Application of the ferrous oxidation-xylenol orange assay for the screening of 5-lipoxygenase inhibitors.
5-Lipoxygenase (5-LO) is the key enzyme involved in leukotriene synthesis and its improper regulation is implicated in several inflammatory diseases. A rapid and sensitive assay for 5-LO activity suitable for high-throughput format is not yet available. In this study, we examined whether the ferrous oxidation-xylenol orange (FOX) assay could be applicable for the high-throughput screening of 5-LO inhibitors. Using insect cell lysates overexpressing rat 5-LO, the effects of cofactors of 5-LO such as ATP, Ca2+, and L-alpha-phosphatidylcholine (PC) on the color development of FOX reagents were investigated. ATP quenched substantially color development by hydroperoxide, an intermediate of 5-LO reaction, and an optimum concentration of ATP with little interference was determined as 20 microM. Ethylenediaminetetraacetate (0.4 mM) also affected the complex formation with FOX reagents. On the other hand, neither Ca2+ nor PC influenced complex formation with FOX reagents. Under optimized assay conditions, zileuton, a 5-LO-specific inhibitor, exhibited inhibitory potency (IC50 values of 0.1-0.2 microM) similar to that determined by the conventional spectrophotometric assay. Taken together, this study shows that the FOX assay with some modifications can be employed for high-throughput assay format for the measurement of 5-LO activity at the stage of primary screening.
Cho
Young Sik
YS
Laboratory of Molecular Pharmacology and Physiology, Medicinal Science Division, Korea Research Institute of Chemical Technology, Jang-dong 100, Yuseong-gu, Taejon 305-600, Republic of Korea.
Kim
Hyo Sun
HS
Kim
Chi Hyun
CH
Cheon
Hyae Gyeong
HG
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005296:Ferrous Compounds / Q000737:chemistry*; D016859:Lipoxygenase Inhibitors / Q000032:analysis*; D010084:Oxidation-Reduction; D010636:Phenols; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D018411:Spodoptera; D013454:Sulfoxides; D014992:Xylenes / Q000737:chemistry*
D017931:DNA Primers; D005296:Ferrous Compounds; D016859:Lipoxygenase Inhibitors; D010636:Phenols; D013454:Sulfoxides; D014992:Xylenes; C016833:xylenol orange