| Assay Method Information | |
| | FAAH Enzyme Inhibition Assay |
| Description: | FAAH inhibition assay was performed using Fatty Acid Amide Hydrolase Inhibitor Screening Assay Kit (Item #10005196), Cayman (1180 E Ellsworth Rd Ann Arbor, MI, USA). The compounds were tested in 10-dose IC50 mode, with 3-fold serial dilution at a starting concentration of 100 μM for compounds (II-2a to II-2m) and a concentration of 5 μM for JZL195 used as a positive control. Manufacturer's protocol was followed to perform the assay. The assay buffer (125 mM Tri-HCl with 1 mM EDTA, pH 9.0) was used to dilute the FAAH human recombinant enzyme. AMC-Arachidonoyl amide, at a concentration of 400 μM was then used as the FAAH substrate. Samples were mixed for 30 s and incubated at 37° C. for 30 min. The fluorescent by-product AMC (7-amino-4-methylcoumarin) released by the FAAH enzyme was detected and quantified at an excitation wavelength of 355 nm and emission wavelength of 460 nm. |
| Affinity data for this assay | |
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