Assay Report Card

ID: ALA3888532

Type: Binding

Description: UPLC/MS Assay: NAAA protein preparation (10 ug) was pre-incubated with various concentrations of test compound or vehicle control in 100 mM NaH2PO4, 100 mM Tri Sodium Citrate Dehydrate, 0.1% Triton-X 100, 3 mM DTT, pH 4.5 for 30 min at 37° C. Duplicate samples were then incubated with 50 uM C17:1 10-cis-heptadecenoylethanolamide (Avanti Polar Lipids, Alabaster, Ala. USA) at 37° C. for 30 minutes. The reaction was terminated by the addition of 0.2 mL of cold methanol containing 1 nmol of heptadecanoic acid (NuChek Prep, Elysian, Minn. USA) as internal standard. Samples were then analyzed by UPLC/MS. Heptadecenoic and heptadecanoic acids were eluted on an Acquity UPLC BEH C18 column (50 mm length, 2.1 mm i.d., 1.7 um pore size, Waters) isocratically at 0.5 mL/min for 1.5 min with a solvent mixture of 95% methanol and 5% water, both containing 0.25% Acetic Acid and 5 mM Ammonium Acetate. The column temperature was 40° C. Electrospray ionization was in the negative mode, capillary voltage was 0.5 kV, cone voltage was 25 kV, desolvation temperature was 500° C. N2 was used as drying gas at a flow rate of 1000 L/hour and a temperature of 500° C. The [M-H]- ion was monitored in the selected-ion monitoring mode (m/z values: heptadecenoic acid 267.37, heptadecanoic acid 269.37). Calibration curves were generated using commercial heptadecenoic acid (NuCheck Prep). Inhibition of NAAA activity was calculated as reduction of heptadecenoic acid in the samples compared to vehicle controls. IC50 values were calculated by non-linear regression analysis of log [concentration]/inhibition curves using GraphPad Prism 5 (GraphPad Software Inc., CA-USA) applying a standard slope curve fitting.