Discovery and characterization of natural products as novel indoleamine 2,3-dioxygenase 1 inhibitors through high-throughput screening

Abstract

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: high-throughput fluorescence-based screening; indoleamine 2,3-dioxygenase 1 inhibitor; natural product library.

Fig. 1

Confirmation of the high-throughput screening assay. a OD values of the blank and control. The Z’ factor was determined to be 0.572. b Fluorescence-based screening of Selleck Customized Library-Z100688 compounds (n = 2430) at 20 μM. NLG919 and INCB024360 are known IDO1 inhibitors

Fig. 2

Screening of the natural product library for inhibitors of recombinant human IDO1. a Screening of the natural product library (n = 2000) at 20 μM. b The effects of 79 natural products on the activity of IDO1 using a method based on Ehrlich’s reagent. c The effects of the nine selected compounds on the activity of IDO1 in HEK 293 cells expressing human IDO1. The dotted line indicates inhibition by 30%

Fig. 3

The activities of compounds 2, 7, and 8 against rhIDO1 and cellular IDO1. a The structures of compounds 2, 7, and 8. b The effects of compounds 2, 7, 8, and INCB024360 on the activity of IDO1 based on the reaction of Ehrlich’s reagent. c The effects of the test compounds on the cellular activity of IDO1 in HEK 293 cells. A CCK8 assay was performed to measure the cell viability at the end of treatment. Data shown are the mean ± SD of biological triplicates from a representative experiment

Fig. 4

Determination of the inhibition modes and kinetic parameters of compounds 2, 7, and 8. The IDO1 activities were determined in the presence of serially diluted INCB024360 a, compound 2 b, compound 7 c, or compound 8 d with L-tryptophan concentrations varying from 7.5 μM to 30 μM. [S]/[V] was plotted against [I]. The data presented are the mean ± SD of biological triplicates from a representative experiment

Fig. 5

Absorbance spectra of ferric or ferrous rhIDO1 in the presence of compounds 2, 7, and 8. Inset shows the absorbance difference spectra. a The absorbance of IDO1 ferric heme at 404 nm in the presence of compound 2, 7, or 8 (20 μM). b The absorbance of IDO1 ferrous heme at 428 nm in the presence of compound 2, 7, or 8 (20 μM)

Fig. 6

Selectivity of compounds 2, 7, and 8 for IDO1 and TDO2. a The effects of compound 2, 7, or 8 on the activity of IDO1 and TDO2 with an Ehrlich’s reagent-based assay. b The effects of compound 2, 7, or 8 on the activity of IDO1 and TDO2 in HEK 293 cells expressing human IDO1 or TDO2, respectively. Data are presented as the mean ± SD of biological triplicates from a representative experiment

Fig. 7

Comparison of the binding modes of 1-CE (orange) and compound 8 (purple) in the active site of IDO1 (PDB ID: 5EK3), where the catalytic pocket of IDO1 is depicted as a gray surface. The image was generated using PyMOL, version 1.3