Discovery of Tryptanthrin Derivatives as Potent Inhibitors of

Article pubs.acs.org/jmc

Discovery of Tryptanthrin Derivatives as Potent Inhibitors of Indoleamine 2,3-Dioxygenase with Therapeutic Activity in Lewis Lung Cancer (LLC) Tumor-Bearing Mice Shuangshuang Yang,†,§ Xishuai Li,†,§ Fangfang Hu,‡ Yinlong Li,† Yunyun Yang,† Junkai Yan,† Chunxiang Kuang,‡ and Qing Yang*,† †

State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Handan Road 220, Shanghai 200433, China ‡ Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092, China S Supporting Information *

ABSTRACT: Indoleamine 2,3-dioxygenase (IDO-1) is emerging as an important new therapeutic target for the treatment of cancer, neurological disorders, and other diseases that are characterized by pathological tryptophan metabolism. However, only a few structural classes are known to be IDO-1 inhibitors. In this study, a natural compound tryptanthrin was discovered to be a novel potent IDO-1 inhibitor by screening of indole-based structures. Three series of 13 tryptanthrin derivatives were synthesized, and the structure−activity analysis was undertaken. The optimization led to the identification of 5c, which exhibited the inhibitory activity at a nanomolar level. In vitro 5c dramatically augmented the proliferation of T cells. When administered to Lewis lung cancer (LLC) tumor-bearing mice, 5c significantly inhibited IDO-1 activity and suppressed tumor growth. In addition, 5c reduced the numbers of Foxp3+ regulatory T cells (Tregs), which are known to prevent the development of efficient antitumor immune responses.

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INTRODUCTION IDO-1 (EC 1.13.11.42) is a monomeric heme-containing enzyme that catalyzes the first and rate-limiting steps in the catabolism of the essential amino acid tryptophan (Trp) along the kynurenine pathway (KP)1 and is widely distributed in various tissues of mammals.2 Dysregulation of the KP, which is associated mainly with the elevation of IDO-1 activity and excitotoxin quinolinic acid (QUIN) production, has been implicated in the pathogenesis of neuroinflammatory disorders, neurodegenerative disorders (Alzheimer’s disease), depression, age-related cataracts, and HIV encephalitis.3−6 Recently, IDO-1 has been shown to play an important role in the process of immune evasion by tumors.7 The expression of IDO-1 is up-regulated in a variety of human malignancies, including ovarian, colorectal, and pancreatic cancers, as well as non-small-cell lung carcinoma.8 Numerous clinical studies have associated the up-regulation of IDO-1 in cancer patients with a less favorable prognosis.9 In various animal models of cancers, systemic blockade of IDO-1 activity with small molecule © 2013 American Chemical Society

inhibitors successfully suppressed the outgrowth of tumors and had a cooperative effect with chemotherapy, radiotherapy, or cancer vaccines to trigger the regression of tumors that were otherwise recalcitrant to treatments.10−12 The IDO-mediated depletion of local tryptophan levels and the production of toxic tryptophan metabolites have resulted in the suppression of T-cell responses and the enhancement of immunosuppression mediated by regulatory T cells (Tregs).13,14 Tregs are emerging as a key component of acquired tolerance to tumors. However, Tregs are not spontaneously suppressive but rather require an activation step before they become functionally inhibitory.15 The most widely used and reliable Treg marker is the forkhead box transcription factor Foxp3, which is specifically expressed in murine CD4+CD25+ Tregs and is a critical regulator of CD4+CD25+ Tregs development and function.16 IDO-1 has been shown to regulate the recruitment, Received: March 5, 2013 Published: October 7, 2013 8321

dx.doi.org/10.1021/jm401195n | J. Med. Chem. 2013, 56, 8321−8331

Journal of Medicinal Chemistry

Article

Figure 1. Tryptanthrins with IDO-1 inhibitory activity. The numbers indicate the notation used throughout.

immunomodulatory30 and anti-inflammatory activity,31 and antitumor activity.32 Tryptanthrin has two characteristic reduction potentials (−0.75 and −1.40 V vs SCE) for the reduction of two carbonyl groups of the five- and the six-membered rings,33 thus having potentials for antileishmanial activity and photoelectronic reception abilities.33,34 In recent years, tryptanthrin has attracted much attention as an anticancer agent.35 Besides its cytotoxicity to a panel of human cancer cell lines in vitro,36,37 tryptanthrin also has cancer chemopreventive activity by preventing azoxymethane-induced intestinal tumor formation in F344 rats.38 Concerning the mechanisms of its antitumor effects, tryptanthrin has been revealed to be an aryl hydrocarbon receptor agonist.39 In addition, tryptanthrin is reported to inhibit hepatocyte growth factor, which is involved in malignant cell transformation and the progression of tumors.40 However, the exact mechanisms underlying antitumor ability of tryptanthrin remain elusive. The objectives of our ongoing long-term project are to screen for optimal IDO-1 inhibitors and to study their applications as potential treatments for diseases with the pathological characteristic of IDO-1-catalyzing Trp catabolism. Herein, by screening of indole-based structures, we identified the natural product tryptanthrin (5a) as a novel potent IDO-1 inhibitor. We designed and synthesized three series of 13 tryptanthrin derivatives, studied their structure−activity relationship, and investigated their redox potentials and effects on T cell proliferation in vitro. With the optimized derivative 5c,

expansion, and activation of Tregs through an APC-dependent mechanism.17−19 As such, the enzyme IDO-1 has emerged as a promising therapeutic target, prompting a search for highly active inhibitors. The landmark competitive inhibitor 1-methyl-L-tryptophan (1-L-MT) was identified in the early 1990s and has a reported Ki of 34 μM. However, 1-L-MT is a rather poor IDO-1 inhibitor when tested in IDO-1 expressing human tumor cells, as more than 200 μM 1-L-MT is required to block 50% of the IDO-1-mediated Trp degradation.20 Since 2006, the X-ray crystal structures of human IDO-1 in a complex with a ligand inhibitor (4-phenylimidazole (PI) or cyanide) have opened the door for the design of new IDO-1 inhibitors.21 Recently, several potent IDO-1 inhibitors have been identified by natural products isolation,22,23 structure-based modification,24,25 and silico drug design.26,27 However, to date, only two molecules have entered clinical trials, 1-methyl-D-tryptophan ( 1-D-MT ) and hydroxylamidine INCB024360 (http://clinicaltrials.gov/ ct2/results?term=1-methyl-D-tryptophan&Search=Search and http://clinicaltrials.gov/ct2/results?term=INCB024360& Search=Search). Therefore, there exists a continuing need for the development of new IDO-1 inhibitors. Tryptanthrin (indolo[2,1-b]quinazolin-6,12-dione) is a natural product of the Chinese medicinal plants Polygonum tinctorium and Isatis tinctoria. It has been reported that tryptanthrin has various biological activities, such as inhibitory activities against a variety of microorganisms and parasites,28,29 8322



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we measured its binding interaction with IDO-1 in vitro and examined its effect on tumor growth, IDO-1 activity, and the expression of IDO-1 and Foxp3 in Lewis lung cancer (LLC) tumor-bearing mice. Our results demonstrate that tryptanthrin and its derivatives are efficient IDO-1 inhibitors, which sheds light on the possible mechanism of their antitumor action.

Table 1. IDO-1 Inhibitory Activity of Tryptanthrins IC50 (μM)

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CHEMISTRY Syntheses of Tryptanthrin Derivatives. The structures of tryptanthrin derivatives are shown in Figure 1. Tryptanthrins (5a−h) were prepared by the condensation of substituted isatoic anhydrides with substituted isatins in toluene by refluxing in the presence of triethylamine (Scheme 1).41 Reaction of tryptanthrin with nitric acid in the presence of sulfuric acid afforded tryptanthrins 5i (Scheme 2).37 Reduced tryptanthrin products (6a−c) were obtained from appropriate tryptanthrins by reduction with NaBH4 (Scheme 3).42 Triazole-containing

a

compd

type of inhibition

Ki (μM)

rhIDO-1

1-L-MT 5a 5b 5c 5d 5e 5f 5g 5h 5i

competitive mixed competitive mixed competitive uncompetitive uncompetitive NDa uncompetitive uncompetitive uncompetitive uncompetitive

42.3 4.81 218.9 0.161 0.389 NDa 5.48 9.65 49.37 0.054

498.7 7.15 121.2 0.534 0.574 864.4 2.72 8.19 74.37 0.103

HEK 293-hIDO-1 18.4 5.37 × NDa 2.30 × 5.39 × NDa 0.466 2.71 × 0.183 1.80 ×

10−2 10−2 10−2

10−2 10−5

Not detected.

IDO-1 inhibitory activity than 5a (IC50 = 7.15 μM) (Table 1). The common characteristic of these tryptanthrin derivatives is that they all possess an electron-withdrawing group (such as fluorine, bromine, or nitro) at the 8-position, which seems to be important for their IDO-1 inhibitory activity. However, with a fluorine at the 2-position, 5e showed poorer activity than 1-LMT. A similar pattern of IDO-1 inhibitory activity was observed for reduced tryptanthrin products (Table 2). The derivative

Scheme 1. General Synthetic Path to Tryptanthrin Derivatives (5a−h, 14, 15)

Table 2. IDO-1 Inhibitory Activity of Reduced Tryptanthrin Products Scheme 2. General Synthetic Path to Tryptanthrin Derivatives (5i)

IC50 (μM)

a

Scheme 3. General Synthetic Path to Reduced Tryptanthrin Products (6)

compd

type of inhibition

Ki (μM)

rhIDO-1

HEK 293-hIDO-1

1-L-MT 6a 6b 6c

competitive uncompetitive NDa uncompetitive

42.3 8.58 NDa 4.54

498.7 16.0 451 6.21

18.4 0.779 NDa 2.43 × 10−2

Not detection.

with an 8-fluoro substituent (6c) had superior activity to the derivative with a 2-fluoro substituent (6b) or no substituent at either position (6a). As 1,2,3-triazoles were found to be a new key pharmacophore of potent IDO-1 inhibitors in our previous study,44 two triazole-containing tryptanthrins (Table 3) were Table 3. IDO-1 Inhibitory Activity of Triazole-Containing Tryptanthrins

tryptanthrin 14 was prepared from a triazole-containing isatin derivative with 5-fluoroisatonic anhydride. Substituting the triazole-containing isatin derivative for 5-fluoroisatin and 5-fluoroisatonic anhydride for triazole-containing isatin anhydride derivative gave tryptanthrin 15.43 Tryptanthrins (5, 6, 14, 15) are known compounds. Structural identities were confirmed by 1 H NMR and ESI-MS methods. All the spectra data were consistent with those previously reported. The tryptanthrins utilized in this study were dissolved at 1−2 mg/mL (3−8 mM) in dimethyl sulfoxide (DMSO) and stored at −80 °C.

IC50 (μM)

a

■

compd

type of inhibition

Ki (μM)

rhIDO-1

HEK 293-hIDO-1

1-L-MT 14 15

competitive NIa uncompetitive

42.3 NIa 63.3

498.7 NIa 39.1

18.4 NIa 0.185

No inhibition.

also synthesized, and their activities were evaluated. Compound 15 containing a fluorine at the 8-position and 4-phenyl-1,2,3triazol at the 2-position showed stronger IDO-1 inhibitory activity than that with 4-phenyl-1H-1,2,3-triazol (IC50 = 143 μM).44 However, a derivative with fluorine placed at the 2-position (14) showed no inhibitory activity. Overall, our data demonstrate that 11 tryptanthrins of the 14 being tested are potent IDO-1 inhibitors with higher activity than that of 1-L-MT. Detailed kinetics analysis, based on plotting a function of the rate (V) against the enzyme amount ([E]), was performed on

RESULTS AND DISCUSSION IDO-1 Inhibitory Activities of Tryptanthrins. To obtain the kinetics parameters (Ki) and IC50 values shown in Tables 1−3, tryptanthrin and its 13 synthesized derivatives (Figure 1) along with 1-L-MT were subjected to IDO-1 inhibition assays. Under the conditions, the IC50 of 1-L-MT was 498.7 μM. Most tryptanthrins displayed higher IDO-1 inhibitory activity than 1-L-MT. The tryptanthrin derivatives 5c, 5d, and 5i exhibited stronger 8323



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Figure 2. Characterization of tryptanthrins as highly potent IDO-1 inhibitors. Kinetics parameters of 1-L-MT and eight tryptanthrins (Ki fluorine > bromine, 5i > 5c > 5d). In conclusion, these results of the structure−activity analysis indicate that an electron-withdrawing group at the 8-position of tryptanthrin is necessary for IDO-1 inhibition. Cellular IDO-1 Inhibitory Activities of Tryptanthrins. To study the cellular IDO-1 inhibitory activity, HEK 293 cells expressing human IDO-1 were created and used for assays with tryptanthrins. The amount of Kyn released from the cells was measured with a microplate reader. All of the tested tryptanthrins displayed much higher inhibitory efficiencies in the cell-based assays than those in the enzyme assays (Tables 1−3). This might result from the complexity of enzyme assays, which utilized a methylene blue ascorbate regeneration system to maintain IDO-1 in an active reduced form, or from unidentified differences between the recombinant IDO-1 used in the enzyme assays and HEK 293 cells. In general, a good correlation between the two assays was observed, confirming that the

those tryptanthrins with more IDO-1 inhibitory potency than 1-L-MT. Eleven tryptanthrins were categorized as reversible inhibitors (Supporting Information Figure 1). As shown in Figure 2A, most tryptanthrins had a kinetics graphical mode that suggested uncompetitive inhibition except 5a and 5b, which were mixed type.45 The inhibitory kinetics parameters (Ki) were evaluated by plotting [S]/V against inhibitor concentration, where [S] represents the substrate concentration, and V represents the reaction velocity. As shown in Table 1, three strong IDO-1 inhibitors, 5c, 5d, and 5i, had Ki values in a nanomolar range while the Ki of 1-L-MT was 42.3 μM. In summary, our results indicated that three general structural features contributed to the IDO-1 inhibition activity of tryptanthrins. First, tryptanthrins containing an electronwithdrawing group at the 8-position (5c, 5d, and 5i) showed higher inhibitory potency relative to the analogue bearing the electron-donative group at the 8-position (5b). Second, tryptanthrins with an electron-withdrawing group at the 8-position (5c, 6c, and 15) increased their potency relative to those at the 2-position (5e, 6b, and 14). Furthermore, compared with 2-fluorinetryptanthrin (5e), 8-fluorinetryptanthrin (5c) exhibited highly potent IDO-1 inhibition activity. Introduction of another 8324



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tryptanthrins with a halogen or nitro at the 8-position were better IDO-1 inhibitors. Effects of Tryptanthrins on T Cell Proliferation. As an immune tolerance-promoting enzyme, IDO-1 contributes to the progression of tumors through its capacity to block T lymphocyte proliferation by depleting Trp locally. It has been reported that 1-L-MT can augment T cell function that is stimulated by LLC tumor cells.46 Here, to determine whether tryptanthrins could improve T cell proliferation in the presence of LLC cells, we performed T cell proliferation assay. As shown in Figure 3, each tryptanthrin derivative (Ki < 10 μM) and 1-L-MT Figure 4. Reduction of the DPPH radical by tryptanthrins and ascorbic acid. Data are the mean ± SEM, from triplicates.

Figure 3. Tryptanthrins enhanced the proliferation of T cells stimulated with LLC tumor cells. Among the eight tryptanthrins (Ki < 10 μM) with highly potent IDO-1 inhibitory activities, compound 5c exhibited the most significant induction of T cell proliferation. Each bar of the graph indicates the mean of three replicate wells with standard error of the mean. The experiment shown is representative of two experiments carried out separately.

Figure 5. SPR assay of the binding between human IDO-1 and 5c. SPR curve for the binding of IDO-1 with 5c is shown. The concentrations of 5c injected over the biosensor chip surface immobilized with IDO-1 protein are indicated. The measurement yielded a KD of 46.8 μM.

at 50 μM could enhance the proliferation of T cells stimulated with LLC cells. In accordance with their IDO-1 inhibitory activity, these eight tryptanthrins also showed more enhancement of T cell proliferation than 1-L-MT. Especially, the increase of T cell proliferation caused by compounds 5c and 5g was 5- to 6-fold more than that by 1-L-MT at the same concentrations. Our results clearly demonstrate that these eight tryptanthrins as highly potent IDO-1 inhibitors can reverse the suppression of T lymphocyte caused by IDO-1 to a great extent, but 1-L-MT has no significant effect on T cell proliferation at the same concentration. Evaluation of the Redox Potentials of Tryptanthrins. A major unresolved question is whether IDO-1 inhibitory activities of tryptanthrins are due to their redox potentials. Therefore, we evaluated the redox potentials of five most potent tryptanthrins by analyzing their radical scavenging properties in the well-recognized diphenylpicrylhydrazyl (DPPH) assay. All the tested tryptanthrins did not reduce the DPPH radical, whereas ascorbic acid acted as expected (Figure 4). Surface Plasmon Resonance Assay of 5c Binding to IDO-1. In order to more precisely validate 5c as potential IDO-1 inhibitor, we utilized surface plasmon resonance (SPR) assay to measure direct interaction between 5c and IDO-1. The equilibrium dissociation constant (KD) between 5c and IDO-1 was around 46.8 μM (Figure 5), which demonstrated its binding activity to purified IDO-1 protein. Effect of 5c on the Tumor Growth in LLC TumorBearing Mice. The combination of low molecular weight, high IDO-1 inhibitory activity, and significant enhancement of T cell proliferation made 5c the best candidate for further in vivo studies. To investigate whether IDO-1 inhibition could similarly reverse immune tolerance in vivo, we treated mice bearing LLC with an IDO-1 inhibitor (5c or 1-L-MT) for 2 weeks. Both 5c

and 1-L-MT retarded the growth of tumors; specifically, 5c reduced 62% of the tumor volume and 1-L-MT reduced 32% (Figure 6A). Mice treated with 5c had a significant reduction of the total tumor weight, while 1-L-MT treated mice showed less effect on the tumor degeneration (Figure 6B). Although 5c was incapable of eliciting tumor regression, it did effectively inhibit tumor growth. These results suggest the therapeutic potential of 5c as a single agent in the treatment of tumor. Effect of 5c on IDO-1 Activity and Expression in LLC Tumor-Bearing Mice. It is known that IDO-1 expression and/or activity is increased in cancer patients,47−49 and Trp degradation and Kyn production are the indicators of IDO-1 activity. Thus, to investigate IDO-1 activity, the Kyn and Trp levels in the plasma harvested from LLC tumor-bearing mice were examined by HPLC, and Kyn/Trp ratios were calculated. As shown in Figure 7A, the Kyn/Trp ratio was significantly lower in 5c treated mice than in control mice. To investigate the IDO-1 expression, the dissected tumor tissues were subjected to PCR, quantitative real-time PCR, and Western blot analysis. A certain amount of IDO-1 mRNA expression was observed in the control tumors, and the levels of IDO-1 mRNA expression were in a rising tendency in IDO-1 inhibitors treated tumors (Figure 7B), especially in 5c treated tumors. IDO-1 mRNA expression in 5c treated tumors was higher than that in the tumors treated by 1-L-MT which had lower IDO-1 inhibitory activity than 5c and was dramatically superior to the gene levels of the control tumors. The Western blot results showed that IDO-1 protein expression was slightly increased in the IDO-1 inhibitor treated tumors, although no prominent changes were detected (Figure 7C). In summary, the increased activity of IDO-1 was relieved by the administration of 5c; 8325



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that is, when partial IDO-1 activity is inhibited, additional IDO1 may be expressed in order to meet special circumstances. Similarly, the influence of 1-D-MT on IDO-1 expression in human cancer cells50 and rhesus macaques has been demonstrated and interpreted.51 However, it was reported that INCB024360, another IDO-1 inhibitor tested in clinical trials, completely blocked IDO-1 enzymatic activity but did not change IDO-1 expression in LPS- or IFN-γ-treated DCs.52 This different impact on IDO-1 expression in these studies may result from the significant difference of IDO-1 expression between in vitro and in vivo systems, given the complicated environment in vivo. Effect of 5c on Foxp3+ Tregs in LLC Tumors. It has been reported that IDO-1-mediated Trp catabolism, which involves both growth-essential Trp degradation and proapoptotic Trp catabolic production, decreased effector T-cell proliferation and increased effector T-cell apoptosis.13,14 In addition, IDO-1mediated Trp catabolism facilitates the conversion of naive T lymphocytes into CD4+CD25+ Tregs,53 which are essential for the active suppression of autoimmunity. Recent studies have shown that the transcription factor forkhead box P3 (Foxp3) is not only a key intracellular marker but also a crucial developmental and functional factor for CD4+CD25+ Tregs.16,54 Therefore, we evaluated Foxp3 mRNA expression in LLC tumorbearing mice in order to elucidate its effect on the Foxp3+ Tregs (Figure 8A). PCR and quantitative real-time PCR analyses revealed that Foxp3 mRNA expression was significantly reduced in 5c treated tumors and less reduced in 1-L-MT treated tumors. Similar results were obtained by Western blot analysis using affinity-purified rabbit anti-mouse Foxp3 antibody. These results indicate that 5c, a highly potent IDO-1 inhibitor, can significantly reduce the numbers of Foxp3+ Tregs in LLC tumor-bearing mice. Immunohistochemistry Analysis for IDO-1 and Foxp3 Expression. IDO-1-mediated Trp degradation is not only restricted to tumor cells; it is also detected in key sites where an immune system encounters tumor antigens, such as tumor draining lymph nodes (TDLNs). A previous study reported that high IDO-1 levels are expressed in a subset of DCs in murine TDLNs.55 To further investigate IDO-1 expression in both tumors and TDLNs, we conducted an immunohistochemical analysis (Figure 9A). As shown, IDO-1 expression was observed in the cytoplasm of these two locations in LLC

Figure 6. Single-agent 5c suppressed tumor growth effectively. C57BL/6 mice bearing LLC tumors were treated orally once daily with vehicle, 5c, or 1-L-MT at a dose of 200 mg/kg once the primary tumor diameter had reached 5 mm. (A) Mean tumor volume of vehicle (rhombuses), 5c-treated mice (rectangles), or 1-L-MT-treated mice (triangles) (n = 9−10 mice/group). The tumor size was measured in regular intervals: (∗) P < 0.05, (∗∗) P < 0.01. (B) Mean tumor weight of each group (n = 9−10). Tumors were dissected 24 h after the last administration: (∗) P < 0.05, (∗∗) P < 0.01.

however, the expression of IDO-1 was unexpectedly increased at the same time. It is interesting to find that the expression of IDO-1 mRNA and protein was increased in both 5c and 1-L-MT treated tumors in vivo. To our knowledge, this is the first report of 1-LMT-mediated effects on IDO-1 expression in vivo. One possible explanation for this observation is that a compensatory counter-regulatory mechanism is activated by IDO-1 inhibitors;

Figure 7. 5c treatment led to decreased IDO-1 activity and increased IDO-1 expression. (A) After 14 days of treatment, plasma was harvested. Kyn and Trp levels were determined by HPLC, and the Kyn/Trp ratios were calculated (n = 9−10): (∗) P < 0.05, (∗∗) P < 0.01. (B) IDO-1 mRNA levels increased significantly in LLC tumors of treated mice: (top) IDO-1 mRNA levels evaluated by polymerase chain reaction (PCR); (bottom) IDO-1 mRNA levels analyzed by quantitative real-time PCR. Column values represent the mean from triplicate measurements with multiple samples. (C) Effects of IDO-1 inhibitors on IDO-1 protein expression, evaluated by Western blot: (top) representative result of three independent experiments; (bottom) expression changes of IDO-1 in tumors shown in the bar graph. 8326



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suppresses tumor growth, and reduces the numbers of Foxp3+ Tregs evidently. Taken together, these results lead us to propose that tryptanthrin derivative 5c is an attractive small molecule immunomodulatory IDO-1 inhibitor that may be used as an immunotherapeutic agent to resist the immune tolerance of the tumor microenvironment and to prevent tumor escape from immune surveillance and destruction.

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EXPERIMENTAL SECTION

General Information. Purity tests by analytical HPLC used a LC10AVP HPLC system equipped with a LC-10AT pump and a SPD10A UV detector (Shimadzu, Kyoto, Japan). The detection wavelength was 254 nm. HPLC analysis of the test compounds was performed using a YMC ODS C18 column (150 mm ± 4.2 mm i.d., 5 μm, Japan) preceded by a C18 guard column (Dikma, China). The column temperature was maintained at 35 °C at a flow rate of 1 mL/min. The mobile phase was a mixture of acetonitrile and 2% ethanoic acid (40:60, v/v). The purity of tested compounds was confirmed as >95%. Syntheses of Tryptanthrin Derivatives. The syntheses were performed as reported previously37,53,55 and described in Supporting Information. IDO-1 Inhibition Assay. The assay was performed as described previously.56 T Cell Proliferation Assay. T cell proliferation assay was performed as previously described46 with some modification. T lymphocytes prepared from splenocytes of BALB/c mice were resuspended in RPMI 1640 containing 10% FBS, L-glutamate, penicillin, and streptomycin. The LLC cells were treated with mitomycin C at a final concentration of 25 mg/L and then incubated at 37 °C for 30 min. After being washed three times, the LLC cells were resuspended in RPMI 1640 containing 10% FBS, L-glutamate, penicillin, and streptomycin. 1 × 105 T lymphocytes (responder cells) and 2 × 104 mitomycin C treated LLC cells (stimulator cells) were added to each well of a 96-well plate in RPMI 1640 containing 10% FBS. Cell proliferation was quantified by WST-1 (KenGEN) method, which is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells to a water-soluble formazen dye. The cells were incubated at 37 °C and 5% CO2 for 4 days; and 10 μL WST-1 was added to each well. At 1 h later, the cells were shaken thoroughly for 1 min on a shaker. The absorbance was measured using a SPECTRAmax 250 microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm. Diphenylpicrylhydrazyl (DPPH) Assay. The antioxidant activities of tryptanthrins were assessed by the literature method,57 with slight modifications. Briefly, an amount of 100 μL of 5, 10, 20, 40, 80, or 160 μM tryptanthrins in DMSO was added to 100 μL of a solution of the stable free radical DPPH in ethanol (100 μM), buffered with acetate to pH 5.5, in a 96-well plate. The absorbance was recorded at 517 nm after a 30 min incubation under gentle shaking in the dark. Ascorbic acid was used as reference compound. All analyses were performed in triplicate. SPR Analysis. The surface plasmon resonance (SPR) was carried out at 25 °C on Biacore T100 instruments using sensor chip CM5 (GE Healthcare). PBS was used as running buffer with 0.1% dodecylmaltoside, 0.05 mM EDTA, and 5% DMSO, pH 7.4. The KD value of the tested compound was determined by BIA evaluation software (GE Healtthcare). Animal Model and Treatments. Eight-week-old C57BL/6 mice used for the study were purchased from Shanghai Laboratory Animal Center, CAS. LLC cells were inoculated sc into the right forelimb at 2 × 106 of each mouse. After the implantation, the mice were randomized into three groups: vehicle, 5c treated, and 1-L-MT treated. Tumor growth was monitored every 2 days. Perpendicular diameters of the tumors were measured using vernier scale calipers, and the tumor volume was calculated as follows: tumor size = long diameter × (short diameter)2/2. When the primary tumor diameter reached 5 mm, therapy was initiated. C57BL/6 mice bearing LLC tumors were treated orally once daily with either 0.5% carboxymethylcellulose sodium (0.5% CMC) or 200 mg/kg IDO-1 inhibitor (5c or 1-L-MT).

Figure 8. 5c treatment led to decreased Foxp3 mRNA and protein expression in LLC tumor-bearing mice. (A) Foxp3 mRNA levels decreased after 5c treatment: (top) Foxp3 mRNA expression evaluated by polymerase chain reaction (PCR); (bottom) Foxp3 mRNA levels analyzed by quantitative real-time PCR. Column values represent the mean from triplicate measurements with multiple samples. (B) Effects of IDO-1 inhibitors on Foxp3 protein expression evaluated by Western blot: (top) representative result of three independent experiments; (bottom) expression changes of Foxp3 in the tumors shown in the bar graph.

tumor-bearing mice. The images shown are representative figures of each group. The IDO-1 expression levels were upregulated in the tumors treated with IDO-1 inhibitors, especially by 5c, which is consistent with the results shown in Figure 7. High expression levels of IDO-1 were also observed in the TDLNs but with no obvious difference between the groups. Additionally, Foxp3+ Tregs were detected in both tumors and TDLNs by positive nuclear staining of Foxp3 (Figure 9B,C). As shown in Figure 7B, the numbers of Foxp3+ Tregs were reduced in the LLC tumors treated with IDO-1 inhibitors, especially with 5c, which may result in the relief of antitumor immune suppression. Foxp3+ Tregs were also detected in the TDLNs with relative fewer amounts than those in the tumors and no significant difference between the groups (Figure 9C).

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CONCLUSIONS Tryptanthrin is a potent indole-based IDO-1 inhibitor, which may present a novel mechanism of its antitumor activity. Modification of tryptanthrin affords a highly potent IDO-1 inhibitor 5c, which exhibits the direct binding to IDO-1, nanomolar IDO-1 inhibitory activity, and significant immunoregulatory activity of promoting T cell proliferation. When administrated to the LLC tumor bearing mice, 5c inhibits IDO-1 activity, 8327



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Figure 9. IDO-1 and Foxp3 expression examined by immunohistochemistry. (A) Fluorescent staining of IDO-1: red, IDO-1+; blue, cell nuclei. IDO-1 was detected in both tumors and TDLNs. IDO-1 expression increased in the tumors treated with the IDO-1 inhibitors, 1-L-MT, and 5c. No obvious difference was observed in the TDLNs between the groups. Original magnification was ×200. (B) Fluorescent staining of Foxp3: green, Foxp3; blue, cell nuclei. Foxp3 was detected in tumors. Less Foxp3 was detected in IDO-1 inhibitor treated tumors, especially in 5c treated tumors. Original magnification was ×200. (C) Fluorescent staining of Foxp3: green, Foxp3; blue, cell nuclei. Foxp3 was detected in TDLNs. No significant difference was observed in the TDLNs between the three groups. Original magnification was ×200. gel with β-actin as an internal standard. For quantitative real-time PCR, the target genes were quantified using SYBR Premix ExTag quantitative PCR kit (Takara, Otsu, Japan) and a BioRad iQ5 system, with β-actin as an internal standard. The samples were analyzed in triplicate, and the expression of target genes was normalized to β-actin. Relative RNA expression levels were determined using the 2−ΔΔCt method. The primer sequences were as follows: Foxp3, forward, 5′-GGGAGCAGTGGACCGTAG-3′, reverse, 5′-CCACAGCCTCAGTCTCATGGT-3′; IDO-1, forward, 5′-CTTCCTCGTCTCTCTATTGG-3′, reverse, 5′-CAGAAGGACATCAAGACTCTG-3′. Western Blot Analysis. Mouse tumors were dissected, pulverized, and resuspended in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate) containing 5 mM EDTA, and a protease inhibitor cocktail from Thermo. Lysates were kept on ice for 30 min, vortexed, disrupted by a 30 s burst of supersonication, and centrifuged at 7500 rpm for 20 min at 4− 8 °C. Supernatants were collected and quantified for protein levels using the bicinchoninic acid method. Briefly, supernatants were boiled in 2× SDS loading buffer, separated by 10% SDS−PAGE, and followed by Western blot using a polyclonal rabbit Foxp3 Ab (catalog no. ab54501, Abcam) and a secondary Ab of anti-rabbit IgG-HRP (Vector Laboratories, Inc.). The expression of IDO-1 was detected by a monoclonal mouse IDO-1 Ab (catalog no. sc-137012, SANTA) and a secondary Ab of anti-mouse IgG-HRP (Vector Laboratories, Inc.).

IDO-1 inhibitors were dissolved in 0.5% CMC. After 14 days of treatment, the mice were sacrificed and the tumors were dissected and weighed. All experimental protocols were approved by the institutional Animal Studies Committee, and all murine experiments were conducted in compliance with institutional guidelines for the use of research animals. The statistical analysis was carried out with SPSS statistical software, version 18.0. The one-way ANOVA method was used to determine the statistical significance of the differences observed between the vehicle and the IDO-1 inhibitor-treated groups of mice. HPLC Analysis. IDO-1 enzyme activity was evaluated by measuring the concentrations of Trp and Kyn in the plasma using a LC-10AVP HPLC system equipped with a LC-10AT pump and a SPD-10A UV detector (Shimadzu, Kyoto, Japan). The mobile phase was 15 mM sodium acetate containing 8% acetonitrile. PCR and Quantitative Real-Time PCR. The expression of the IDO-1 and Foxp3 mRNAs in the tumors was evaluated by polymerase chain reaction (PCR) and quantitative real-time PCR. After the tumors were dissected, total RNA was isolated using the Trizol reagent (Invitrogen, Japan). Subsequently, reverse transcription was performed to obtain cDNA. cDNA was then amplified by PCR using specific primers: IDO-1, forward, 5′-CTTCCTCGTCTCTCTATTGG-3′, reverse, 5′-CAGAAGGACATCAAGACTCTG-3′; Foxp3, forward, 5′-GGGCGATGATGTGCCTGCTA-3′, reverse, 5′-GGTTGTGAGGGCTCTTTGACTGG-3′. The PCR products were separated on a 1% agarose 8328



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Blotting for β-actin using a mouse mAb of corresponding specificity (catalog no. 60008, Proteintech) served as a loading control. Immunohistochemistry Analysis. Tumors and lymph nodes that were dissected from mice were fixed in formalin and embedded with paraffin. The tissue sections were cut into 5 μm slices, and heat mediated antigen retrieval was performed prior to staining at room temperature. To test the sections, rabbit Foxp3 Ab (dilution 1/100; catalog no. ab54501, Abcam) or mouse IDO-1 Ab (dilution 1/50; catalog no. sc-137012, SANTA) was first added for 1 h and then Alexa Fluor secondary Ab (Invitrogen, Carlsbad, CA) for 30 min. Images were taken at ×200 magnification.

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ASSOCIATED CONTENT

In vitro biology assay protocols and syntheses procedures. This material is available free of charge via the Internet at http:// pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: +86-21-65643446. Fax: 86-21-65643446. E-mail: [email protected]. Author Contributions §

S.Y. and X.L contributed equally to this work. S.Y. performed all of the biochemical, cellular, and animal experiments. X.L. contributed to animal experiments, DPPH assay, and SPR analysis and partially performed cellular experiments. Y.L. contributed to the SPR experiment. F.H. and C.K. synthesized tryptanthrins and analyzed chemistry and structure-related results. Y.Y. and J.Y. partially contributed to animal experiments. Q.Y. initiated the research, led the project team, designed experiments, analyzed results, and wrote the manuscript.

Notes

The authors declare no competing financial interest.

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REFERENCES

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S Supporting Information *

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ACKNOWLEDGMENTS

We thank Yang Li and Juanjuan Li for help with the DPPH assay. This work was sponsored by the National Natural Science Foundation of China (Grant 81373396), the National Basic Research Program of China (973 Program) (Grant 2010CB912600), and the Key Biomedical Program of Shanghai (Grant 12431900204).

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ABBREVIATIONS USED IDO-1, indoleamine 2,3-dioxygenase; Tregs, regulatory T cells; Trp, tryptophan; Kyn, kynurenine; KP, kynurenine pathway; QUIN, quinolinic acid; 1-L-MT, 1-methyl-L-tryptophan; PI, 4-phenylimidazole; LLC, Lewis lung cancer; HEK 293, human embryonic kidney 293 cells; PDB, Protein Data Bank; 1-D-MT, 1-methyl-D-tryptophan; LPS, lipopolysaccharide; IFN-γ, interferon γ; DC, dendritic cell; Foxp3, forkhead box P3; TDLN, tumor draining lymph node; WST-1, 2-(4-iodophenyl)-3-(4nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt; FBS, fetal bovine serum; DPPH, diphenylpicrylhydrazyl; SPR, surface plasmon resonance; CMC, carboxymethylcellulose sodium; Tris-HCl, tris(hydroxymethyl)aminomethane hydrochloride; NaCl, sodium chloride; EDTA, ethylenediaminetetraacetic acid; Ab, antibody; HRP, horseradish peroxidase 8329



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