| Assay Method Information | |
| | Determining Endocannabinoid Hydrolase Activity |
| Description: | In the Experimental example, endocannabinoid hydrolases used were Fatty Acid Amide Hydrolase (FAAH) and N-acylethanolamide hydrolyzing acid amidase (NAAA), which were prepared by the method described in the document (PMCID: PMC3423427, PMC3723234, PMC2692831, PMC3382457). The preparation method was as followed: a plasmid (pCDNA3.1/NAAA or pCDNA3.1/FAAH) carrying a whole NAAA/FAAH gene was constructed, wherein the plasmid carried Cytomegalovirus (CMV) promoter and Neomycin selectable gene; the plasmid was transformed into HEK-293 cell via lipid medium, stable cell lines expressing NAAA/FAAH at a high level were obtained by G418 screening and Western-blot method. HEK-293 recombinant cells were cultured and collected, washed with PBS for 2-3 times, and ultrasonically treated in 20 mM Tris-HCl containing 0.32 M sucrose, then repeatedly frozen and thawed twice, and then centrifuged at 4° C., 800 g for 15 min. The supernatant (i.e., the desired protein) was collected, the protein concentration was determined by BCA method, and the protein was diluted to a concentration of 1 mg/mL, and sub-packaged and stored in a refrigerator at −80° C. for further use.In the Experimental example, PBS solution used was prepared as followed: 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 were dissolved in 1 L ultrapure water, and the resultant solution was subjected to moist heat sterilization and stored at 4° C.30 μL (1 mg/mL) endocannabinoid hydrolase was added to a sample vial, and 2 μL DMSO (Blank control group) or a different concentration of a test compound (Compounds 1-46 prepared in the Examples according to the invention) was further added. The reaction was carried out at 37° C. for 10 min. 170 μL buffer (the buffer consisted of 50 mM disodium Hydrogen Phosphate, 0.1% Triton X-100, 3 mM DTT, 150 μL) containing enzymatic hydrolysis substrate (the substrate was a heptadecenoyl ethanolamine containing a double bond and 17 carbon atoms, abbreviated as 17:1 FAE) was further added, wherein the concentration of 17:1 FAE was 5 μM. The reaction was carried out at 37° C. for 30 min, and 200 μL methanol solution containing internal standard (the internal standard was margaric acid, at a concentration of 1 nmol) was then added to stop the reaction. LC-MS was used to determine the yield of the hydrolysate 17:1 FA (i.e., a heptadecenoic acid containing a double bond) of 17:1 FAE, then a graph was plotted with Graphpad Prism 5. Thereby, the IC50 of the test compound on endocannabinoid hydrolase was determined.By the method above, the inhibitory effects of the Compounds 1-46 prepared in the invention on NAAA and FAAH were determined. The results were shown in Table 1, wherein IC50 (NAAA) represents a concentration that inhibits NAAA activity to 50% of the activity prior to inhibition, IC50 (FAAH) represents a concentration that inhibits FAAH activity to 50% of the activity prior to inhibition, and >100 μM represents that IC50 of a compound on a corresponding enzyme is above 100 μM, indicating that the compound has no inhibitory effect on the enzyme. |
| Affinity data for this assay | |
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