Cell experiment:

To study the cellular hIDO2 inhibition of candidate compounds, recombinant plasmid pcDNA3.1(+)-hIDO2 is constructed and transfected into human glioblastoma U87 MG cells which had no IDO1 expression (confirmed by RT-PCR and western blot) therefore eliminated the interference of IDO1. U87 MG cells are cultivated in DMEM containing 50 U/mL penicillin, 50 mg/mL streptomycin, 4500 mg/L glucose, and 10% inactivated FBS at 37℃ with 5% CO2 and 95% humidity. When a cell density of 80% confluent monolayer is reached, U87 MG cells are transfected with pcDNA3.1(+)-hIDO2 using the transfection reagent Lipofectamine 2000 according to the manufacturer's instructions. An empty pcDNA3.1(+) expression vector is served as control. After 18 h of incubation, the transfected cells are seeded in 96-well culture plates at a density of 2.5×104 cells/well in a final volume of 200 μL supplemented with 200 μM L-Trp. A serial dilution of the tested compounds is added to the culture medium after an additional 6 h of incubation. The reaction is terminated by addition of 30% (w/v) trichloroacetic acid (10 μL for 140 μL of the reaction mixture) 24 h later. The plates are incubated at 65℃ in water bath for 15 min to facilitate the transformation of N-formylkynurenine to L-kynurenine, followed by centrifugation at 13,000× g for 10 min to remove the sediments. 100 μL of the supernatant are then transferred to another 96-well plate and mixed with a same volume of 2% (w/v) 4-dimethylaminobenzaldehyde in acetic acid. The percentages of inhibition of tryptophan degradation or kynurenine production by the compounds are calculated by measuring the absorption at 492 nm using a microplate reader. Cellular IC50s are determined via non-linear regression analysis using GraphPad Prism 5.0[1].

8-Nitrotryptanthrin is a potent human indoleamine 2,3-dioxygenase 2 (hIDO2) inhibitor which significantly reduces IDO2 activity with Ki of 0.97 μM.

The typtanthrin derivative 8-Nitrotryptanthrin (5i) is found to be a potent hIDO2 inhibitor with superior efficiency far better than that of the most frequently-used inhibitor L-1-MT. The IC50 values show that all nine tryptanthrin compounds display hIDO2 inhibitory activities, especially, the compound 8-Nitrotryptanthrin demonstrates much stronger inhibition (1.87 μM) than both L-1-MT (82.53 μM) and D-1-MT (262.75 μM). 8-Nitrotryptanthrin exhibits significant antitrypanosomal activity with EC50 of 0.82 μM[2]. 8-Nitrotryptanthrin has a microplate Alamar Blue assay (MABA) minimum inhibitory concentration (MIC) value of 0.032 μg/mL. 8-Nitrotryptanthrin also has a LORA MIC value of 2.4 μg/mL, while the majority of analogues lack LORA activity[3].

[1]. Li J, et al. Establishment of a human indoleamine 2, 3-dioxygenase 2 (hIDO2) bioassay system and discovery of tryptanthrin derivatives as potent hIDO2 inhibitors. Eur J Med Chem. 2016 Nov 10;123:171-9. [2]. Scovill J, et al. Antitrypanosomal activities of tryptanthrins. Antimicrob Agents Chemother. 2002 Mar;46(3):882-3. [3]. Hwang JM, et al. Design, synthesis, and structure-activity relationship studies of tryptanthrins as antitubercular agents. J Nat Prod. 2013 Mar 22;76(3):354-67.