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Published as doi: 10.1096/fj.07-101477.
 (The FASEB Journal. 2008;22:2072-2083.)
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Thioredoxin reductase inhibition by antitumor quinols: a quinol pharmacophore effect correlating to antiproliferative activity

Eng-Hui Chew*, Jun Lu*, Tracey D. Bradshaw{dagger} and Arne Holmgren*,1

* Department of Medical Biochemistry and Biophysics, Medical Nobel Institute for Biochemistry, Karolinska Institutet, Stockholm, Sweden; and

{dagger} School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, UK

1Correspondence: Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-17177 Stockholm, Sweden. E-mail: arne.holmgren{at}ki.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Novel heteroaromatic-substituted 4-hydroxycyclohexa-2,5-dienones (quinols) demonstrate potent in vitro antiproliferative activity and in vivo antitumor activity in tumor xenografts. The mechanism of action of these promising novel anticancer agents, however, remains to be fully elucidated. The thioredoxin (Trx) system comprising Trx, thioredoxin reductase (TrxR), and NADPH participates in a broad range of cellular functions involved in cell survival and proliferation. Accumulating evidence has indicated that the selenocysteine-containing mammalian TrxR is a valid molecular target for development of novel cancer therapeutics. In this study, we demonstrate that structural analogs containing a quinol pharmacophore inhibited TrxR with potencies correlated with their antiproliferative and cytotoxic efficacies. Benzenesulfonyl-6F-indole-substituted quinol (compound 6) irreversibly inhibited TrxR most strongly with a half-maximal inhibitory concentration of 2.7 µM after 1 h of incubation with recombinant rat TrxR. The inhibition was shown to be concentration-, time-, and NADPH-dependent and mediated through a direct quinol attack on the penultimate C-terminal selenocysteine residue. Moreover, TrxR activity in lysates of HCT 116 cells treated with apoptosis-inducing doses of quinols was significantly reduced. From the results obtained, we propose that TrxR inhibition is a critical cellular event that contributes to the proapoptotic effects of quinols.—Chew, E. H., Lu, J., Bradshaw, T. D., Holmgren, A. Thioredoxin reductase inhibition by antitumor quinols: a quinol pharmacophore effect correlating to antiproliferative activity.


Key Words: Michael acceptors • anticancer agents • antitumor mechanism of action • selenocysteine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CANCER REMAINS ONE OF THE LEADING causes of death worldwide. In addition, drug resistance is causing many cancers to become resistant to conventional chemotherapeutic regimens. Persistent efforts to uncover novel molecular targets and new anticancer agents are therefore of utmost importance.

The thioredoxin system, comprising thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH, is a major thiol redox system that plays key roles in numerous cellular signaling pathways involved in cell survival and proliferation (1 , 2) . TrxR is the only known enzyme to catalyze the NADPH-dependent reduction of the active site disulfide (Cys32 and Cys35) of oxidized Trx; reduced Trx performs biological functions such as the following: 1) protection of cellular proteins against oxidative insult by reducing protein disulfides; 2) an antioxidant acting as an electron donor to peroxide-scavengers peroxiredoxins; 3) redox control of transcription factors such as nuclear factor-{kappa}B (NF-{kappa}B), activator protein-1 (AP-1), and hypoxia-inducible factor-1{alpha} (HIF-1{alpha}); and 4) as an electron donor for the enzyme ribonucleotide reductase (RNR) involved in DNA synthesis (1 2 3 4) . Essentially, these genes are not functionally redundant, as demonstrated in studies (5 6 7 8) where deletion of cytosolic and mitochondrial isoforms of Trx and TrxR leads to embryonic lethality in homozygous mice.

On the other hand, in cancer, the properties and biological effects of the Trx system contribute to tumor growth and progression (9) . Accumulating evidence indicates that the Trx system serves as a valid therapeutic target. Overexpression of the Trx system is reported in several human cancers (10 11 12) ; it is reported that increased Trx expression is associated with resistance to docetaxel in primary breast cancer (13) and decreased survival in colorectal cancer patients (14) . In recent years, the essential role of TrxR in tumorigenesis has been further assessed: mouse lung carcinoma (LLC1) cells stably transfected with a TrxR small interfering RNA construct achieve a reversal of their tumor phenotype to those of normal cells, and when injected into a mouse model, tumor growth and metastasis were largely reduced (15) . In the field of anticancer research, novel strategies based on targeting Trx and TrxR are thus pertinent with regards to the goal of achieving improved clinical outcome among patients with intractable malignancies.

As part of the drug discovery program, generation of novel and structurally diverse chemical oxidation products of bioactive phenols with potential biological properties has been actively pursued. This has led to the syntheses of heteroaromatic-substituted 4-hydroxycyclohexa-2,5-dienones (quinols) possessing selective and potent antiproliferative activity against colon, renal, and certain breast carcinoma cell lines [median growth inhibition (GI50)<0.5 µM; refs. 16 17 18 19 ]. Previous work has provided in vitro evidence of quinol binding to cysteine residues in cytosolic thioredoxin-1 (Trx1) protein and of quinol-mediated dose-dependent inhibition of TrxR/Trx signal transduction [half-maximal inhibitory concentration (IC50)<6 µM], suggesting that Trx is a molecular target of quinols (20) . Owing to a highly accessible C-terminal -Gly-Cys-Sec-Gly- active site and the low pKa of the free selenol group (-SeH) in the selenocysteine residue (Sec) that ensures full ionization to selenolate (Se–) at physiological pH, mammalian TrxR is known to be targeted by electrophilic compounds for inhibition (4 , 21) . Given that quinols are Michael acceptors with electrophilic propensity, we wanted to find out whether they could target TrxR. We addressed this hypothesis by testing a series of quinol analogs to analyze possible inhibition of mammalian TrxR and found that analogs containing a quinol pharmacophore inhibited mammalian TrxR with orders of potencies correlating to their cell growth inhibitory and cytotoxicity potencies. We found that active quinols exhibited a concentration-, time-, and NADPH-dependent selective inhibition of mammalian TrxR and that the mechanism would involve direct irreversible modification of the C-terminal Sec residue. Consistent with the previous finding that glutathione (GSH) protects cells from the cytotoxic effects of quinols (22) , we show here that GSH attenuated quinol-mediated TrxR inhibition to a similar magnitude. Based on the evidence obtained from cell-free and cell-based assays undertaken in this study, we propose that TrxR is a mechanistic target of antitumor quinols.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and cell culture
Recombinant rat TrxR was prepared as described previously (23) . The enzyme exhibited 50% specific activity of wild-type TrxR as determined by 5,5'-dithiobis-(2-nitrobenzoic acid; DTNB) assay. Preparation of recombinant Escherichia coli Trx and TrxR and double mutant human C62S/C73S Trx was as described previously (24 , 25) . Yeast glutathione reductase (GR), insulin, and glutathione disulfide (GSSG) were purchased from Sigma (St. Louis, MO, USA). Biotin-conjugated iodoacetamide (BIAM) was from Molecular Probes (Eugene, OR, USA). Quinol compounds 1 to 10 were kindly obtained from Malcolm Stevens (University of Nottingham, Nottingham, UK), of which the chemical syntheses of compounds 1 to 9 have been described previously (16 17 18 19) . Quinol stocks (10 mM) were prepared in dimethyl sulfoxide (DMSO), stored, and protected from light at 4°C.

Colon carcinoma HCT 116 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin and incubated at 37°C in an humidified atmosphere of 95% air-5% CO2.

TrxR activity by DTNB reduction assay
TrxR activity was determined using previously described methods (25 , 26) . Quinols of different concentrations were incubated with 110 nM recombinant rat TrxR and 200 µM NADPH in a volume of 100 µl of 50 mM Tris-HCl and 1 mM EDTA, pH 7.5 (TE buffer), for different time points in 96-well plates at room temperature. Then, 100 µl of TE buffer containing DTNB and NADPH was added (final concentration: 5 mM and 200 µM, respectively), and the linear increase in absorbance at 412 nm during the initial 2 min was measured with a VERSA microplate reader (Molecular Devices, Sunnyvale, CA, USA). TrxR activity was calculated as a percentage of enzyme activity of that of DMSO vehicle treated sample.

TrxR activity by spectrophotometric insulin reduction assay
Assays were carried out in 96-well plates using published methods with modifications (25 , 26) . In a volume of 100 µl of 50 mM Tris-HCl, 1 mM EDTA, pH 7.5 (TE buffer), and 200 µM NADPH, 50 nM recombinant rat TrxR and 2 µM human Trx1 were incubated with quinols of concentrations 1–50 µM for 30 min. An aliquot of 100 µl of TE buffer containing insulin and NADPH was then added (final concentration for both: 200 µM), and the absorbance at 340 nm was followed. In a separate experiment, human Trx1 (2 µM) was excluded from the 30 min incubation and then added together with insulin before the reaction was followed at 340 nm. TrxR activity was calculated as the linear change in absorbance at 340 nm per min of the initial 10 min. Experiments were performed with similar procedures to assess inhibition of the E. coli Trx system, where quinols of various concentrations were incubated with 25 nM E. coli TrxR, 2 µM E. coli Trx1, and 200 µM NADPH.

Preparation of reduced TrxR
For experiments to isolate NADPH- or dithiothreitol (DTT) -reduced TrxR free of excess reductant, 110 nM TrxR was preincubated in TE buffer containing 200 µM NADPH or 1 mM DTT and then desalted by passing through PD-10 desalting columns (Amersham Biosciences, Uppsala, Sweden) equilibrated with TE buffer. The eluted enzyme fractions were used immediately for quinol inhibition assays, and TrxR enzyme activity was determined using the DTNB and spectrophotometric insulin reduction assays, respectively.

Cell viability assay
HCT116 cells were seeded into 96-well plates at a density of 3' 103 per well and allowed to attach for 24 h before drugs at various concentrations prepared as serial dilutions in medium from DMSO stocks were introduced. After 72 h of drug treatment, cell viability was determined by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as described previously (20) .

Preparation of cell lysate
HCT116 cells (3'106) were plated onto 100 mm plates and incubated for 24 h to allow exponential growth before treatment with quinols for 16 h. Whole cell lysates of DMSO control or quinol-treated cells were prepared by lysing cell pellets (comprised of attached and floating cells) in reducing agent-free lysis buffer comprised of 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2.5 mM EDTA, 2.5 mM EGTA, 20 mM NaF, 1 mM Na3VO4, 20 mM sodium β-glycerophosphate, 10 mM sodium pyrophosphate, and 0.5% Triton X-100 containing freshly added protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Lysates were precleared by centrifugation before use, and protein concentrations were determined using a modified Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) as described in the manufacturer’s manual.

Immunoblotting
For Western blot analysis, equal amounts of protein (50 µg) in each lysate sample were separated by SDS-PAGE. The following antibodies were used: goat anti-human TrxR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-human Trx (IMCO Corporation, Stockholm, Sweden; www.imcocorp.se), and goat anti-β-actin (Santa Cruz). All Western blots shown represent three independent experiments.

Determination of TrxR and Trx activity in cell lysates
TrxR and Trx activity in cell lysates was measured in 96-well plates using an end point insulin assay as described previously (27) . For TrxR activity measurement, 25 µg of cell lysate was incubated in a final reaction volume of 50 µl containing 85 mM HEPES (pH 7.6), 0.3 mM insulin, 660 µM NADPH, 2.5 mM EDTA, and 5 µM human C62S/C73S Trx (24) for 20 min at room temperature. Controls were set up alongside with incubation of cell lysates in reaction solutions without the presence of human Trx. The reaction was quenched by adding 250 µl of 1 mM DTNB and 240 µM NADPH in 6 M guanidine hydrochloride and 200 mM Tris-HCl, pH 8.0 solution, where the amount of free thiols generated from insulin reduction was determined by DTNB reduction measured at 412 nm (extinction coefficient for 2-nitro-5-thiobenzoic acid 13.6 mM–1·cm–1). TrxR activity was represented as the absorbance at 412 nm subtracted from that of control. For Trx activity measurement, procedures were the same as for the TrxR activity assay, where in control wells, cell lysates (25 µg) were incubated in reaction solutions without 600 nM recombinant rat TrxR. Trx activity for each lysate sample was obtained as the absorbance at 412 nm subtracted from that of the corresponding control.

GR assay
Quinols of concentrations 1–50 µM were incubated with 10 nM yeast GR and 200 µM NADPH in a volume of 100 µl TE buffer for 30 min in 96-well plates at room temperature. When substrate GSSG and fresh NADPH (final concentration: 1 mM and 200 µM, respectively) were added, GR activity was determined by measuring the decrease in absorbance at 340 nM during the initial 3 min and expressed as a percentage of enzyme activity of that of the DMSO vehicle treated sample.

BIAM-labeling assay
The biotin labeling of free Sec in mammalian TrxR was used to determine whether the Sec residue is susceptible to quinol treatment. Briefly, a 100 µM BIAM stock solution in 100 mM Tris-HCl and 1 mM EDTA, pH 6.5, was freshly prepared for each experiment. In a tube, NADPH-reduced recombinant rat TrxR (0.9 µM) was incubated with compound 1 or 6 (100 µM) or DMSO vehicle in 20 mM Tris-HCl, 1 mM EDTA, pH 7.5, and 200 µM NADPH at room temperature. At different time points, a 3 µl aliquot was withdrawn and mixed with 20 µM BIAM in 100 mM Tris-HCl and 1 mM EDTA, pH 6.5 (final volume=30 µl), for 15 min at 37°C. Alkylation of free selenol was quenched by adding freshly made iodoacetamide solution (final concentration=50 mM). The protein samples were then denatured in SDS sample buffer and subjected to SDS-PAGE and Western blotting. The biotin was detected by horseradish perioxidase (HRP) -conjugated streptavidin using the enhanced chemiluminescence system (Perkin-Elmer Life Sciences). Membranes were stripped with stripping buffer (0.15 M glycine, pH 2.5, 0.4% SDS) and reprobed with polyclonal antirat TrxR1 antibody purified from rabbit antiserum against rat liver TrxR1 (28) ; protein levels were determined using the same detection system. At each time point, 40 µl of TrxR protein mixture was also removed for TrxR activity determination using the DTNB assay.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Quinols with an active pharmacophore inhibit TrxR in a dose- and time-dependent manner
A novel series of quinols with heteroaromatic or (arylsulfonyl)indole substitutions at the 4 position has been reported to exhibit potent in vitro antitumor activity (16 17 18 19) . To study the structure-activity relationship of these quinol compounds on mammalian TrxR inhibition, eight compounds (1 to 8) containing a quinol pharmacophore with various heterocyclic substituents and two structural derivatives of compound 1 lacking a quinol pharmacophore (9 and 10 contain a phenol and 4-hydroxy-cyclohexane moiety, respectively) were selected for evaluation (Fig. 1 ; Table 1 ). The compounds were incubated at different concentrations with NADPH-reduced recombinant rat TrxR (110 nM) for various time points at room temperature, followed by TrxR activity determination using DTNB reduction assay. Quinol compounds 1 to 8 caused a dose-dependent (Fig. 2 A) and time-dependent (Fig. 2B ) decrease in TrxR activity, while compounds 9 and 10 showed no enzyme inhibition even at a high dose of 150 µM. As illustrated in Table 1 , compounds 5 and 6 with a benzenesulfonyl indole substitution that possess the most potent in vitro antiproliferative activity also exhibited the strongest TrxR inhibitory activity with a respective IC50 value of 2.9 and 2.7 µM at 1 h of incubation. Their respective more polar propionic acid derivatives, compounds 7 and 8, display an overall decrease in in vitro activity against TrxR (IC50=8.4 and 9.8 µM, respectively, at 1 h incubation) and cell growth. Benzothiazole-substituted quinol compounds 1 and 2 showed >10-fold enhancement in TrxR inhibitory activity (IC50=6.5 and 6.3 µM, respectively, at 1 h incubation) compared to thiophen- and benzoxazole-substituted quinols (compounds 3 and 4; IC50=104.3 and 76.1 µM, respectively, at 1 h of incubation; Table 1 ). Among the compounds tested, it was observed that the trend of TrxR activity inhibition, a property attributed to the presence of a quinol pharmacophore, was positively correlated to antitumor potency.


Figure 1
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  		Figure 1. Chemical structures of quinols with heteroaromatic or (arylsulfonyl)indole substitution at the 4 position.


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  		Table 1. Mammalian TrxR inhibitory and antiproliferative activities of quinol analogs


Figure 2
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  		Figure 2. Inhibitory activity of quinols on mammalian TrxR in vitro. A) Quinol compounds 1–8, containing a quinol pharmacophore, display a concentration-dependent inhibition of TrxR activity. TrxR activity was evaluated by DTNB reduction assay (see Materials and Methods) after 1 h incubation of compounds with 110 nM recombinant rat TrxR. Compounds 9 and 10, lacking a quinol pharmacophore, showed no enzyme inhibition at all concentrations indicated. B) Illustration of time-dependence inhibition of quinols 1, 2, and 5–8 (15 µM) and compunds 3 and 4 (100 µM) on mammalian TrxR in contrast to compounds 9 and 10 (100 µM), which failed to inhibit TrxR at all time points indicated. All data points are means ± SD of 2 independent experiments.

TrxR inhibition by quinols in cells
The effect of four of the more potent TrxR inhibitory quinols (compounds 1, 2, 5, and 6) on the Trx system in intact cells was assessed. As previously reported, quinols induce apoptosis within 12 to 24 h in quinol-sensitive cell lines, and the effect is most profound in colon carcinoma HCT116 cells (22) . Here, we exposed HCT116 cells to a similar range of drug concentrations for 16 h, after which collected cell lysates were assayed for TrxR and Trx activity and protein expression. As illustrated in Fig. 3 A, quinols at apoptosis-inducing doses of 3 and 5 µM brought about a marked decrease in TrxR activity to ~60% that of DMSO control cells, whereas at sublethal concentrations (0.5 and 1 µM), an elevation in TrxR activity to as high as 130% was observed. These low quinol doses resulted in a similar marginal rise in cellular Trx activity (a possible consequence of the increase in cellular TrxR activity), but the higher doses (3 and 5 µM) did not have a significant effect on the activity of Trx (Fig. 3B ). Western blot results showed that TrxR expression was induced in cells treated with quinols at 0.5 and 1 µM and was at comparable levels as in DMSO control cells when exposed to concentrations ≥3 µM (Fig. 3C ). Trx protein levels remained unchanged for all drug concentrations tested (data not shown). Taken together, these results indicated that the quinol-mediated decrease in cellular TrxR activity was due to an inhibition of enzyme function and not a result of a decrease in protein synthesis or an increase in protein degradation.


Figure 3
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  		Figure 3. Effects of quinols on TrxR activity and protein levels in HCT116 cells. Cells were exposed to indicated quinol compounds (Cpd) for 16 h at concentrations as indicated, and cell lysates were collected for enzyme activity and protein expression determination. TrxR (A) and Trx activity (B) was expressed as a percentage of DMSO control. *Statistically significant difference (P<0.05) in enzyme activity from the control. All data points are means ± SE of 4 independent experiments. C) Aliquots of cell lysates with equal protein content were analyzed by Western blotting with the indicated antibodies.

Quinols irreversibly inhibit TrxR in a NADPH-dependent manner
Experiments were carried out to determine the influence of NADPH on TrxR inhibition by selected quinol compounds 1, 2, 5, and 6. As compounds 1 and 2, as well as compounds 5 and 6, share similar activity inhibition profiles, only results of assays performed on compounds 1 and 6 are shown in Fig. 4 . Mammalian TrxR was preincubated with quinols in the presence or absence of NADPH for 30 min, before enzyme activity was determined by the DTNB reduction assay. The oxidized enzyme remained unaffected, while the reduced form was inhibited. A sample of NADPH-reduced TrxR removed of excess NADPH by desalting over a PD-10 column was inhibited to a similar extent with comparable IC50 values (Fig. 4A, B ), suggesting that NADPH is not directly involved in the quinol-mediated enzyme inhibition. The effect of reductant DTT on quinol inhibition of mammalian TrxR was also investigated. Interestingly, DTT-reduced TrxR with either excess DTT removed over a PD-10 desalting column (Fig. 4A, B ) or not removed (results not shown) did not show susceptibility to quinol inhibition. It may be appreciated that the enzyme activity herein was determined using the insulin reduction assay to follow the reaction rate of NADPH consumption at 340 nm over a 10 min period. Thus enzyme inhibition produced at higher quinol concentrations (compound 1: 50 µM, Fig. 4A ; compound 6: 25 and 50 µM, Fig. 4B ) was not negligible. Taken together, these results highlight the NADPH dependence of TrxR inhibition by quinols.


Figure 4
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  		Figure 4. NADPH dependence of the inhibition of mammalian TrxR by quinols. A 110 nM concentration of oxidized recombinant rat TrxR was either not reduced with NADPH, reduced with NADPH (200 µM), or reduced with NADPH (200 µM) and stripped of excess NADPH by desalting over a PD-10 column; and 50 nM of oxidized recombinant rat TrxR was reduced with DTT (1 mM) and stripped of excess DTT by desalting over a PD-10 column. The enzyme was next exposed to quinol compounds 1 (A) and 6 (B) at concentrations as indicated for 30 min. For reactions involving incubation of enzyme with or without NADPH, TrxR activity was then measured by DTNB reduction assay (see Materials and Methods). For reactions involving enzyme reduction by DTT, TrxR activity was measured by insulin reduction assay, in which the absorbance at 340 nm was followed after adding insulin, human Trx1, and NADPH (see Materials and Methods). All data points are means of 2 independent experiments. Assays were also carried out on compounds 2 and 5, and similar activity inhibition profiles were observed as for compounds 1 and 6, respectively (results not shown).

To investigate whether the inhibition was reversible or irreversible, NADPH-reduced mammalian TrxR (0.9 µM) was incubated with selected quinol compounds 1 and 6 (100 µM) and when complete inhibition was achieved (TrxR activity of a withdrawn aliquot determined by DTNB assay), quinols were removed by passing the enzyme sample through a PD-10 column. At various time points over a 24 h period, aliquots of the eluted enzyme were assayed for activity in the presence of NADPH. No TrxR activity was recovered (data not shown), indicating that mammalian TrxR was irreversibly inhibited by quinols.

GSH modulation of quinol-mediated inhibition of mammalian TrxR
The modulatory role of GSH in quinol-induced cytotoxicity has been investigated in a previous study (22) that reports a 6- to 10-fold increased sensitivity of GSH-depleted cells to quinols, whereas GSH monoethyl ester supplementation reduces quinol potencies by 2–3x. We were interested to investigate the effect of GSH on quinol inhibition of mammalian TrxR. Recombinant rat TrxR was preincubated with NADPH, different concentrations of quinol compounds 1, 2, 5, and 6 and with or without the presence of GSH for 30 min. Again, compounds 2 and 5 gave activity profiles similar to compounds 1 and 6, respectively; only assay results of compounds 1 and 6 are presented in Fig. 5 . Addition of GSH at physiological concentrations 0.5 and 1 mM impeded inactivation of mammalian TrxR with resulting IC50s raised by 2- to 5-fold (Fig. 5A, B ). These results concurred with the magnitude of influence GSH had on quinol-induced cytotoxicity, which, taken together, underpin the protective role the GSH system plays in cell survival against quinol-mediated TrxR inactivation.


Figure 5
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  		Figure 5. GSH modulation of quinol-mediated inhibition of mammalian TrxR. A 50 nM concentration of recombinant rat TrxR in the presence or absence of reduced GSH (0.5 and 1 mM) was incubated with 200 µM NADPH and quinol compounds 1 (A) and 6 (B) at concentrations as indicated for 30 min. TrxR activity was determined by insulin reduction assay, in which absorbance at 340 nm was followed after adding insulin, human Trx1, and NADPH (see Materials and Methods). All data points are means of 4 independent experiments. Assays were also carried out on compounds 2 and 5, and similar activity inhibition profiles were observed as for compounds 1 and 6, respectively (results not shown).

Quinols are selective for mammalian TrxR inhibition and target the penultimate Sec residue
In the TrxR inhibition reaction, quinols did not induce increased NADPH oxidase activity (data not shown), suggesting that quinols are not likely substrates of the flavoenzyme. Moreover, presence of superoxide dismutase (25 µg/ml) and catalase (30 µg/ml) failed to protect TrxR from quinol-mediated inactivation (data not shown).

To test the specificity of mammalian TrxR inhibition by quinol compounds, we determined the effect of compounds 1, 2, 5, and 6 on bacterial TrxR and yeast GR. Mammalian TrxR differs structurally from bacterial TrxR: the former possesses an additional redox active site at the C terminus, the penultimate Sec residue of which is indispensable for its Trx reduction activity (30 31 32) . The absence of this C-terminal -Cys-Sec- active site is also apparent in GR, to which mammalian TrxR shares closer similarity (compared to E. coli TrxR) on the basis of sequence homology, domain structure architecture (33 , 34) , and reaction mechanism (35) . Similarities in inhibitory profiles were obtained for compounds 1 and 2 and compounds 5 and 6, and only results obtained for compounds 1 and 6 are presented in Fig. 6 . Compounds 5 and 6, the most potent mammalian TrxR inhibitors, exhibited no inhibitory effect on bacterial TrxR and GR (Fig. 6B ). Compounds 1 and 2, next in line on the TrxR inhibition potency tally (Table 1) , did not inhibit GR and, interestingly, at higher concentrations of 25 and 50 µM caused a respective 20 and 30% inactivation of E. coli TrxR (Fig. 6A ). Although relatively insignificant in comparison to the degree of inhibition observed with mammalian TrxR, the difference in the behavior between benzothiazole-substituted (compounds 1 and 2) and (arylsulfonyl)indole-substituted (compounds 5 and 6) quinols suggests the possibility to rationally design and develop structural derivatives of the former class of quinols as inhibitors of bacterial TrxR with minimal inhibition of its mammalian counterpart. Furthermore, the inhibitory effect of quinols on the whole Trx system was compared with that on TrxR only (independent of Trx) using the insulin reduction assay. The compounds were found to inhibit the catalyzed reduction of insulin disulfide to the same extent with comparable IC50s (Fig. 6A, B ), suggesting that the presence of Trx did not affect the inhibitory efficiency and that TrxR was indeed the specific target of quinols.


Figure 6
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  		Figure 6. Effect of quinols on TrxR and GR activity. Recombinant rat (r) TrxR (50 nM) alone or in the presence of 2 µM human (h) Trx1 or E. coli TrxR (25 nM) alone or in the presence of 2 µM E. coli Trx was preincubated with 200 µM NADPH and quinol compounds 1 (A) and 6 (B) at concentrations as indicated for 30 min. TrxR activity was determined by insulin reduction assay (see Materials and Methods). Yeast GR (10 nM) was preincubated with 200 µM NADPH and quinol compounds 1 (A) and 6 (B) at concentrations as indicated for 30 min. GR activity was determined by glutathione reduction assay, in which absorbance at 340 nm was followed after adding GSSG and NADPH (see Materials and Methods). All data points are means of 2 independent experiments. Assays were also carried out on compounds 2 and 5, and similar activity inhibition profiles were observed as with compounds 1 and 6, respectively (results not shown).

Results obtained so far have revealed the susceptibility of mammalian TrxR to quinol inactivation, while bacterial TrxR and GR activity remained unaltered. Given that the selenol in the penultimate C-terminal Sec residue has a low pKa of 5.2 (36) that results in reduced mammalian TrxR existing as a deprotonated selenol (Se–) species at physiological pH, we hypothesized that the Sec residue was targeted by quinols. To confirm this hypothesis, we made use of the BIAM-labeling reaction at low pH to monitor the extent of free Sec modified by the presence of quinols. Previous studies (37 38 39) have reported the selective alkylation of the Sec at pH 6.5 due to its low pKa that renders the deprotonated species more susceptible to alkylation; a Sec that is oxidized or chemically modified would be unavailable for BIAM labeling, which could then be monitored by streptavidin blot analysis. As shown in the bottom panel of Fig. 7 , the HRP-streptavidin-blotted bands of the 55 kDa TrxR monomer depicting free selenol decreased in intensity along increasing incubation time with compounds 1 and 6. This observed decrease in free selenolate available for BIAM labeling was consistent with the decline in enzyme activity over time (Fig. 7 , top), strongly suggesting that quinols target the Sec residue to bring about mammalian TrxR inhibition.


Figure 7
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  		Figure 7. Quinols target the Sec residue of mammalian TrxR. A 0.9 µM concentration of recombinant rat TrxR was incubated with 200 µM NADPH and 100 µM quinol compounds 1 and 6. At different time points, an aliquot of enzyme mixture was withdrawn for TrxR activity measurment and BIAM labeling. Top: time course of TrxR activity of the mixture. Bottom: HRP-conjugated streptavadin detection of BIAM labeling of free selenol in the mixture at different incubation times. Results shown are representative of 3 independent experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TrxR is a homodimeric enzyme belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases that also includes GR (40) . Two distinct types of TrxR exist: the low molecular mass (35 kDa subunit) TrxR is present in bacteria, archaea, plants, and yeast and uses a -Cys-Ala-Thr-Cys- catalytic motif for a narrow substrate spectrum; a large subunit (55 kDa) TrxR is present in higher eukaryotes and has both an N-terminal redox active dithiol/disulfide in common with GR and a second C-terminal active site that accounts for its broad substrate specificity, including Trx, DTNB, protein disulfide isomerase, NK-lysin, peroxides, GSH peroxidase, selenite, and others (2 , 4 , 41) . Generally, mammalian TrxR1 localized in the cytosol (31 , 32 , 34 , 42) , TrxR2 in the mitochondria (38 , 43) , and thioredoxin/glutathione reductase (TGR) in the testis (44) have a common domain structure comprised of a flavin adenine dinucleotide (FAD) and NADPH-binding domain and an interface domain that is similar to GR; also conserved in mammalian TrxRs are the N-terminal -Cys59-Val-Asn-Val-Gly-Cys64- dithiol/disulfide active site contained in the FAD-binding domain and the second -Gly-Cys497-Sec498-Gly- active site that resides in the 16-residue extension in the C terminus. The essential role of the penultimate C-terminal Sec residue for TrxR catalytic activity has been well studied (32 , 37 38 39 , 45 , 46) . The catalytic mechanism of mammalian TrxR, as supported by biochemical and X-ray crystallographic studies, involves the transfer of reducing equivalents from NADPH to bound FAD and then to the N-terminal active site disulfide; the resultant dithiol subsequently reduces the C-terminal selenenylsulfide of the other subunit of the homodimer, and the selenolthiol motif finally reduces a substrate of the enzyme, for example, the active site disulfide within oxidized Trx (34 , 35 , 42 , 43 , 47) . On NADPH reduction, the flexible C-terminal tail moves such that the selenothiol motif becomes surface exposed (34 , 42) . This unique feature of reduced mammalian TrxR, the highly accessible Sec and high reactivitiy of the nucleophilic selenolate moiety, has made the enzyme a selective target of many electrophilic compounds (4 , 21) .

Novel heteroaromatic-substituted quinols exhibit in vitro antiproliferative activity against tumor cell lines and in vivo antitumor activity in xenograft models (16 17 18 19) . However, the precise mechanisms of antitumor action of these promising novel anticancer agents remain elusive. A previous study (20) using mass spectrometry has shown in vitro binding of quinol to cysteine residues in Trx1 protein, suggesting that protein thiol inactivation contributes at least in part to the proapoptotic and antiproliferative effect of quinols. Quinols are Michael acceptors, which are chemical compounds containing an {alpha},β-unsaturated carbonyl moiety where the electrophilic β carbon atom may interact with nucleophiles such as sulfhydryl groups. Michael acceptors are known to have sulfhydryl reactivities (48) . Compared to cysteine, Sec is a stronger nucleophile due to a larger atomic radius and a lower pKa to ensure full ionization under physiological conditions (36) . Given that compounds reported to inhibit mammalian TrxR such as quinones (49) , curcumin (50) , and flavonoids quercetin and myricetin (51) are known Michael acceptors, we sought to investigate whether quinols could inhibit mammalian TrxR. We screened a number of quinol analogs with different heteroaromatic or (arylsulfonyl)indole substitutions at the 4 position (Fig. 1) for their effects on mammalian TrxR activity. A structure-activity relationship was established, which positively correlated with the cell-growth inhibition and cytotoxicity profile of each analog. The presence of a quinol pharmacophore was accountable for TrxR inactivation. A benzenesulfonyl indole substitution resulted in the most potent analogs (compounds 5 and 6), but coupling a propionate group to this substituent (compounds 7 and 8) led to a detrimental effect on potency; benzothiazole substitutions gave rise to more potent analogs (compounds 1 and 2) than thiophen (compound 3) or benzoxazole (compound 4) substitutions.

Here, we have shown that quinols irreversibly inhibited mammalian TrxR in a concentration-, time-, and NADPH-dependent manner. Earlier reports (35 , 50 51 52 53) have shown that most inhibitors fail to inactivate oxidized TrxR in the absence of NADPH. Similarly, oxidized mammalian TrxR, which contains a nonreactive C-terminal Cys497-Sec498 selenenylsulfide bridge (47) , was resistant to inactivation by quinols. Across a range of cytotoxic doses, potent quinols, for instance, compound 6, selectively inhibited mammalian TrxR with an IC50 of 4.3 µM (at 30 min incubation; Table 1 ) but had no effect on GR and bacterial TrxR. Indeed, a time-related decrease in BIAM labeling of free selenol (-SeH) corresponding to a decline in catalytic activity (Fig. 7) indicated that quinols target the Sec in the enzyme. Collectively, we demonstrated that mammalian TrxR is a mechanistic target for quinols based on several lines of evidence: 1) a correlating trend of quinol cytotoxicity with the in vitro TrxR inhibition potency, 2) cell lysates of HCT116 cells treated with quinol concentrations and for a time point that was previously found to induce apoptosis (22) showed a reduction in TrxR activity, and 3) the results of our in vitro experiment where the presence of physiological concentrations of GSH impeded quinol-mediated TrxR inactivation, which is in agreement with the finding that depletion or supplementation of intracellular GSH has modulated quinol-mediated cytotoxicity (previously reported in ref. 22 ).

Quinol-modified TrxR did not show induction of NADPH oxidase activity, suggesting that quinols are not substrates of TrxR. Based on the obtained results, a proposed mechanism of mammalian TrxR inactivation by quinols is outlined in Fig. 8 : the quinol electrophilic β carbon atom directly attacks the selenolate moiety in NADPH-reduced TrxR, and the resultant enzyme is chemically modified to lose catalytic activity.


Figure 8
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  		Figure 8. Proposed mechanism of inhibition of mammalian TrxR by quinols. The electrophilic β carbon atom in active quinols, for example, compound 6, directly attacks the Sec residue in NADPH-reduced TrxR. The C-terminal-modified enzyme is unable to reduce oxidized Trx.

With the use of in vitro experimental approach, a previous study (20) suggested Trx as a molecular target of quinols; it remains to be determined whether quinols inhibit Trx directly in intact cells. In this present study, although a decrease in TrxR activity was detected in lysates of cells exposed to apoptosis-inducing doses of quinols, Trx activity remained unaffected (Fig. 3A, B ). We also performed the insulin reduction assay where the whole Trx system was preincubated with quinols, and the results obtained were consistent with observations in the work of Bradshaw et al. (20) that show quinol inhibition of TrxR/Trx protein disulfide reduction. We thus investigated whether quinols would inhibit TrxR independent of Trx by performing the assays where TrxR alone was preincubated with quinols; the results indeed showed that quinols caused the same degree of TrxR/Trx disulfide reduction. As supported by in vitro evidence of quinol binding to cysteines in Trx (20) , quinols have a propensity toward nucleophiles such as reactive cysteines in cellular proteins. However, in intact cells, within which numerous reactive cellular proteins are contained, we would surmise from the evidence presented here that quinols at therapeutic doses preferentially target TrxR instead of Trx.

The Trx and GSH systems exert redox control over many cellular processes involved in cell proliferation and apoptosis (54) . Accumulating evidence has shown that components of the Trx system are overexpressed in tumors and are involved in the carcinogenic process (10 11 12 , 15) . Numerous chemotherapeutic agents such as nitrosoureas (35 , 55) , cisplatin (53) , cyclophosphamide (56) , motexafin gadolinium (57) , and arsenic trioxide (58) have been reported to inhibit TrxR, suggesting that mammalian TrxR is an important molecular target for anticancer drug development. Trx is involved in DNA synthesis/repair and redox balance through respective reduction of RNR and peroxiredoxins, as well as in the modulation of gene expression of cellular proteins through redox regulation of transcriptional activity of NF-{kappa}B, AP-1, p53, and HIF-1{alpha} (1 2 3 4) . TrxR inhibition leads to Trx oxidation and culminates in disruption or deregulation of Trx-regulated cellular activities and responses, such as DNA synthesis and repair. Reduced Trx is known to inhibit apoptosis by binding to apoptosis signal regulating kinase 1 (ASK1); oxidized Trx dissociates from the complex, leading to ASK1 activation and ultimately to apoptosis induction (59) . Moreover, studies have shown that TrxR modified at the Sec residue by alkylating agents (60) or endogenous lipid electrophiles (61) is the species (not the full-length Sec-containing enzyme) that is necessary for inducing apoptosis. We therefore suggest that cytotoxicity due to quinol inhibition of TrxR is the result of the catastrophic events after Trx oxidation and quinol-modified TrxR directly inducing apoptosis.

In summary, we have demonstrated that quinols irreversibly inhibit mammalian TrxR by targeting the penultimate C-terminal Sec residue. Particularly, we have identified compound 6, a quinol with a benzenesulfonyl-6-fluoroindolyl substitution, to exhibit TrxR inhibition with remarkable potency that correlates well with its superior antitumor potency against sensitive cancer cell lines. In the context of anticancer research focused on developing novel cancer therapeutics against novel molecular targets, quinols as TrxR inhibitors certainly make themselves promising clinical candidates.


   ACKNOWLEDGMENTS
 
We are grateful to Malcolm Stevens, School of Pharmacy, University of Nottingham, for kindly providing the quinol compounds. We thank Elias Arner, Qing Cheng, and Olle Rengby (Medical Nobel Institute for Biochemistry, Karolinska Institutet) for kind provision of recombinant rat TrxR and R. Eliasson for expert technical assistance in the affinity purification of polyclonal antirat TrxR1 antibodies. This work was supported by Swedish Cancer Society Grant 961, Swedish Research Council Medicine Grant 13x-3529, the K.A. Wallenberg Foundation, and the Karolinska Institutet.

Received for publication October 29, 2007. Accepted for publication November 29, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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