Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity

Abstract

Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan-catabolizing enzyme whose expression by a broad range of clinical tumors is associated with immunosuppression and poor patient outcome. Here we describe a new fluorescence assay for measuring IDO1 activity suitable for high-throughput screening of compound libraries for novel IDO1 inhibitors. This assay is easy to perform, requiring the addition of only one reagent prior to readout. In place of measuring kynurenine, it uses the in situ formation of an N-formylkynurenine-derived fluorophore (NFKPIP) measured at an excitation wavelength of 400 nm and an emission wavelength of 500 nm. The fluorescence intensity of the NFKPIP formed is directly related to the amount of enzyme activity, and the signal is stable over 8 h. This assay has a lower limit of detection, equating to 153 nM N-formylkynurenine, which is over 30-fold lower than the limits of detection of existing assays for IDO1 activity. When we compared the performance of the new assay with that of the published colorimetric absorbance assay in screening the National Cancer Institute Diversity Set III of 1,597 compounds for IDO1 inhibitors, we obtained an identical list of the 25 most active compounds in the two assays. Although 93 compounds (aldehydes, ketones, and aromatic amines) in the library interfered with the absorbance readout, only 18 compounds (conjugated systems and fused cycles) interfered with the readout of the new fluorescence assay. IC(50) values determined using the new assay for three known IDO1 inhibitors-1,4-naphthoquinone, 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-carboximidamide and 4-phenyl-1H-imidazole-were consistent with their literature values, further validating the new assay for measuring IDO1 activity.

High-Throughput Fluorescence-Based Screening Assays for Tryptophan-Catabolizing Enzymes

“…The NFK Green assay is a homogeneous mix-and-measure assay that avoids heating and addition of very acidic or toxic reagents, common for other IDO/TDO assays. [19][20][21][22][23] We Table S1.…”

“…22 NFK can also be directly detected by reaction with piperidine to produce a fluorophore. 23 However, while both these assays have better selectivity than pDMAB, 22 they have one or more steps at extremely acidic pH (pH 1.0) or extremely basic pH (pH 11.0) and high temperature (55-65 °C). Furthermore, piperidine is a toxic chemical and has a nasty smell, hampering its use in large HTS campaigns.…”

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

“…To identify novel IDO1 inhibitors with diverse structures, we used a fluorescent assay for measuring IDO1 activity that was suitable for high-throughput screening [ 19 ]. The Z ’ factor was determined to be 0.572, confirming the stability of the high-throughput screening assay (Fig.…”

“…The fluorescent enzymatic assay was carried out in 384-well plates as described previously [ 19 ] with minor modifications. The reaction was carried out in the presence of IDO1 holoenzyme, which consumed no more than ~30% of the initial L -Trp over 30 min and was terminated by the addition of piperidine (200 mM, final concentration).…”

Discovery and Characterisation of Dual Inhibitors of Tryptophan 2,3-Dioxygenase (TDO2) and Indoleamine 2,3-Dioxygenase 1 (IDO1) Using Virtual Screening

“…The important role of accelerated tryptophan catabolism in escaping immune surveillance makes IDO1 and TDO2 prime targets for development of small molecule drugs to augment cancer immunotherapy. Advanced biochemical assay methodologies [7,8,9] and virtual screening approaches [10,11] have played a key part in discovery of a diverse range of IDO1-specific inhibitors [1,12,13]. Two IDO1 inhibitors, Epacadostat (Incyte Corporation) and Linrodostat (Bristol-Myers-Squibb), have reached Phase 3 clinical trials [14,15,16].…”