Auckland Cancer Society Research Centre and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand.
Anal Bioanal Chem. 2013 Mar;405(8):2515-24. doi: 10.1007/s00216-012-6650-y. Epub 2013 Jan 12.
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.
吲哚胺 2,3-双加氧酶 1（IDO1）是一种色氨酸分解代谢酶，其在广泛的临床肿瘤中的表达与免疫抑制和患者预后不良有关。在这里，我们描述了一种新的荧光测定法，用于测量 IDO1 活性，适用于新型 IDO1 抑制剂化合物库的高通量筛选。该测定法易于操作，在读取之前仅需添加一种试剂。它不测量犬尿氨酸，而是使用原位形成的 N-甲酰犬尿氨酸衍生荧光团（NFKPIP）进行测量，激发波长为 400nm，发射波长为 500nm。形成的 NFKPIP 的荧光强度与酶活性的量直接相关，并且信号在 8 小时内保持稳定。该测定法的检测下限为 153 nM N-甲酰犬尿氨酸，比现有 IDO1 活性测定法的检测下限低 30 多倍。当我们将新测定法与已发表的比色吸光度测定法在筛选国立癌症研究所多样性集 III 中的 1597 种化合物中用于 IDO1 抑制剂的性能进行比较时，我们在两种测定法中获得了相同的 25 种最活跃化合物的列表。虽然库中 93 种化合物（醛，酮和芳香胺）干扰吸光度读数，但只有 18 种化合物（共轭系统和融合循环）干扰新荧光测定法的读数。使用新测定法确定三种已知 IDO1 抑制剂-1,4-萘醌，4-氨基-N-（3-氯-4-氟苯基）-N'-羟基-1,2,5-恶二唑-3-甲脒和 4-苯基-1H-咪唑的 IC50 值与文献值一致，进一步验证了新测定法用于测量 IDO1 活性。
