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

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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. IC50 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.

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

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Acknowledgments

This work was funded by grants from the Cancer Society of New Zealand (Auckland Division), the Auckland Medical Research Foundation, and the Maurice Wilkins Centre for Molecular Biodiscovery.

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Authors and Affiliations

  1. Auckland Cancer Society Research Centre and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, 85 Park Road, Auckland, 1023, New Zealand

    Petr Tomek, Brian D. Palmer, Jack U. Flanagan, Sai-Parng S. Fung, David J. A. Bridewell & Lai-Ming Ching

  2. Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, 2109, Australia

    Joanne F. Jamie

  3. Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand

    Lai-Ming Ching

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  1. Petr Tomek
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Tomek, P., Palmer, B.D., Flanagan, J.U. et al. Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity. Anal Bioanal Chem 405, 2515–2524 (2013). https://doi.org/10.1007/s00216-012-6650-y

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