[CH:1]1[CH:2]=[CH:3][C:4]2[NH:9][CH:8]=[C:7](C[C@@H](N)C(O)=O)[C:5]=2[CH:6]=1.O=C1O[C@H]([C@H](CO)O)C([O-])=C1O.C[N:29](C1C=CC2N=C3C(=CC(C=C3)=[N+](C)C)SC=2C=1)C.C1C=C(C(C[C@H](N)C(O)=O)=O)C(NC=O)=CC=1>P([O-])([O-])([O-])=O.[K+].[K+].[K+]>[NH:9]1[C:4]2[C:5](=[CH:6][CH:1]=[CH:2][CH:3]=2)[CH:7]=[C:8]1[NH2:29]Human indoleamine 2,3-dioxygenasae (IDO) with an N-terminal H is tag was expressed in E. coli and purified to homogeneity. IDO catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N′-formylkynurenine. The assays were performed at room temperature as described in the literature using 95 nM IDO and 2 mM D-Trp in the presence of 20 mM ascorbate, 5 μM methylene blue and 0.2 mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5). The initial reaction rates were recorded by continuously following the absorbance increase at 321 nm due to the formation of N′-formylkynurenine. See: Sono, M., Taniguchi, T., Watanabe, Y., and Hayaishi, O. (1980) J. Biol. Chem. 255, 1339-1345 Compounds of the invention having an IC50 less than about 100 μM were considered active.
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