Effects of Various Phytochemicals on Indoleamine 2,3-Dioxygenase 1 Activity: Galanal Is a Novel, Competitive Inhibitor of the Enzyme

Indoleamine 2,3-dioxygenase (IDO) 1, that catalyzes the first and rate-limiting step in the degradation of L-tryptophan, has an important immunomodulatory function. The activity of IDO1 increases in various inflammatory diseases, including tumors, autoimmune diseases, and different kinds of inflammation. We evaluated the suppressive effect of plant extracts or phytochemicals on IDO1 induction and activity; sixteen kinds of plants extracts and fourteen kinds of phytochemicals were examined. As a result, the methanol extracts of Myoga flower buds, which are traditional Japanese foods, and labdane-type diterpene galanal derived from Myoga flowers significantly suppressed IDO1 activity. The Lineweaver-Burk plot analysis indicated that galanal is a competitive inhibitor. Galanal attenuated L-kynurenine formation with an IC50 value of 7.7 µM in the assay system using recombinant human IDO1, and an IC50 value of 45 nM in the cell-based assay. Further, mechanistic analysis revealed that galanal interfered with the transcriptional function of the nuclear factor-κB and the interferon-γ signaling pathway. These effects of galanal are important for immune response. Because the inhibitory effect of galanal on IDO1 activity was stronger than that of 1-methyl tryptophan, a tryptophan analog, galanal may have great potential as the novel drug for various immune-related diseases.

The metabolism of L-Trp via IDO1 is accompanied by the production of a series of immunoregulatory metabolites, collectively known as ''kynurenines,'' which can suppresses the proliferation and differentiation of effector T cells [4], and markedly enhance the suppressor activity of regulatory T cells [5]. As a result, IDO1 controls and fine-tunes both innate and adaptive immune responses [6] under a variety of conditions, including pregnancy [7], transplantation [8], infection [9], chronic inflammation [10], autoimmunity [11], neoplasia, and depression [12]. Owing to the outstanding immune-modulate properties of IDO1, IDO1 inhibitors have been searched for in many fields, to control various inflammatory diseases. Thus, it is hoped that the inhibitor of IDO1 becomes the new therapeutic target for drugs corresponding to various inflammatory diseases [13] [14].
Previous researches have given direct evidence of the crucial role of natural products from plants, animals, and microorganisms as potential sources of various modern pharmaceuticals. Currently, phytochemical research is being considered an effective approach in the discovery of novel chemical entities, with potential as drug leads. Previous reports have shown that some food compounds such as epigallocatechin gallate (EGCg; CID 65064) and curcumin (CID 969516) inhibit the induction of IDO1 [15] [16]. Therefore, we extracted various compounds from traditional Japanese foods and plants. The purpose of this study was to find a novel effective inhibitor of IDO1 from food and plant compounds. We examined the inhibitory effects of fourteen kinds of plant extracts and sixteen kinds of phytochemicals on the induction of IDO1. Among these compounds, we found that galanal (CID 3050416) isolated from the methanol extract of Myoga flower buds was the most effective inhibitor of IDO1.

Cell culture
Human acute leukemic cells, THP-1, and Human embryonic kidney, HEK293, were maintained in RPMI-1640 or DMEM medium supplemented with 10% FCS, at 37uC in a humid atmosphere of 5% CO 2 . Cells (1610 6 ) were treated with phytochemicals (10 mM) or plant extracts (30 mg/ml), and LPS (50 ng/ml) for 24 hrs. Measurement of L-Kyn L-Kyn in each conditioned medium was measured by the method using high-performance liquid chromatography (HPLC) with a spectrophotometric detector (SHIMADZU, Prominence UFLC), as described in our previous reports [18] [19].

Expression and purification of recombinant IDO1
The human IDO1 cDNA was expressed in E. coli, and purified by a Ni 2 -column by affinity-binding to the N-His-tag of recombinant IDO1, as described in our previous reports [20]. The resultant IDO1 was enzymatically active when assayed using L-Trp as a substrate. Therefore, this purified IDO1 was used for monitoring IDO1 activity. It is stored at 280uC until use.
Enzyme assay for rIDO1 IDO1 activity was determined by the methylene blue/ascorbate assay as previously described [3]. The reaction mixture contained 50 ml of rIDO and 50 ml of substrate solution. The composition of the substrate solution was 100 mM potassium phosphate buffer (pH 6.5), 50 mM methylene blue, 20 mg of catalase, 50 mM ascorbate, and 0.4 mM L-Trp. After incubating the reaction mixture at 37uC for sixty minutes, samples were acidified with 3% perchloric acid and centrifuged at 70006g for 10 min at 4uC. The concentrations of the enzymatic products were measured using HPLC. The type of IDO1 inhibition by galanal was determined from the plot of enzyme kinetics.

RNA Extraction and semi-quantitative analysis of RT-PCR products
The total RNA was extracted from cell lines with ISOGEN (Nippon GENE, Tokyo, Japan) and the RNA concentration was determined spectrophotometrically at 260 nm. Reverse transcription-PCR was performed by using Revetra Ace Kits (TOYOBO, Japan). Primers used in semi-quantitative analysis were as follows-GAPDH: 59-accacagtccatgccatcac (sense) and 59-tccaccaccctgttgctgta (antisense); IDO1: 59-cctgacttatgagaacatggacgt (sense) and 59-atacaccagaccgtctgatagctg (antisense). Semi-quantitative analysis of RT-PCR products was performed by using NIH ImageJ 1.34 s software and normalized to GAPDH. When the same experiment was repeated three times, the three experiments showed similar results. Therefore, the results obtained from one of the three experiments were shown.

Protein phosphorylation
Protein phosphorylation was examined with using Phospho-Tracer ELISA kit (Cambridge, UK). Each experiment was performed according to the manufacture protocols.

Statistical analysis
Values obtained, except the kinetic study, are expressed as means 6 SD. Statistically significant differences (p,0.05) were analyzed by one-way ANOVA, followed by post-hoc test with Tukey-test.
The enzymatic assay of IDO1 in the presence or absence of either each plant extract or each phytochemical was performed in duplicate or triplicate; each assay was repeated at least two or three times. Based on the replicate assays of IDO1 activity in the presence or absence of galanal, the average values of the activities obtained in the replicate assays were shown in all Figures. Each value of IDO1 activity in the replicate assays varied within less than 5% of the average value of the activities.

Effects of plant extracts and phytochemicals on induction of IDO1
First, the effects of fourteen kinds of plants extracts and sixteen kinds of phytochemicals on IDO1 induction were examined, that is, the expression of IDO1 at its mRNA level was examined in LPS stimulated THP-1 cells treated with and without fourteen kinds of plants extracts and sixteen kinds of phytochemicals ( Figure 1A). Among them, five kinds of plants extracts and five kinds of phytochemicals were selected because they had an inhibitory effect on the expression of IDO1 at its mRNA level ( Figure 1A). The selected five phytochemicals were luteolin, quercetin, andrographolide, curcumin and galanal. The selected five plant extracts were the extracts of angelica, chrysanthemums, burock, zeodoary, and andrographis. Second, the inhibitory effects of the selected five plant extracts and five phytochemicals on the production of L-Kyn from L-Trp in LPS-stimulated THP-1 cells were examined by measuring the concentration of L-Kyn using HPLC. As shown in Figure 1B, the levels of L-Kyn produced via IDO1 in LPSstimulated THP-1 cells were reduced extensively by some compounds such as quercetin, luteolin, curcumin, andrographolide and galanal. Thus, andrographolide and galanal present in the extract of Myoga were able to inhibit IDO1 induction. Galanal, especially, had a strong inhibitory effect on IDO1 induction.
Because quercetin, luteolin, curcumin, andrographolide and galanal clearly showed an inhibitory effect on IDO1 induction, we further examined these compounds using IDO1-overexpressed HEK293 cells, to see whether they inhibited the enzymatic activity of IDO1 or not ( Figure 1C). Among the five compounds, galanal showed a significant inhibition of the enzymatic activity of IDO1 in IDO1-overexpressed in HEK293 cells.

Inhibitory effect of galanal on IDO1 activity in different cell species
The inhibitory effect of galanal on IDO1 activity was examined in human IDO1-overexpressed cells and mouse IDO1-overexpressed cells in order to clarify the specificity of the compound on IDO1 species. When human IDO1-overexpressed cells and mouse IDO1-overexpressed cells were treated with various concentrations of galanal (1-5 mM), galanal inhibited both enzymatic activities of human IDO1 and mouse IDO1 in a dose-dependent manner ( Figure 2).

Kinetic analysis for IDO1 inhibition by galanal
Results of the kinetic studies of IDO1 using two concentrations (0 and 30 mM) of galanal were plotted on the Michaelis-Menten model; galanal lowered the Vmax 0.88 folds determinations when Trp was varied, but it had pronounced effects on the apparent Michaelis constants in Figure 3A (Km = 1/2 Vmax, Control; 52.6 mM, galanal; 74.5 mM).
The mode of IDO1 inhibition by galanal was examined using the Lineweaver-Burk plot ( Figure 3B). Galanal inhibited the activity of the purified recombinant human IDO1 with an increase in its concentration and caused an almost complete inhibition of the enzymatic activity at 30 mM; the IC 50 value of galanal was 7.7 mM, proving that it inhibited IDO1 activity in a competitive manner ( Figure 3C).

Effect of galanal on the NFk-B signal transduction pathway
As shown in Figure 4, galanal inhibited IDO1 induction in LPSstimulated THP-1cells, contingent on the dose. The inhibitory ability of galanal was confirmed by measuring the levels of both L-Kyn production and IDO1 mRNA expression ( Figure 4A and B). Galanal decreased the expression of IDO1 mRNA in a dosedependent manner, suggesting that galanal could inhibit signal transduction by LPS stimulus. It is known that LPS stimulus upregulates IDO1 expression through the NFk-B signal transduction pathway [2,3]. To confirm the effect of galanal on the NFk-B signal transduction pathway, IkB-a expression and the phosphorylation of IKK-a, IkB-a, and NFk-B were examined. Galanal did not decrease the IkB-a expression in LPS-stimulated THP-1 cells ( Figure 4C). The phosphorylation of IKK-a, IkB-a, and NFk-B were down-regulated by Galanal ( Figure 4D). Galanal was thus found to interfere with the transcriptional function of the NFk-B signaling pathway by reducing the degradation of signal transducer IkB-a.

Effect of galanal on the IFN-c signal transduction pathway
Galanal inhibited both L-Kyn production and IDO1 mRNA induction in IFN-c-stimulated THP-1cells ( Figure 5A and B). The phosphorylation of stat1 was down-regulated by Galanal ( Figure 5C). Furthermore, galanal interfered with the transcriptional function of the IFN-c dependent pathway.

Inhibition of cellular IDO1 activity by galanal
Cellular IDO1 inhibition by galanal was examined in LPSstimulated THP-1 cells. As shown in Figure 6, galanal caused a clear inhibition of the cellular IDO1 activity and when the percentage of inhibition of IDO1 activity by galanal in LPSstimulated THP-1 cells against control cells was plotted, the IC 50 value was found to be 45 nM.

Discussion
IDO1 is recognized as one of the prominent mediators of immune regulation by metabolic pathways. Therefore, the regulation of the enzymatic activity of IDO1 is useful in the treatments of tumor, inflammatory diseases, and depression. Previous data have suggested that IDO1 inhibition can modulate immune response, delay tumor growth, and enhance dendritic cell vaccines [21] [22]. A frequently used IDO1 inhibitor is 1-methyltryptophan (1-MT). Several studies have provided evidence that IDO1 inhibition with 1-MT or other small molecule inhibitors can exert antitumor effects [23] [24]. Initial evidence, in 2002, showed that IDO1 inhibitor 1MT could partly retard growth of mouse melanoma cells engrafted onto a syngeneic host [25]. A recent report showed that 1-MT with an anticancer drug, improved tumor prognosis [26]. It seems that D-1-methyl-tryptophan (D-1- MT) selected for phase I trials has immune modulating activity in the United States [24]. However, some reports have shown that D-1-MT inhibits IDO2, a recently identified IDO1 isoform, more selectively than IDO1 [27] [28] [29]. The mechanisms by which D-1-MT inhibits IDO1 and IDO2 have yet to be cleared.
Considerable attention has been paid to the research of effective IDO1 inhibitors [30][31] [32]. The IDO1 inhibitor search was based on indole-based structures, combination of docking-based pharmacophore model development, and several natural products. Among the IDO1 inhibitors studied so far, it was seen that phytochemicals such as EGCG and curcumin inhibited the activity of IDO1 [15,16]. To find a new, effective IDO1 inhibitor, we examined the ability of various phytochemicals to inhibit IDO1 activity: galanal was found to have a strong ability to inhibit IDO1 activity in a competitive manner. Galanal is extracted from Myoga, a perennial herb with pungently aromatic flower buds, native to Eastern Asia. In Japan, the flower buds of Myoga are used as a spice and eaten as pickles. The labdane-type diterpene trialdehydes, 12(E)-lab-dene-15,16,(8b),17-trial(miogatrial), galanal A, and galanal B, are found in Myoga flower buds and exhibit certain biological activities [17] [33].
Though many inhibitors for IDO1 have been found, there are few inhibitors with competitive inhibition. In the present study, galanal was found to possess more IDO1 inhibitory potency than either the most-commonly used 1-MT or other previouslypublished inhibitors (  [43]. Since the IC 50 value of inhibitor depends on the IDO1 enzyme activity in each experiment, we could not unconditionally compare the previous results. However, these results showed that galanal is an effective IDO1 inhibitor because a very low dosage of it showed inhibitory activity in the cell-based assay.
IDO1 is known to be induced by both the IFN-c -dependent pathway and the IFN-c-independent pathway, i.e., the NF-kBdependent pathway [2] [3]. Galanal inhibited not only the enzymatic activity of IDO1 but also suppressed the signal transduction associated with the expression of IDO1 mRNA. Namely, a very low dose of galanal inhibited the expression of IDO1 mRNA induced by the NF-kB-dependent pathway clearly. In addition, it was suggested that galanal could inhibit the expression of IDO1 mRNA induced by the IFN-c -dependent pathway. NFk-B is activated by various stimuli such as free radicals, inflammatory stimuli, cytokines, carcinogens, tumor promoters, endotoxins, radiation, and ultraviolet light [44]. Upon activation, NFk-B induces the expression of more than 200 genes known to suppress apoptosis and also induces cellular transformation, proliferation, invasion, metastasis, chemoresistance, radioresistance, and inflammation [45]. Many of the target genes that are activated are critical to the establishment of inflammation and tumor [46]. Therefore, control of the NFk-B signal transduction may be useful for treatments of tumor and inflammatory diseases.
Our results have shown that galanal is an effective compound in immunomodulation, because the compound inhibits both the certain signal transduction and enzymatic activity of IDO1 at a very low dose.

Conclusion
The results of the present study indicate that galanal is a competitive IDO1 inhibitor. The results also indicate that a very low dose of galanal inhibits not only the enzymatic activity of IDO1 but also the expression of IDO1 mRNA induced by the NF-kB-dependent pathway. Furthermore, the results suggest that galanal could inhibit the expression of IDO1 mRNA induced by the IFN-c -dependent pathway. Therefore, galanal and its derivatives may be useful for the treatment of inflammatory diseases.