A cell-based assay for screening lipoxygenase inhibitors.
Lipoxygenases (LOX) form a family of lipid peroxidizing enzymes within the plant and animal kingdoms. In humans, six functional lipoxygenase isoforms have been identified. 5-LOX, "platelet-type" 12-LOX (p12-LOX) and 15-LOX type 1 (15-LOX1), originally identified in leukocytes, platelets, and reticulocytes, respectively, generate lipid mediators involved in host cellular functions and in the pathophysiology of asthma, cardiovascular diseases, and cancer. The pharmaceutical industry has reinvigorated their programs to develop novel LOX inhibitors in view of recent findings. However, high throughput LOX screening assays to test novel agents against these intracellular enzymes are limited. We describe a cell-based 96-well microplate fluorescence assay tested against several existing LOX inhibitors, and validate the assay by comparing known IC(50) values and HPLC analysis, which may provide a useful screen for novel LOX inhibitors.
Nair
Dileep G
DG
Department of Physiology and Biochemistry, Queen's University, Kingston, ON, Canada.
Funk
Colin D
CD
D000081:Acetamides / Q000494:pharmacology; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005449:Fluorenes / Q000494:pharmacology; D006801:Humans; D006918:Hydroxyurea / Q000031:analogs & derivatives / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D008084:Lipoxygenase / Q000235:genetics / Q000378:metabolism; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D009637:Masoprocol / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D010647:Phenyl Ethers
C106248:6,11-dihydro-5-thia-11-aza-benzo(a)-fluorene; D000081:Acetamides; D005449:Fluorenes; D016859:Lipoxygenase Inhibitors; D010647:Phenyl Ethers; C068663:N-hydroxy-N-(1-(4-(phenylmethoxy)phenyl)ethyl)-acetamide; C082739:A 78773; D009637:Masoprocol; D008084:Lipoxygenase; D006918:Hydroxyurea