| Assay Method Information | |
| | In Vitro Inhibition on FAAH and MAGL Activity Assay |
| Description: | The inhibitory effect of compounds of Formula (I-IV) on the activity of human recombinant FAAH was studied using a commercial inhibitor screening kit (Cayman Chemicals, Godlewski et al., 2010 [19]). In brief, FAAH hydrolyzes AMC arachidonoyl amide resulting in the release of the fluorescent product, while the inhibitor will inhibit the FAAH activity and thus reduce the fluorescent signal. The resulting fluorophore can be analyzed using an excitation wavelength of 340-360 nm and an emission wavelength of 450-465 nm by a plate reader (Synergy H1, Biotek). Inhibition is calculated as a percentage of the treated sample over the non-treated control based on the signal values, and where possible, a range of concentrations of compounds was tested to find the IC50 values (i.e. the concentration of compound that inhibited 50% FAAH activity).The inhibitory effect of the compounds on activity of human recombinant MAGL was also studied using a commercial inhibitor screening kit (Cayman Chemicals). In brief, MAGL hydrolyzes 4-nitrophenylacetate resulting in a yellow product, 4-nitrophenol with an absorbance at 405-412 nm, while the inhibitor will inhibit the MAGL activity and thus reduce the yellow signal. The absorbance of the yellow product can be analyzed using the plate reader. |
| Affinity data for this assay | |
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