Inhibition of Fatty Acid Amide Hydrolase (FAAH) by Macamides
Authors: Alasmari Moudi M. , Böhlke M. , Kelley C. , Maher T. , Pino‐Figueroa A. 
Published: June 2021

PurposePentane extract of the Peruvian plant Lepidium meyenii (Maca) has been demonstrated to possess neuroprotective activity in previous studies. This extract contains several macamides that might act on the endocannabinoid system. The aim of this study is to characterize the inhibitory activity of four maccamides (N‐benzylstearamide, N‐benzyloleamide, N‐benzyloctadeca‐9Z,12Z‐dienamide, and N‐benzyloctadeca‐9Z,12Z,15Z‐trienamide) on fatty acid amide hydrolase (FAAH), an enzyme that is responsible for the endocannabinoid degradation in the nervous system. The enhancement of endocannabinoid activity by inhibition of FAAH enzyme results in analgesic, anxiolytic, anti‐depressant and neuroprotectant effects, avoiding unwanted side effects of cannabinoid receptor activation such as weight gain and motor cognitive impairments.MethodsThe four compounds at concentrations from 1 to 100 μM were tested using a FAAH inhibitor screening assay method. This assay is based on fluorescence produced upon the hydrolysis of a substrate, 7‐amino‐4‐methylcoumarin‐arachidonamide (AMC‐AA) by the FAAH enzyme to the 7‐amino‐4‐methylcoumarin (AMC) which is measured 60 min following substrate addition at 37ºC with excitation and emission of 360 and 460 nm, respectively. To study the time dependence effect on the FAAH enzyme inhibition, the previous assay was repeated using different pre‐incubation times (0, 60 and 120 min) before substrate addition at 37ºC. To evaluate whether macamides are purely inhibitors or might be substrates of FAAH, LC/MS/MS was used to analyze the 60 min incubation of N‐benzyloctadeca‐9Z,12Z‐dienamide with and without FAAH enzyme at 37ºC. The benzylamine was used as a standard to determine the possible degradation of N‐benzyloctadeca‐9Z,12Z‐dienamide by FAAH enzyme.ResultsThe four macamides studied demonstrated concentration‐dependent FAAH inhibitory activity. Double bonds appeared to have an important impact on FAAH inhibitory activity. The macamides containing oleic, linoleic and linolenic acids produced FAAH inhibition of 64, 73, and 54%, respectively, while the macamide containing stearic acid and lacking a double bond produced only 13% inhibition at 100 μM. N‐Benzyloctadeca‐9Z,12Z‐dienamide (two double bonds) demonstrated the greatest inhibitory activity, indicating that two double bonds significantly influence the inhibition of FAAH. Pre‐incubation studies showed significant differences in the FAAH inhibitory activity of N‐benzyloctadeca‐9Z,12Z‐dienamide, N‐benzyloleamide, and N‐benzylstearamide, indicating that the inhibitory mechanism of these compounds is time‐dependent. Incubation of N‐benzyloctadeca‐9Z,12Z‐dienamide with FAAH enzyme for 60 min resulted in a 44% degradation, when analyzed by LC/MS/MS. This result indicates that N‐benzyloctadeca‐9Z,12Z‐dienamide likely acts as a slow substrate for the FAAH enzyme.ConclusionN‐Benzyloctadeca‐9Z,12Z‐dienamide is the most active macamide. The presence of double bonds in macamides significantly increases FAAH inhibition, which could improve the potential neuroprotective and analgesic activities. These results will provide useful information regarding the development of new compounds with FAAH inhibitory activity.Support or Funding InformationKing Saud ben Abdulaziz University for Health Sciences (KSAU‐HS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.