qHTS Assay for Inhibitors of 12-hLO (12-human lipoxygenase)
Human lipoxygenase 12hLO is a member of the closely related lipoxygenase family of enzymes which catalyze the site-specific oxidation of arachidonic acid to various hormone precursor molecules and as such is a candidate for drug development in a variety of disease areas, such as cancer and inflammation. ..more
BioActive Compounds: 181
Assay Provider: Holman, T.R., University of California, Santa Cruz
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
Human lipoxygenase 12hLO is a member of the closely related lipoxygenase family of enzymes which catalyze the site-specific oxidation of arachidonic acid to various hormone precursor molecules and as such is a candidate for drug development in a variety of disease areas, such as cancer and inflammation.
Inhibition of 12hLO activity was screened by utilizing arachidonic acid as a substrate. The extent of hydroperoxide product formation was measured by a secondary chromogenic reaction in which xylenol orange (XO, Sigma-Aldrich, cat. number 39,818-7) reacts with the ferric ions produced from the reaction between the hydroperoxide and ferrous ions. The Fe(III)-XO complex is characterized with red-shifted absorbance at 560 nm. A purified preparation of human 12hLO was supplied by Professor Ted Holman, University of California, Santa Cruz.
Buffer: 25 mM Hepes pH 7.0, 0.01% Triton X-100.
Buffer in columns 3 and 4 as negative control (no enzyme).
Substrate solution: 40 uM arachidonic acid (Sigma-Aldrich, St. Louis, MO) final concentration dispensed throughout the plate.
Enzyme: 150 nM 12hLO final concentration in columns 1, 2, 5-48. Column 1 is neutral (100% activity). Column 2 contained pin-transferred titration of NDGA (nordihidroguaiaretic acid, Sigma-Aldrich, N 5023, top concentration 20 mM in DMSO, then 1:3 dilution in duplicate). (Dilution factor: 23 nL into 4 uL.)
Chromogenic detection reagent (divalent iron/xylenol orange, Fe-XO): 200 uM xylenol orange plus 300 uM ferrous ammonium sulfate prepared freshly in 50 mM sulfuric acid.
Three uL of enzyme were dispensed to 1536-well Greiner black clear bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 15 min at room temperature, and then 1 uL of substrate solution was added to start the reaction. After room temperature incubation for 30 minutes, 4 uL of Fe-XO solution was added to each well and the plates were incubated for 30 min. The absorbance at 405 and 573 nm were measured using ViewLux (Perkin-Elmer) High-throughput CCD imager and absorbance protocol settings. The 573-to-405 absorbance ratio was used to compute reaction progress.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. LogAC50 was used for determining relative score and was scaled to each curve class' score range. Acitves in the assay ranged from a score of 40-88.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)