A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases

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• 作者：Jun Qi^a ; ^b ; Karina Kizjakina^a ; ^b ; Reeder Robinson^a ; ^b ; Karishma Tolani^a ; ^b ; Pablo Sobrado^a ; ^b ; ^c ; ^{psobrado@vt.edu}
• 关键词：Flavin-dependent monooxygenases ; Kynurenine monooxygenase ; Ornithine hydroxylase ; Lysine hydroxylase ; Siderophore ; Huntington&rsquo ; s and Alzheimer&rsquo ; s diseases
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：1 June, 2012
• 卷：425
• 期：1
• 页码：80-87
• 文件大小：640 K

摘要

N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington鈥檚 and Alzheimer鈥檚 diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K_d value of 0.60 卤 0.05 渭M and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K_d values of 2.1 卤 0.2 and 4.0 卤 0.2 渭M, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z鈥?/em> factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration.