| Assay Method Information | |
| | MAGL Enzyme Inhibition Assay |
| Description: | The compounds II-2a to II-2m and controls in Table II were tested in 10-dose IC50 mode, with 3-fold serial dilution at a starting concentration of 100 μM for compounds (II-2a-m) and a concentration of 10 μM for JZL184 used as a positive control. The assay buffer (10 mM Tris-HCl with 1 mM EDTA, pH 7.2) was used to dilute the FAAH human recombinant enzyme (Reaction Biology Corp., Malvern, 19355, PA, USA) at a final concentration of 10 nM. The substrate, 4-nitrophenylacetate, was used as a final concentration of 250 μM. The plate was mixed for 30 s and incubated at room temperature for 30 min. The plate was read at an absorption wavelength of 405 nm to detect the release of the by-product 4-nitrophenol. The measurement of IC50 for compound II-2d was repeated in presence 10 mM of dithiothreitol to check the reversibility of the MAGL inhibition by our compounds. |
| Affinity data for this assay | |
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