AID	Name	Type	Comment
887	qHTS Assay for Inhibitors of 15-hLO (15-human lipoxygenase)	confirmatory	primary qHTS assay for 15hLO-1
2169	Probe Development Summary of Inhibitors of 15-hLO-1 (15-human lipoxygenase 1)	summary

Assay Provider: Holman, T.R., University of California, Santa Cruz
Screening Center PI: Austin, Christopher P.
Screening Center: NIH Chemical Genomics Center

Human lipoxygenase 15hLO-1 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. The enzyme activity of 15hLO-1 was determined by a direct measurement of product formation by monitoring the absorbance at 234 nm in a 2 mL cuvette. IC50 values of inhibitors were obtained by measuring the enzymatic rate at a variety of concentrations (Carroll et al, Segraves et al). A purified preparation of human 15hLO-1 was supplied by Professor Ted Holman, University of California, Santa Cruz.

This assay is includes some confirmation and follow-up compounds for related primary screening assay AID 887. The follow-up compounds focus on analogues of current lead 15hLO-1 inhibitor series.

For control experiments, 2 mL of substrate buffer (7 uM arachidonic acid / 25 mM HEPES / 0.01% (v/v) Triton X-100, pH 7.5) was aliquoted in a cuvette with a magnetic stir bar. After equilibrium was ensured, an aliquot of inhibitor solvent was added (DMSO or MeOH), and equilibrium was once again assured. The reaction was started by adding enzyme to the cuvette and the reaction was followed until completed. The inhibition experiments were performed as above, except the actual inhibitory compound was added instead of vehicle. To achieve an IC50, typically 5 concentrations of the inhibitor were studied. If the inhibitor concentration was constant, then five different reaction volumes were used.

Step \ Parameter \ Value \ Description
1; UV-Vis; 234 nm; Equilibration
2; Reagent; 2 mL; Substrate buffer
3; UV-Vis; 234 nm; Equilibration
4; Reagent; varies; Compound
5; UV-Vis; 234 nm; Equilibration
6; Reagent; 50-200 nM; Enzyme 15hLO-1
7; UV-Vis; 234 nm; Enzymatic reaction

Step \ Notes
1. Blank cuvette to air.
2. 7 uM AA, 25 mM HEPES, 0.01% Triton X-100, pH 7.5.
3. No change in absorbance ensured.
4. Compound vehicle or compound in various concentrations depending on potency.
5. No change in absorbance ensured.
6. Enzyme concentration to achieve reaction rate between 0.02 and 0.04 AU/s.
7. Enzymatic reaction visualized until completion.

Keywords: NIH Roadmap, MLSCN, MLPCN, MLI, MLSMR, qHTS, NCGC, human
lipoxygenase, 15hLO1, 15-hLO-1

Compound Ranking:

For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given based on -Log[Potency*10^-6], scaled to 100 and 0.

Grant Number: MH081283-01