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

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

Research output: Contribution to journalArticleResearchpeer-review

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.

LanguageEnglish
Pages2515-2524
Number of pages10
JournalAnalytical and bioanalytical chemistry
Volume405
Issue number8
DOIs
Publication statusPublished - Mar 2013

Fingerprint

Indoleamine-Pyrrole 2,3,-Dioxygenase
Fluorophores
Assays
Fluorescence
Libraries
Limit of Detection
Oxadiazoles
Kynurenine
National Cancer Institute (U.S.)
Screening
Enzymes
N'-formylkynurenine
Ketones
Aldehydes
Tryptophan
Immunosuppression
Inhibitory Concentration 50
Amines
Wavelength
Enzyme activity

Cite this

Tomek, Petr ; Palmer, Brian D. ; Flanagan, Jack U. ; Fung, Sai Parng S ; Bridewell, David J A ; Jamie, Joanne F. ; Ching, Lai Ming. / Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity. In: Analytical and bioanalytical chemistry. 2013 ; Vol. 405, No. 8. pp. 2515-2524.
@article{6042270a40af45b7b87ed230e700a146,
title = "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. 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.",
author = "Petr Tomek and Palmer, {Brian D.} and Flanagan, {Jack U.} and Fung, {Sai Parng S} and Bridewell, {David J A} and Jamie, {Joanne F.} and Ching, {Lai Ming}",
year = "2013",
month = "3",
doi = "10.1007/s00216-012-6650-y",
language = "English",
volume = "405",
pages = "2515--2524",
journal = "Analytical and bioanalytical chemistry",
issn = "1618-2642",
publisher = "Springer, Springer Nature",
number = "8",

}

Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity. / Tomek, Petr; Palmer, Brian D.; Flanagan, Jack U.; Fung, Sai Parng S; Bridewell, David J A; Jamie, Joanne F.; Ching, Lai Ming.

In: Analytical and bioanalytical chemistry, Vol. 405, No. 8, 03.2013, p. 2515-2524.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Formation of an N-formylkynurenine-derived fluorophore and its use for measuring indoleamine 2,3-dioxygenase 1 activity

AU - Tomek, Petr

AU - Palmer, Brian D.

AU - Flanagan, Jack U.

AU - Fung, Sai Parng S

AU - Bridewell, David J A

AU - Jamie, Joanne F.

AU - Ching, Lai Ming

PY - 2013/3

Y1 - 2013/3

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84878401284&partnerID=8YFLogxK

U2 - 10.1007/s00216-012-6650-y

DO - 10.1007/s00216-012-6650-y

M3 - Article

VL - 405

SP - 2515

EP - 2524

JO - Analytical and bioanalytical chemistry

T2 - Analytical and bioanalytical chemistry

JF - Analytical and bioanalytical chemistry

SN - 1618-2642

IS - 8

ER -