Development of a fluorescent intensity assay amenable for high-throughput screening for determining 15-lipoxygenase activity.
15-Lipoxygenase-1 catalyzes the introduction of molecular oxygen into polyunsaturated fatty acids to form a lipid hydroperoxide. The authors have developed an assay for the detection of lipid hydroperoxides formed by human 15-lipoxygenase (15-LO) in enzyme or cellular assays using either a 96-well or a 384-well format. The assays described take advantage of the ability of lipid hydroperoxides to oxidize nonfluorescent diphenyl-1-pyrenylphosphine (DPPP) to a fluorescent phosphine oxide. Oxidation of DPPP yields a fluorescent compound, which is not sensitive to temperature and is stable for more than 2 h. The assay is sensitive toward inhibition and robust with a Z' value of 0.79 and 0.4 in a 96- and 384-well format, respectively, and thus amenable for high-throughput screening. The utility of DPPP as a marker for 15-lipoxygenase activity was demonstrated with both enzyme- and cell-based assays for the identification of hits and to determine potency by IC(50) determinations.
Dahlström
Märta
M
In Vitro Pharmacology, Orexo AB, Uppsala, Sweden. Marta.Dahlstrom@orexo.com
Forsström
Daniel
D
Johannesson
Malin
M
Huque-Andersson
Yasmin
Y
Björk
Marie
M
Silfverplatz
Erik
E
Sanin
Andrei
A
Schaal
Wesley
W
Pelcman
Benjamin
B
Forsell
Pontus K A
PK
D000975:Antioxidants / Q000494:pharmacology; D001093:Arachidonate 15-Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D001681:Biological Assay; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D057075:Enzyme Assays; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D008054:Lipid Peroxides / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D011721:Pyrenes / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology
D000975:Antioxidants; D008054:Lipid Peroxides; D009943:Organophosphorus Compounds; D011721:Pyrenes; D054852:Small Molecule Libraries; C066322:diphenyl-1-pyrenylphosphine; D001093:Arachidonate 15-Lipoxygenase