High-throughput fluorescence-based screening assays for tryptophan-catabolizing enzymes

Area of Science:

• Biochemistry
• Enzymology
• Drug Discovery

Background:

• Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are enzymes catalyzing tryptophan metabolism.
• IDO1 is a validated cancer drug target, while TDO is a potential target for cancer immunotherapy and neurodegenerative diseases.

Purpose of the Study:

• To develop a high-throughput screening (HTS) assay for IDO1 and TDO.
• To compare the selectivity of known IDO1 and TDO inhibitors using the new assay.
• To assess the utility of the assay for cellular tryptophan metabolism and inhibitor potency measurements.

Main Methods:

• Development of a novel HTS assay utilizing the chemical probe NFK Green for N-formylkynurenine (NFK) detection.
• Side-by-side comparison of published IDO1 and TDO inhibitors.
• Screening of a large chemical library (87,000 substances) in 384- and 1536-well formats.
• Application of the assay to measure cellular tryptophan metabolism and inhibitor potency.

Main Results:

• The NFK Green probe forms a green fluorescent molecule upon reaction with NFK (Ex 400 nm, Em 510 nm).
• A previously studied IDO1 inhibitor, Compound 5l, demonstrated significant cross-reactivity with TDO.
• The selectivity of other inhibitors was confirmed, and the assay's HTS suitability was validated.
• The assay successfully measured cellular tryptophan metabolism and inhibitor potency.

Conclusions:

• A novel, robust HTS assay for IDO1 and TDO has been established using NFK Green.
• The assay is valuable for evaluating inhibitor selectivity and cellular activity, highlighting potential cross-reactivity issues.
• This tool facilitates drug discovery efforts targeting IDO1 and TDO in various diseases.