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Discovery and Characterization of Natural Products As Novel Indoleamine 2,3-Dioxygenase 1 Inhibitors Through High-Throughput Screening

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Discovery and Characterization of Natural Products As Novel Indoleamine 2,3-Dioxygenase 1 Inhibitors Through High-Throughput Screening www.nature.com/aps ARTICLE Discovery and characterization of natural products as novel indoleamine 2,3-dioxygenase 1 inhibitors through high-throughput screening Wei Guo1,2, Sheng Yao3,4, Pu Sun1,2, Tian-biao Yang4, Chun-ping Tang3,4, Ming-yue Zheng4, Yang Ye3,4,5 and Ling-hua Meng1,2 Indoleamine 2,3-dioxygenase 1 (IDO1) is emerging as a promising therapeutic target for the treatment of malignant tumors characterized by dysregulated tryptophan metabolism. However, the antitumor efficacy of existing small-molecule IDO1 inhibitors is still unsatisfactory, and the underlying mechanism remains largely undefined. To identify novel IDO1 inhibitors, an in-house natural product library of 2000 natural products was screened for inhibitory activity against recombinant human IDO1. High- throughput fluorescence-based screening identified 79 compounds with inhibitory activity > 30% at 20 μM. Nine natural products were further confirmed to inhibit IDO1 activity by > 30% using Ehrlich’s reagent reaction. Compounds 2, 7, and 8 were demonstrated to inhibit IDO1 activity in a cellular context. Compounds 2 and 7 were more potent against IDO1 than TDO2 in the enzymatic assay. The kinetic studies showed that compound 2 exhibited noncompetitive inhibition, whereas compounds 7 and 8 were graphically well matched with uncompetitive inhibition. Compounds 7 and 8 were found to bind to the ferric-IDO1 enzyme. Docking stimulations showed that the naphthalene ring of compound 8 formed “T-shaped” π–π interactions with Phe-163 and that the 6-methyl-naphthalene group formed additional hydrophobic interactions with IDO1. Compound 8 was identified as a derivative of tanshinone, and preliminary SAR analysis indicated that tanshinone derivatives may be promising hits for the development of IDO1 inhibitors. This study provides new clues for the discovery of IDO1/TDO2 inhibitors with novel scaffolds. Keywords: indoleamine 2,3-dioxygenase 1 inhibitor; natural product library; high-throughput fluorescence-based screening Acta Pharmacologica Sinica (2020) 41:423–431; https://doi.org/10.1038/s41401-019-0246-4 INTRODUCTION macrophages, myeloid-derived suppressor cells, and dendritic Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygen- cells. In contrast, TDO2 is predominantly expressed in the liver, ase 2 (TDO2) are intracellular heme-containing metalloproteins where it has a key role in maintaining the systemic homeostasis of that initiate the first and the rate-limiting steps of tryptophan the tryptophan levels [3]. The expression of IDO1 is greatly breakdown along the kynurenine pathway [1]. IDO is ubiquitously elevated in multiple types of human cancer and is correlated with expressed in many types of cells and tissues, with the highest a poor prognosis [4–6]. Recently, IDO1 has been shown to play an expression occurring in antigen-presenting cells, such as macro- important role in the process of immune evasion by tumors [7]. phages and dendritic cells. Two homologs, IDO1 and IDO2, have The IDO-mediated depletion of local tryptophan levels and been found in the IDO family, and IDO1 is by far the better the production of toxic metabolites results in the suppression of characterized of the two, and it has a central role in mediating T-cell responses and the enhancement of immunosuppression immune privilege and preventing T-cell-driven rejection of mediated by regulatory T cells [8, 9]. allogeneic fetuses during pregnancy [2]. Although the sequence As such, IDO1 has emerged as a promising therapeutic target, similarity between human IDO1 and TDO2 is poor (16%), the high prompting searches for highly active inhibitors. The landmark similarity of their catalytic domains has been revealed by protein competitive inhibitor 1-methyl-L-tryptophan (1-L-MT) was identi- crystallography studies. Although TDO2 is specific for metaboliz- fied in the early 1990s and has a reported Kiof34μM. However, ing tryptophan to kynurenine, IDO1 recognizes a broad range of 1-L-MT is a rather poor IDO1 inhibitor when tested in IDO1- indole-containing substrates, including the neurotransmitter expressing human tumor cells, as 1-L-MT concentrations >200 μM melatonin. IDO1 is ubiquitously expressed in many tissues are required to block IDO1-mediated tryptophan degradation by and cells, including endothelial cells and cells that in part 50% [10]. The X-ray crystal structure of human IDO1 in a complex constitute the tumor microenvironment, such as fibroblasts, with an inhibitor (4-phenylimidazole or cyanide) was revealed in 1Division of Anti-tumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 2University of Chinese Academy of Sciences, Beijing 100049, China; 3Department of Natural Products Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; 4State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China and 5School of Life Science and Technology, ShanghaiTech University, Shanghai 201203, China Correspondence: Yang Ye ([email protected]) or Ling-hua Meng ([email protected]) These authors contributed equally: Wei Guo, Sheng Yao Received: 25 January 2019 Accepted: 5 May 2019 Published online: 13 June 2019 © CPS and SIMM 2019 Discovery of natural products as novel IDO1 inhibitors W Guo et al. 424 2006 and facilitated the discovery of new IDO1 inhibitors [11]. kynurenine was measured at 492 nm using a SpectraMax Plus Recently, several potent IDO1 inhibitors have been identified by 384 microplate reader (Molecular Devices, Sunnyvale, CA). The unbiased screening [12, 13], structure-based modification [14, 15], data were analyzed using the enzyme kinetics module in and in silico drug design [15, 16]. However, only six compounds, SigmaPlot, version 10. 1-methyl-D-tryptophan (1-D-MT), hydroxylamidine INCB024360, The fluorescent enzymatic assay was carried out in 384-well NLG802, BMS-986205, SHR9146, LY3381916, and PF-06840003, are plates as described previously [19] with minor modifications. currently in clinical trials. Therefore, the number and structures The reaction was carried out in the presence of IDO1 holoenzyme, of IDO1 inhibitors in clinical trials are very limited, and new which consumed no more than ~30% of the initial L-Trp inhibitors with diverse structures and improved activities are of over 30 min and was terminated by the addition of piperidine great interest. (200 mM, final concentration). The plate was incubated at 65 °C for Herein, we screened an in-house natural product library for 15 min and left at room temperature for 60 min prior to novel inhibitors of IDO1. The screening led to the discovery that measurement with a SYNERGY H1 microplate reader (Bioteck, compounds 2, 7, and 8 showed inhibitory potencies in the Winooski, VT, USA). The inhibitory rate was calculated using the micromolar range. Detailed kinetic studies were performed and formula (ODcontrol−ODblank)−(ODtreatment−ODblank)/(ODcontrol− revealed that compound 2 exhibited noncompetitive inhibition, ODblank) × 100%. The IC50 values were determined by nonlinear whereas compounds 7 and 8 showed uncompetitive inhibition. regression analysis with Prism 4 software (GraphPad Software Inc., Compounds 7 and 8 were shown to interact with the ferric form of San Diego, CA, USA). The Z’ factor of the assay was calculated IDO1. Preliminary SARs were drawn from the analogs of using the formula [(ODmean control−3SDcontrol)−(ODmean blank + compound 8 and corroborated the putative binding orientation 3SDblank)]/(ODmean control−ODmean blank). suggested by molecular docking. Cell-based assay of IDO1 and TDO2 activities HEK 293 cells were seeded in a six-well plate at a density of 5 × 105 MATERIALS AND METHODS cells/well. The next day, the HEK 293 cells were transfected with Materials pcDNA3.1-hIDO or pcDNA3.1-hTDO using Lipofectamine 2000 Methylene Blue hydrate (#28514), L-tryptophan (#T0254), DMSO according to the manufacturer’s instructions. The cells were (#D2650), and p-dimethylaminobenzaldehyde (#D2004) were seeded in 96-well plates at a density of 2.5 × 104 cells/well 24 h purchased from Sigma (St. Louis, MO, USA). Catalase, bovine after transfection and treated with tested compounds. After an 1234567890();,: hemin, and ascorbic acid were purchased from Sangon (Shanghai, additional 12 h of incubation, 200 μL of the culture medium from China). Piperidine and TCA were obtained from SCRC (Shanghai, each well was transferred to a 96-well plate and mixed with 100 μL China). INCB024360 and Selleck Customized Library–Z100688 of 30% trichloroacetic acid. The plate was incubated at 65 °C for were purchased from Selleck Chemicals (Houston, TX, USA). The 15 min to hydrolyze the N-formylkynurenine produced by the natural product library was constructed by the Department of catalytic reaction of IDO1 or TDO2. The reaction mixture was then Natural Products Chemistry, Shanghai Institute of Materia Medica, centrifuged at 12 000 r/min for 10 min. Then, 100 μL of the Chinese Academy of Sciences. Cell Counting Kit-8(#CK04) was pur- supernatant was transferred to another 96-well plate and mixed chased from Dojindo (Kumamoto, Japan). Recombinant IDO1 and with 100 μL of 2% (w/v) p-dimethylaminobenzaldehyde in acetic TDO2 were expressed and purified in our laboratory as described acid. The yellow color derived from kynurenine was measured at previously [17]. The plasmids pET28a-hIDO1, pET28a-hTDO2, 492 nm using a SpectraMax Plus 384 microplate reader (Molecular pcDNA3.1-hIDO1, and pcDNA3.1-TDO2 were synthesized by Devices, Sunnyvale, CA, USA). Synbio Technologies (Suzhou, China). Determination of the inhibition kinetics of IDO1 inhibitors Cell culture The inhibition kinetics were determined as described previously HEK 293 cell line was obtained from ATCC (Manassas, VA, USA). [20]. In brief, the enzymatic activity of IDO1 was determined with a Cells were grown in Dulbecco’s modified Eagle’s medium serial dilution of the test compounds with L-tryptophan at (Corning, New York, USA) supplemented with 10% fetal bovine concentrations ranging from 7.5 μMto30μM. A function of S/V serum (Gibco, New York, USA) in 5% CO2 at 37 °C.
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