0101 in the IDO1 active site. Heme is rendered as a
gray stick, IDO1 residues are shown as purple sticks, and compounds are shown as yellow sticks. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
enzyme assays. Knockdown of IDO1 expression did not improve ZC0101
treatment’s anti-proliferation effect; in contrast, the effect was improved
in cells with reduced TrxR1 expression (Fig. 6C). Taken together, our
loss-of-function data suggested that TrxR1 and IDO1 are cellular targets
of ZC0101, and the anti-proliferation effect of ZC0101 is related to its
inhibitory effect to TrxR1.
4

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
Table 1
relationships and mechanisms between cancer immunotherapy and
TrxR-targeted therapy. However, structural optimization of ZC0101 and
the synergistic effects of dual IDO1/TrxR inhibitors with immune
checkpoint inhibitors (e.g., PD-1 antibodies) remain to be further
investigated.
Cytotoxicity screening of PL and ZC0101.
Cell lines
IC50
PL
( M)
μ
ZC0101
Normal lung epithelial cells
BEAS-2B
A549
17.12 ± 0.30
9.43 ± 0.12
9.46 ± 0.11
5.28 ± 0.11
6.30 ± 0.34
6.63 ± 0.09
46.90 ± 1.21
6.79 ± 0.03
3.77 ± 0.10
3.54 ± 0.09
6.68 ± 0.11
5.37 ± 0.30
8.66 ± 0.13
29.64 ± 0.74
11.35 ± 0.21
7.34 ± 0.24
7.65 ± 0.11
1.11 ± 0.01
2.95 ± 0.12
6.35 ± 0.16
42.00 ± 0.27
0.44 ± 0.005
2.70 ± 0.55
2.75 ± 0.07
0.55 ± 0.24
1.31 ± 0.59
3.00 ± 0.07
13.49 ± 0.38
Non-small cell lung cancer cells
A549/DDP
H1299
HCC827
PC-9
4
. Experimental section
4
.1. Chemistry
Normal colon epithelial cells
Colon cancer cells
FHC
HCT-8
Reactions were monitored via thin-layer chromatography on silica
HCT-8/5-Fu
HCT15
HCT116
HT29
gel plates (60F-254) visualized under UV light. Melting points were
determined on a Mel-TEMP II melting point apparatus without correc-
1
13
3
tion. H NMR and C NMR spectra were recorded in CDCl on a Bruker
SW620
HeLa
Avance 300 MHz spectrometer at 300 MHz and 75 MHz, respectively.
Cervix cancer cells
Chemical shifts (δ) are reported in parts per million (ppm) from tetra-
a
3
methylsilane (TMS) using the residual solvent resonance (CDCl : 7.26
The data (IC50) were obtained through MTT assay and are expressed as the
mean ± SD of three independent experiments.
1
13
ppm for H NMR, 77.16 ppm for C NMR. Multiplicities are abbreviated
as follows: s = singlet, d = doublet, t = traplet, q = quartet, m =
multiplet). IR spectra were recorded on a Nicolet iS10 Avatar FT-IR
spectrometer using KBr film. MS spectra were recorded on a LC/MSD
TOF HR-MS Spectrum. Flash column chromatography was performed
with 100–200 mesh silica gel, and yields refer to chromatographically
and spectroscopically pure compounds.
2
.7. ROS level analysis in HCT-116 and hela cells
The major function of TrxR1 is to maintain Trx in a reduced state,
and thus defend against oxidative stress. IDO1 is also related to ROS
production [23,37]. Having confirmed that ZC0101 is a potent TrxR1
and IDO1 dual inhibitor, we next determined the ROS levels in HCT-116
and HeLa cells. ROS levels were assessed by flow cytometry (Fig. 7A ,B
and E) and cell imaging (Fig. 7C and D) using the redox-sensitive fluo-
Reactions and chromatography fractions were monitored on Merck
silica gel 60F-254 glass TLC plates. All of the solvents were reagent grade
and, when necessary, were purified and dried using standard methods.
′
′
rescent probe 2 -,7 -dichlorofluorescein diacetate (DCFH-DA). As shown
in Fig. 7, treatment with ZC0101 for 4 h induced elevated ROS levels
within HCT-116 and HeLa cells in a dose-dependent manner, indicating
that ZC0101 had the ability to promote cellular ROS accumulation.
Knocking down either TrxR1 or IDO1 expression could help to improve
the ROS accumulation effect of ZC0101 treatment. These results
demonstrated that the ROS accumulation effect of ZC0101 was related
to its inhibitory effects to TrxR1 and IDO1.
4
.1.1. Synthesis of 2-oxo-5,6-dihydropyridine-1 (2H) -tert-butyl carbonate
4).
First, 2-oxo-piperidine-1-tert-butyl carbonate 1 (3.98 g, 0.02 mol)
(
was dissolved in anhydrous THF (20 mL), protected with N
2
, cooled to
◦
ꢀ
78 C, after which LDA (20 mL) was slowly added in a dropwise
◦
manner, and stirred at ꢀ 78 C for 30 min. Diphenyl disulfide (4.36 g,
.02 mol) was dissolved in anhydrous THF (40 mL). Under N protec-
0
2
tion, the mixture obtained in the first step was slowly added thereto, and
◦
reacted at ꢀ 78 C for 1.5 h. After the reaction was complete, a saturated
2
.8. Kynurenine/tryptophan metabolism study in mice
NH Cl solution was slowly added dropwise to the reaction system in
4
order to quench the reaction. The solution was extracted three times
Inhibition of IDO1 activity in vivo could reduce plasma Kyn levels. To
with diethyl ether (3 × 100 mL) and saturated with NH Cl solution.
4
investigate whether ZC0101 could inhibit IDO1 in vivo, we performed a
kynurenine/tryptophan metabolism study in C57BL/6 mice. Once daily
oral dosing of Epacadostat or ZC0101 at 60 mg/kg reduced plasma
kynurenine levels in mice, while the tryptophan levels were slightly
upregulated (Fig. 8B). Additionally, the kynurenine/tryptophan ratio
was also reduced (Fig. 8A). Taken together, these results indicated that
ZC0101 was able to effectively suppress IDO1 activity in vivo.
Then, the organic layers were combined and dried over anhydrous
MgSO , filter. The solvent was centrifuged to obtain the crude product,
4
and column chromatography (PE/EA = 1: 10) was performed. The
1
product 2 (3.10 g) was obtained as a colorless oil with a yield of 50%. H
NMR (DMSO‑d ): δ (400 MHz, CDCl ) 1.52 (9H, s), 1.79–1.87 (1H, m),
6
3
1.97–2.12 (2H, m), 2.15–2.22 (1H, m), 3.67–3.74 (1H, m), 3.76–3.83
(1H, m), 3.85 (1H, t, J = 6.0 Hz), 7.28–7.32 (3H, m), and 7.52–7.55 (2H,
m) ppm; 13C NMR (DMSO‑d
6
C 3
): δ (100 MHz, CDCl ) 169.9, 153.3, 133.5,
3
. Conclusions
129.1, 128.3, 83.2, 51.6, 45.9, 28.2, 20.8 ppm. HRMS (ESI): m/z
+
+
calculated for C16
H
21NO
3
SH (M + H ): 308.1315; found: 308.1320.
In conclusion, due to the advantages of combining IDO1-targeted
Next, 2-oxo-3-(phenylthio) piperidine-1-tert-butyl carbonate 2 (6.15
tumor immunotherapy and TrxR-targeted therapy, we hybridized PIM
and PL into a single chemical entity. We synthesized and evaluated
ZC0101, the first dual IDO1/TrxR inhibitor. ZC0101 exhibited better
IDO1 and TrxR inhibitory activities than PIM and PL in both enzyme and
cellular experiments. It also exhibited better cytotoxic activity than PL
in all of the tested cancer cell lines and could significantly induce
apoptosis in HCT116 cells, which was correlated with its strong in vitro
TrxR inhibitory activity. In addition, ZC0101 also selectively killed
colorectal cancer cells, while sparing normal colorectal cells. Further-
more, it induced ROS accumulation in cancer cells through its inhibitory
effects to TrxR1 and IDO1. Considering the consistent results obtained
from the cell-free and cell-based assays, this proof-of-concept study
provides a novel strategy for the development of new antitumor agents.
Thus, ZC0101 has potential as a promising lead compound for drug
development. It can also be used a valuable probe to clarify the
g, 0.02 mol) was dissolved in DCM (220 mL), and a saturated NaHCO
3
◦
solution (41.5 mL) was added, then cooled to 0 C. After that, m-CPBA
(3.46 g, 0.02 mol) was added to the reaction system once and stirred at
◦
0 C for 2 h. At the end of the reaction, the organic layer was separated,
and the aqueous phase was extracted three times with DCM (3 × 50 mL).
4
The organic layers were combined, dried over anhydrous MgSO ,
filtered, and the solvent was rotary evaporated to obtain intermediate 3.
◦
This was dissolved in toluene (10 mL) and refluxed at 80 C for 3 h. After
the reaction was completed, the solution was rotary evaporated, and the
solvent was distilled off to obtain a crude product. Product 4 (2.98 g)
1
was a colorless oily liquid with a yield of 76%. H NMR (DMSO‑d
6
): δ
(400 MHz, CDCl
3
) 1.54 (9H, s), 2.40 (2H, td, J = 9.8, 1.9 Hz), 6.77 (1H,
dt, J = 6.4 Hz, 1.8 Hz), 5.95 (1H, dt, J = 9.8, 1.9 Hz), 6.77 (1H, dt, J =
+
9.8, 4.2 Hz) ppm; HRMS(ESI): m/z calculated for C10
H
5
NO
3
H
(M +
+
H ): 198.1125; found: 192.1118.
5

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
Fig. 4. Inhibitory effects of ZC0101 to IDO1 and TrxR1 in cancer cells. (A) ZC0101 exhibited better activity than PL in the downregulation of TrxR1 protein
expression in HCT-116 and HeLa cells. (B) ZC0101 reduced IDO1 expression in HCT-116 and HeLa cells at high concentrations. Cells were treated with the indicated
concentrations of ZC0101 or PL for 24 h, after which the cell extracts were prepared and analyzed by western blotting with an antibody against TrxR1. GAPDH was
used as a loading control. TrxR1 proteins were quantified using ImageJ software and normalized to GAPDH. Data are expressed as the mean ± standard deviation of
three independent experiments. (C, D) Imaging of TrxR activity in living HCT-116 (C) or HeLa (D) cells. Cells were treated with the indicated concentrations of
ZC0101 for 24 h followed by further treatment with TRFS-green (10
μ
M) for 4 h. Phase-contrast (top) and fluorescence (bottom) images were acquired by fluo-
rescence microscopy (EVOS FL). PL (20 M) and DMSO were used as the positive and negative controls. (E) TRFS-green mean fluorescence density was quantified
μ
using ImageJ. Mean fluorescence density = Integral fluorescence density /area. Data are expressed as the mean ± SD of three independent experiments. Bars = SD.
*P < 0.05, ***P < 0.005. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
6

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
Fig. 5. ZC0101 showed more potency than PL in inducing cell apoptosis. (A) Analysis of apoptosis by Annexin-V/PI double-staining assay. HCT-116 cells were
treated with ZC0101 or PL at indicated concentrations for 24 h. DMSO was used as the negative control. (B) The percentage of cell apoptosis was graphed by
GraphPad Prism 5. The cells in Q2-2 and Q2-4 were considered apoptotic. Data are presented as the mean ± SD values from at least three independent experiments.
Bars = SD. ***P < 0.005.
4
.1.2. Synthesis of 5,6-dihydropyridine-2 (1H) -one (5)
4.01–4.06 (2H, m), 4.60 (1H, s), 4.77 (1H, s), 6.02–6.05 (1H, m),
+
The 2-oxo-5,6-dihydropyridine-1 (2H) -tert-butyl carbonate 4 (2.87
6.93–6.99 (1H, m). HRMS(ESI): m/z calculated for C
7
H
9
BrNO
2
H
(M +
+
g, 0.014 mol) obtained in the previous step was dissolved in dry DCM
H) : 217.9817; found: 217.9826.
◦
(
18.2 mL) and cooled to 0 C. TFA (14.35 mL, 0.12 mol) was slowly
added dropwise. After the dropwise addition was completed, the ice
bath was removed and the reaction was performed at room temperature
for 2 h. Toluene (about 20 mL) was added to the reaction system, after
which rotary evaporation was performed to remove toluene, DCM, and
TFA. The residue was dissolved in DCM (50 mL), the organic phase was
4.1.4. Synthesis of (2-oxo-2- (3,6-dihydropyridine-6-one) ethyl)
triphenylphosphonium bromide (7)
Under the protection of argon, compound 6 (70 mg, 0.32 mmol) was
dissolved in THF (1.25 mL), and then added to a THF system (0.75 mL)
in which triphenyl phosphorus (83 mg, 0.32 mmol) was dissolved. This
was then stirred at room temperature for 3–5 h. After the reaction was
completed, the filtrate was discarded, and the filtered solid was washed
with n-hexane and dried. The progress of the reaction was monitored by
washed with saturated K
repeatedly extracted (4 × 50 mL) four times with DCM and methanol
approximately 10:1). The organic layers were combined and dried over
anhydrous sodium sulfate (Na SO ), followed by filtration and concen-
tration by column chromatography (methanol: DCM = 1: 20) yielded
2 3
CO (40 mL), and the aqueous phase was
(
2
4
TLC (the developing solvent was EA and n-hexane in a ratio of 1:3). The
1
yield was 70%. H NMR (DMSO‑d
6
): δ (400 MHz, CDCl
3
) 2.59 (s, 2H),
1
product 5 (1 g) as a white solid with a yield of 72%. H NMR (DMSO‑d
6
):
) 2.32 (2H, tdd, J = 7.2, 4.2, 1.9 Hz), 3.40 (2H, td, J =
.2, 2.7 Hz), 5.87 (1H, dq, J = 9.9, 1.9 Hz), 6.62 (1H, dt, J = 9.9, 4.2 Hz),
.65 (1H, dq, br s) ppm; 13C NMR (DMSO‑d
(100 MHz, CDCl
66.7, 141.6, 124.9, 39.6, 23.9 ppm. HRMS (ESI): m/z calculated for
3.87–3.91 (m, 2H), 5.97 (d, J = 8.0 Hz, 1H), 6.14 (d, J = 8.0 Hz, 2H),
δ (400 MHz, CDCl
3
6.99 (t, J = 4.0 Hz 1H), 7.73–7.92 (m, 15H). HRMS(ESI): m/z calculated
+
7
6
1
C
for C25
H
23BrNO
2
P + H(M + H) : 480.0728; found: 480.0716.
6
): δ
C
3
)
4.1.5. Synthesis of 2- (1H-imidazole-4-) benzaldehyde (10).
+
+
H
5 7
NOH (M + H ): 98.0600; found: 98.0559.
Next, 2-formylphenylboronic acid (0.6 g, 4 mmol), 4-iodo-1H-imid-
azole (0.485 g, 2.5 mmol), and tetrakis (triphenylphosphine) palla-
dium (0.1445 g, 0.125 mmol) were dissolved in DMF (37.5 mL), then
4
.1.3. Synthesis of 1- (2-bromoacetyl) ꢀ 5,6-dihydropyridine-2 (1H) -one
(
6)
placed in a single-necked flask under the protection of argon. Saturated
◦
5
,6-Dihydropyridone (97 mg, 2 mmol) was placed in a single-necked
Na
2
CO
3
(12.5 mL) was added and stirred at 110 C. The progress of the
flask under argon protection, dissolved in THF (6 mL), and 1.5 times
reaction was monitored by TLC (the developing agent was 4:1 EA and n-
hexane). After the reaction was completed, the reaction mixture was
cooled to room temperature, after which 70 mL of water was added, and
then extracted with 80 mL of EA three times. The aqueous phase was
removed, and the organic phases were combined. After separation and
purification on silica gel column chromatography (developing solvent
was MeOH and DCM with a ratio of 75:2000), the reaction product was
easy to tail in the developing agent. Adding an appropriate amount of
◦
equivalent NaH (0.12 g) was added at 0 C, 3 mmol), then reacted for 1
h. Next, 1.2 times equivalent of bromoacetyl chloride (0.196 mL, 2.4
mmol) was added dropwise to the system. After the dropwise addition
was completed, the mixture was returned to room temperature over-
night. The progress of the reaction was monitored by TLC the next day
(
the developing solvent was EA and n-hexane in a ratio of 1:2). After the
reaction was completed, 30 mL of saturated NH Cl was added, and then
4
extracted three times with 30 mL of EA. After that, the aqueous phase
was removed, and the organic phases were combined, dried over
triethylamine to the developing agent improved this phenomenon. The
1
final product was 0.27 g with a yield of 40%. H NMR (DMSO‑d
6
): δ
MgSO
4
, filtered with suction, and concentrated. The concentrate was
(400 MHz, CDCl
3
) 7.33–7.43 (m, 1H), 7.66–7.78 (m, 4H), 7.82 (s, 1H),
separated and purified by silica gel column chromatography (devel-
10.53 (s, 1H), 12.46 (s, 1H). HRMS(ESI): m/z calculated for C10
H
8
N
2
O
1
+
oping solvent was EA and PE in a ratio of 1:4), and the yield was 20%. H
+ H(M + H) : 173.0715; found: 173.0721.
NMR (DMSO‑d
6
): δ (400 MHz, CDCl
3
) 2.47 (2H, d, J = 4.2 Hz),
7

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
Fig. 6. The effects on ZC0101 activity after knockdown of TrxR1 or IDO1 expression. (A, B) Knockdown of TrxR1 and IDO1 expression improved the cellular enzyme
inhibitory activity of ZC0101 treatment in HCT-116 (A) and HeLa (B) cells. Insets: the expression of TrxR1 or IDO1 was analyzed by western blotting. (C) Cytotoxicity
of ZC0101 on HCT-116 and HeLa small interference RNA (siRNA)-transfected cells. The cells were transfected with indicated siRNAs for 36 h prior to ZC0101
exposure at the indicated concentrations for 24 h. Cell proliferation was determined by CCK-8 assay. Data are presented as the mean ± SD values from at least three
independent experiments. Bars = SD.
8

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
times the equivalent of DMAP (0.02 g, 0.16 mmol), and 1.5 times the
3
equivalent of Et N (0.33 mL, 2.36 mmol) were dissolved in 23.5 mL of
toluene, placed in a single-necked flask, and stirred at room tempera-
ture. The progress of the reaction was monitored by TLC (the developing
solvents were EA and n-hexane in a ratio of 1:2). After the reaction was
completed, the solvent was spin-dried. It was then separated and puri-
fied by silica gel column chromatography with EA and PE as the eluent
1
at 1:4. The final product was 0.276 g with a yield of 65%. H NMR
(
DMSO‑d
6
): δ (400 MHz, CDCl
3
) 1.62 (s, 9H), 7.5 (t, J = 4.8 Hz, 1H),
7
1
2
.72 (t, J = 4.8 Hz, 1H), 7.8 (t, J = 5.8 Hz, 2H), 8.15 (s, 1H), 8.38 (s, 1H),
+
0.41 (s, 1H). HRMS(ESI): m/z calculated for C15
H
16
N
2
O
3
+ H(M + H) :
73.1239; found: 273.1252.
4
.1.7. Synthesis of (E)-1-(3-(2-(1H-imidazol-4-yl)phenyl)acryloyl)-5,6-
dihydropyridin-2(1H)- one (ZC0101)
Compound 11 (17.2 mg, 0.1 mmol) and two equivalents of com-
3
pound 7 (100 mg, 0.2 mmol) were dissolved in 1 mL of CHCl , after
which three times the equivalent of DMAP (36.6 mg, 0.3 mmol) was
added and stirred at room temperature for 4 h. Then, the system was
◦
stirred at 60 C. The progress of the reaction was monitored by TLC (the
developing solvent was EA and n-hexane in a ratio of 1:3). After the
reaction, it was concentrated and subjected to silica gel column chro-
matography (the developing solvent was EA and n-hexane in a ratio of
1
:6), and the yield was 50%. Compound 12 was dissolved in dry DCM (5
◦
mL) and cooled to 0 C. TFA was slowly added dropwise. After the
dropwise addition was completed, the ice bath was removed and the
reaction was performed at room temperature for 2 h. Toluene (about 10
mL) was added to the reaction system, and then rotary evaporation was
performed to remove toluene, DCM, and TFA. The residue was dissolved
2 3
in DCM (10 mL), the organic phase was washed with saturated K CO
(
10 mL), and the aqueous phase was repeatedly extracted (4 × 10 mL)
four times with DCM and methanol (approximately 10:1). The organic
layers were combined and dried over anhydrous sodium sulfate
(
Na
tography (methanol: DCM = 1:20) resulted in ZC0101 (20 mg) as a
white solid with a yield of 72%. 1H NMR (DMSO‑d
): δ (400 MHz,
CDCl
2 4
SO ), followed by filtration and concentration by column chroma-
6
3
) 2.49–2.50 (m, 2H), 4.06 (t, J = 4.8 Hz, 2H), 6.07 (d, J = 4.8 Hz,
1
1
2
1
1
H), 6.94–6.98 (m, 1H), 7.46 (t, J = 4.8 Hz, 1H), 7.49 (d, J = 7.2 Hz,
H), 7.50 (t, J = 7.8 Hz, 2H), 7.74 (d, J = 5.6 Hz, 2H), 8.15–8.19 (m,
1
3
6 c 3
H). C NMR (DMSO‑d ): δ (100 MHz, CDCl ) 173.66, 170.66, 147.03,
44.58, 144.47, 141.05, 140.38, 135.56, 135.46, 133.96, 129.67,
27.12, 126.91, 122.54, 110.78, 46.79, 29.55 ppm. HRMS(ESI): m/z
+
calculated for C22
.2. Materials
Dimethyl sulfoxide (DMSO), L-Tryptophan, L-Kynurenine, sodium
H
23
N
3
O
4
+ H(M + H) : 293.1164; found: 293.1168.
4
ascorbate, methylene blue, catalase from bovine liver, potassium phos-
phate monobasic, sodium hydroxide, trichloroacetic acid, 4-(Dimethy-
′
lamino)benzaldehyde, human IFN-γ, NADPH, insulin, 5, 5 -dithiobis-2-
nitrobenzoic acid (DTNB), ethylene diamine tetraacetic acid (EDTA),
thioredoxin reductase from rat liver, and recombinant human thio-
redoxin (expressed in E.coli Trx) were purchased from Sigma-Aldrich
Fig. 7. ZC0101 induced intracellular ROS generation through its inhibitory
effects to TrxR1 and IDO1. (A, B, C, D) Intracellular ROS generation induced by
increasing doses of ZC0101. Cells were stained with 10 M DCFH-DA and ROS
μ
(
Darmstadt, Germany); a Cell Counting Kit-8 (CCK-8) was purchased
generation was improved by pre-transfection with siTrxR1 or siIDO1 for 56 h
before exposure to ZC0101. Intracellular ROS generation in HCT-116 (A) or
HeLa (B) cells was measured by flow cytometry. Fluorescence microscopy was
also used to reveal the difference in ROS generation between HeLa (C) and
HCT-116 (D) cells. (E) DCFH-DA mean fluorescence density was quantified by
flow cytometry. Data are expressed as the mean ± SD of three independent
experiments. Bars = SD. *P < 0.05, **P < 0.01, ***P < 0.005.
from Dojindo Laboratories (Kumamoto, Japan); and rabbit polyclonal
anti-TrxR1 antibody was purchased from Proteintech (#11117–1-AP;
Wuhan, Hubei, China). Rabbit monoclonal anti-IDO1 antibody was
purchased from Abcam (#ab211017, Cambridge, MA, USA). Mouse
monoclonal anti-GAPDH antibody, HRP conjugated goat anti-rabbit and
goat anti-mouse IgG (H + L) Secondary Antibodies were purchased from
Thermo Fisher Scientific (#MA515738, 31,460 and 31430, respectively;
Waltham, MA, USA). ROS assay kit and RIPA lysis buffer were purchased
from Beyotime Institute of Biotechnology (#S0033 and P0013B; Hai-
men, Jiangsu, China). FITC-Annexin V Apoptosis Detection Kit I was
purchased from BD Biosciences (#556547; San Jose, CA, USA). The
Protease and Phosphatase Inhibitor Cocktail and BCA Protein Assay Kit
4
.1.6. Synthesis of tert-butyl 4-(2-formylphenyl)-1H-imidazole-1-
carboxylate (11)
Subsequently, 2- (1H-imidazole-4-) benzaldehyde (0.27 g, 1.57
2
mmol), 1.5 times the equivalent of Boc O (0.514 g, 2.36 mmol), 0.1
9

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
Fig. 8. ZC0101 efficiently suppressed Kyn in wild-type mice. (A, B) Wild-type male C57BL/6 mice were orally single administered Epacadostat or ZC0101 at 60 mg/
kg, and blood was harvested at the indicated times. Plasma from five mice was analyzed for Kyn/Trp ratio (A), Kyn or Trp levels (B) by LC/MS/MS. The results were
plotted as mean ± SD.
were purchased from Thermo Fisher Scientific (#78440 and 23225,
respectively; Waltham, MA, USA).
supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino
acids, 1 mM sodium pyruvate, and 10% (v/v) FBS. All of the cells except
SW620 were maintained in a humidified atmosphere with 5% CO
7˚C .
2
at
3
4
.3. Cell culture
All of the human cell lines except A549/DDP and HCT-8/5-Fu were
4.4. IDO1 enzyme assay
purchased from the American Type Tissue Culture Collection (ATCC,
Manassas, VA, USA); A549/DDP was purchased from the China Infra-
structure of Cell Line Resource (Beijing, China); and HCT‑8/5‑Fu was
purchased from the Advanced Research Center of Central South Uni-
versity (Changsha, China). BEAS-2B cells were grown with BEGM me-
dium (#CC-3170; Lonza, Walkersville, MD, USA) in the flasks pre-coated
with a mixture of 0.01 mg/mL fibronectin (#F1056; Sigma-Aldrich),
Recombinant human IDO1 (residues 5–403) with an N-terminal His
tag (pET-28a-IDO1) was expressed in the E. coli BL21 (DE3) strain and
purified by immobilized metal affinity chromatography (IMAC) with a
protein purification system (AKTA pure 5, GE health, Uppsala, Sweden).
◦
The assays were performed at 37 C using 0.625
μ
M IDO1 and 0.2 mM L-
M methylene blue and 0.1
mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5) for 1 h.
Then, 140 L of the supernatant per well was transferred to a new 96
well plate. Subsequently, 10
Trp in the presence of 10 mM ascorbate, 10
μ
0
0
.03 mg/ml bovine collagen type I (#A1048301; Thermo Fisher), and
.01 mg/mL bovine serum albumin (BSA, #A1933; Sigma-Aldrich). FHC
μ
cells were grown in DMEM/F12 medium (#30–2006; ATCC) with a final
concentration of 25 mM HEPES, 10 ng/mL cholera toxin (#C8052;
Sigma-Aldrich), 0.005 mg/mL insulin (#91077C; Sigma-Aldrich), 0.005
mg/mL transferrin (#T8158; Sigma-Aldrich), 100 ng/ml hydrocortisone
μ
L of 6.1 N trichloroacetic acid were mixed
◦
into each well and incubated at 60 C for 30 min to hydrolyze the N-
formylkynurenine produced by IDO1 to kynurenine. The reaction
◦
mixture was then centrifuged at 0 C for 10 min with 10000 rpm to
(
#HY-N0583; MCE, Monmouth Junction, NJ, USA), 20 ng/mL human
remove sediments. After that, 100
μ
L of the supernatant per well were
recombinant EGF (#PHG0311; Thermo Fisher), and 10% (v/v) fetal
bovine serum (FBS, #10099141C; Thermo Fisher). A549 and A549/DDP
cells were grown in F12K medium; H1299, HCC827, PC-9, HCT-8, HCT-
transferred to another 96 well plate and mixed with 100
μ
L of 2% (w/v)
4-(Dimethylamino)benzaldehyde in acetic acid. The yellow color
derived from kynurenine was measured at 480 nm using a Cytation 5
microplate reader (BioTek). L-Kynurenine, which was used as the stan-
dard, was prepared in a series of concentrations (200, 100, 50, 25, 12.5,
8
/5-Fu, HCT-15, and HCT-116 cells were grown in RPMI-1640 medium;
HT-29 cells were grown in McCoy’s 5a modified medium; while SW620
cells were grown in Leibovitz’s L-15 medium supplemented with 10%
6.25, 3.12, and 1.56
μ
M) in 100 L potassium phosphate buffer and
μ
(
v/v) FBS. HeLa cells were grown in Eagle’s minimum essential medium
analyzed through the same procedure. The percent inhibition at
1
0

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
individual concentrations was determined. The data were processed
using nonlinear regression to generate IC50 values (Prism Graphpad 5).
plates overnight, and then treated with the drugs (PL and ZC0101: 100
μ
M, 2-fold dilution for 12 concentrations). Complete, drug-free medium
was added to the blank wells. Control cells were treated with DMSO
4
.5. TrxR1 enzyme assay
only. After incubation for 24 h, 10
μ
L CCK-8 was added to each well and
◦
cells were incubated at 37 C for 4 h. Then, the medium was removed
The TrxR activity was determined at room temperature using a
and 150 L DMSO was added, followed by gentle shaking. The absor-
μ
microplate reader. The NADPH-reduced rat liver TrxR (0.24
incubated with different concentrations of ZC0101, PL, or Aurofin for 1
h at room temperature (the final volume of the mixture was 50 L) in a
6-well plate. A master mixture in TE buffer (50 mM Tris-HCl pH 7.5, 1
μ
g) was
bance (AB) at 450 nm was measured by a Cytation 5 microplate reader
(BioTek), and the cell cytotoxicity inhibition rate was calculated using
the following formula: Growth inhibition rate = (AB value of control -
AB value of test)/(AB value of control - AB value of blank) × 100%.
μ
9
mM EDTA, 50
μ
L) containing DTNB and NADPH was added (final con-
M, respectively), and the linear increase in
centration: 2 mM and 200
μ
4.9. Imaging TrxR activity in HCT-116 and HeLa cells using TRFS-green
absorbance (AB) at 412 nm during the initial 3 min was recorded. The
same amounts of DMSO (0.1%, v/v) were added to the control experi-
ments, and the TrxR1 inhibitory rate was calculated using the following
formula: TrxR1 inhibitory rate = [1- (AB value of test at 3 min - AB value
of test at 0 min)/(AB value of control at 3 min - AB value of control at 0
min)] × 100%.
Cells were treated with the indicated concentrations of ZC0101 or PL
for 24 h followed by further treatment with TRFS-green (10 M) for 4 h.
μ
Phase contrast (top panel) and fluorescence (bottom panel) images were
acquired by fluorescence microscopy (EVOS FL). Ten cells were
randomly selected, and the fluorescence intensity in each individual cell
was quantified using ImageJ software (version 1.8; NIH).
4
.6. HeLa cell-based IDO1 activity assay [38–41]
4
.10. Western blot analysis
HeLa cells were seeded in a 96-well culture plate at a density of 5 ×
3
1
0 per well and grown overnight. On the next day, a final concentration
HCT-116 and HeLa cells were treated with ZC0101 or PL at the
of 10 ng/mL human IFN-γ, 15
of compounds in a total volume of 200
added into cells. After an additional 48 h of incubation, 140
supernatant per well was transferred to a new 96-well plate. Then, 10
μ
g/mL L-Tryptophan, and serial dilutions
L culture medium per well were
L of the
indicated concentrations for 24 h. The whole cells were lysed with RIPA
lysis buffer. Protein quantification was performed using a BCA Protein
Assay kit (#23225, Thermo Fisher Scientific), following which equal
quantities of proteins (20 µg) were separated via a 4–20% gradient SDS-
PAGE gel, and transferred onto a PVDF membrane. The membranes
were blocked with 5% BSA at room temperature for 2 h. TrxR1 and IDO1
antibodies were used at a 1:2000 dilution and GAPDH antibody was
used at a 1:5000 dilution in 5% BSA together with the membranes, and
incubated at 4˚ C overnight. Following three washes in TBS/0.1% Tween
20, the membranes were each probed with secondary horseradish
peroxidase-conjugated antibodies at a 10000-fold dilution in 5% BSA.
Following six washes with TBS/0.1% Tween 20, the immune complexes
were incubated with ECL reagents (#34577, Thermo Fisher Scientific),
and detected using the ChemiDoc™ Touch Imaging System (Bio-Rad
Laboratories, Hercules, CA, USA). The resulting bands on the mem-
branes were calculated and normalized to GAPDH in each sample using
ImageJ software.
μ
μ
μL
of 6.1 N trichloroacetic acid were mixed into each well and incubated at
◦
6
0
C for 30 min to hydrolyze the N-formylkynurenine produced by
◦
IDO1 into kynurenine. The reaction mixture was then centrifuged at 0 C
for 20 min at 4000 rpm to remove sediments. After that, 100
supernatant per well were transferred to another 96-well plate and
mixed with 100 L of 2% (w/v) 4-(Dimethylamino)benzaldehyde in
acetic acid. The yellow color derived from kynurenine was measured at
80 nm using a Cytation 5 microplate reader (BioTek). L-Kynurenine,
which was used as the standard, was prepared in a series of concentra-
tions (200, 100, 50, 25, 12.5, 6.25, 3.12, and 1.56 M) in 100 L HeLa
μL of the
μ
4
μ
μ
cell culture media and analyzed using the same procedure. The percent
inhibition at individual concentrations was determined. The data were
processed using nonlinear regression to generate IC50 values (Prism
Graphpad 5).
To detect the knockdown efficiency of TrxR1 or IDO1, cells were pre-
transfected with siTrxR1 or siIDO1 for 60 h prior to lysis.
4
.7. Cellular TrxR1 activity assay
4
.11. Cell apoptosis assay
After HCT-116 cells were treated with different concentrations of
ZC0101, PL, and Aurufin for 24 h, the cells were harvested and washed
twice with PBS. Total cellular proteins were extracted by RIPA buffer for
HCT-116 cells were plated in six-well plates at an initial density of
5
2.4 × 10 cells per well for 8 h in complete RPMI-1640 media. Cells were
starved in serum-free RPMI-1640 medium for 16 h, followed by ZC0101
or PL treatment for 24 h. Cells were harvested, washed twice with
phosphate-buffered saline (PBS, Thermo Fisher Scientific, Waltham,
MA, USA), evaluated for apoptosis using the FITC-Annexin V Apoptosis
Detection Kit I, and analyzed using the Novocyte flow cytometer (ACEA
Biosciences, San Diego, CA, USA) with NovoExpress 1.2.4 software.
3
0 min on ice and quantified using the BCA procedure. TrxR1 activity in
cell lysates was measured by the endpoint insulin reduction assay. In
brief, the cell extract containing 20 g of total proteins was incubated in
a final reaction volume of 50 L containing 100 mM Tris-HCl (pH 7.6),
M NADPH, 3 mM EDTA, and 1.3 M E. coli Trx for
L of 1 mM
μ
μ
0
3
.3 mM insulin, 660
μ
μ
◦
0 min at 37 C. The reaction was terminated by adding 200
μ
DTNB in 6 M guanidine hydrochloride, pH 8.0. A blank sample, con-
taining everything except Trx, was treated in the same manner. The
absorbance (AB) at 412 nm was measured, and the blank value was
subtracted from the corresponding absorbance value of the sample. The
same amounts of DMSO were added to the control experiments, and the
TrxR1 inhibitory rate was calculated using the following formula: TrxR1
inhibitory rate = [1- (AB value of sample - AB value of blanksample)/(AB
value of control - AB value of blanksample)) × 100%.
4.12. RNA interference analysis
TrxR1 siRNA, IDO1 siRNA, and a scramble nontargeting siRNA
(siNC) were purchased from Biomics Biotech (Biomics Biotechnologies
Co., Ltd., Nan Tong, China). The siRNA were transfected into cells using
Lipofectamine 3000 reagents (Thermo Fisher, Waltham, MA, USA) ac-
cording to the manufacturer’s instructions. The siRNA sequences are as
follows:
′
′
4
.8. Cell cytotoxicity screening
siTrxR1 sense: 5 - GCAUCAAGCAGCUUUGUUAdTdT-3
′
′
siTrxR1 antisense: 5 - UAACAAAGCUGCUUGAUGCdTdT-3
′
′
The effects of drug treatments on cell cytotoxicity were quantified
siIDO1 sense: 5 - CCUACUGUAUUCAAGGCAAdTdT-3
′
′
using the CCK-8 assay. There were 5000 cells per well seeded in 96-well
siIDO1 antisense: 5 - UUGCCUUGAAUACAGUAGGdTdT-3
1
1

Q.-Z. Fan et al.
Bioorganic Chemistry 105 (2020) 104401
For loss-of-function analysis, HCT-116 or HeLa cells were pre-
Development Project of Anhui Province (Grant No. 202004a07020041),
the University Natural Science Research Project of Anhui Province
(Grant No. KJ2018A0259), the Major University Natural Science
Research Project of Anhui Province (Grant No. KJ2019ZD30), the
Foundation for Excellent Talents in Higher Education of Anhui Province
(Grant No. gxbjZD18), the Specialized Research Fund for the Doctoral
Program of Wannan Medical College (Grant No. rcqd201607), and the
Key Research Cultivation Foundation of Wannan Medical College (Grant
No. WK2017Z03).
transfected with siNC, siTrxR1, or siIDO1 for 36 h prior to ZC0101
exposure, followed by the same process as the CCK-8 assay, cellular
TrxR1 and IDO1 activity assay.
4
.13. Measurement of reactive oxygen species generation
Cellular ROS content was measured by flow cytometry and fluores-
cence microscopy for quantitative and qualitative evaluation. HCT-116
5
or HeLa cells were plated in six-well plates at a density of 2.0 × 10
cells/well and allowed to attach overnight, and then exposed to various
concentrations of ZC0101 for 4 h (for HCT-116 cells: 0, 0.25, 0.5, and 1
Appendix A. Supplementary data
μ
M; for HeLa cells: 0, 2.5, 5, and 10
μ
M). Cells were stained with 10
μ
M
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2020.104401.
◦
DCFH-DA (Beyotime Biotech, Nantong, China) at 37 C for 30 min, and
then washed three times in serum-free medium. For flow cytometry,
cells were collected and fluorescence was analyzed at excitation and
emission wavelengths of 488 nm and 525 nm, respectively. Additionally,
the mean value of DCFH-DA fluorescence intensity was utilized for
quantitative analysis. For fluorescence microscopy, cells were collected
and photos were obtained using the EVOS FL Imaging System (Thermo
Fisher Scientific, Waltham, MA, USA). In the same experiments, cells
were pretreated with siTrxR1 or siIDO1 for 56 h prior to ZC0101
exposure and ROS generation analysis.
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Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgements
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This study was supported by the National Natural Science Founda-
tion of China (Grant No. 81272485), the Natural Science Foundation of
Anhui Province (Grant No. 1808085QH262), the Key Research and
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