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

作者：Qi Jun; Kizjakina Karina; Robinson Reeder; Tolani Karishma; Sobrado Pablo^*

来源：Analytical Biochemistry, 2012, 425(1): 80-87.

DOI：10.1016/j.ab.2012.03.002

• Flavin-dependent monooxygenases
• Kynurenine monooxygenase
• Ornithine hydroxylase
• Lysine hydroxylase
• Siderophore
• Huntington%26apos
• s and Alzheimer%26apos
• s diseases

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%26apos;s and Alzheimer%26apos;s 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 +/- 0.05 mu M and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K-d values of 2.1 +/- 0.2 and 4.0 +/- 0.2 mu 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%26apos; factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration.

• 出版日期2012-6-1
• 单位Virginia Tech; 美国弗吉尼亚理工大学(Virginia Tech)