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

Abstract

Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are two structurally different enzymes that have a different tissue distribution and physiological roles, but both catalyze the conversion of tryptophan to N-formylkynurenine (NFK). IDO1 has been clinically validated as a small-molecule drug target for cancer, while preclinical studies indicate that TDO may be a target for cancer immunotherapy and neurodegenerative disease. We have developed a high-throughput screening assay for IDO1 and TDO based on a novel chemical probe, NFK Green, that reacts specifically with NFK to form a green fluorescent molecule with an excitation wavelength of 400 nm and an emission wavelength of 510 nm. We provide the first side-by-side comparison of a number of published inhibitors of IDO1 and TDO and reveal that the preclinical IDO1 inhibitor Compound 5l shows significant cross-reactivity with TDO, while the relative selectivity of other published inhibitors was confirmed. The suitability for high-throughput screening of the assays was demonstrated by screening a library of 87,000 chemical substances in 384-or 1536-well format. Finally, we demonstrate that the assay can also be used to measure the capacity of cells to metabolize tryptophan and to measure the cellular potency of IDO1 and TDO inhibitors.

Binding Properties of Different Categories of Ido1 Inhibitors: A Microscale Thermophoresis Study

“…MST analysis of compounds 10 and 11 reveals K d of 45.3 ± 11.7 μM and K d of 3.30 ± 0.41 μM to IDO1 (Table 1, Supplementary Figures S5, S6), respectively. At a first glance, these results are in agreement with the rank order of IC 50 potency that is reported in the literature for 4-PI (10, IC 50 = 48 μM) and 11 (IC 50 = 0.1 μM or 0.038 μM) [31,54]. However, it cannot be ruled out that the imidazoleisoindole analog (11) also binds to the reduced active ferrous (Fe 2+ ) state of hIDO1, with this latter interaction accounting for the nanomolar inhibition potency in biochemical assay.…”

Targeting key dioxygenases in tryptophan–kynurenine metabolism for immunomodulation and cancer chemotherapy

“…Incyte reported that INCB024360 (also known as compound 5L [60]) displayed specificity against IDO1, with little inhibition of TDO or IDO2 activity at 10 or 5 mM, respectively, and no effect on tryptophan transport at 30 mM. Interestingly, a 2014 study described INCB024360 cross-reactivity against TDO in a cell-free assay system [61]; however, because molecular modelling has not been performed on hydroxyamidine-based drugs with either IDO2 or TDO, a mechanism for reported selectivity of INCB024360 (IDO1 > IDO2 > TDO) has yet to be proposed.…”

Malpighian Tubules as Novel Targets for Mosquito Control

“…High-throughput assays for discovering small-molecule inhibitors of mammalian thioredoxin reductase, tryptophan 2,3-dioxygenase, and kynurenine 3-monoxygenase, which are human drug targets, have already been developed [30,31,32]. Moreover, inhibitors of mosquito tryptophan 2,3-dioxygenase and 3-hydroxykynurenrine transaminase have already been discovered, some of which exhibit larvicidal activity [33,34].…”