Fatty acid amide hydrolase inhibition and N‐arachidonoylethanolamine modulation by isoflavonoids: A novel target for upcoming antidepressants

Abstract Modulation of the endocannabinoid system (ECS) is a novel putative target for therapeutic intervention in depressive disorders. Altering concentrations of one of the principal endocannabinoids, N‐arachidonoylethanolamine, also known as anandamide (AEA) can affect depressive‐like behaviors through several mechanisms including anti‐inflammatory, hormonal, and neural circuit alterations. Recently, isoflavonoids, a class of plant‐derived compounds, have been of therapeutic interest given their ability to modulate the metabolism of the endogenous ligands of the ECS. To determine the therapeutic potential of isoflavonoids, we screened several candidate compounds (Genistein, Biochanin‐A, and 7‐hydroxyflavone) in silico to determine their binding properties with fatty acid amide hydrolase (FAAH), the primary degrative enzyme for AEA. We further validated the ability of these compounds to inhibit FAAH and determined their effects on depressive‐like and locomotor behaviors in the forced swim test (FST) and open field test in male and female mice. We found that while genistein was the most potent FAAH inhibitor, 7‐hydroxyflavone was most effective at reducing immobility time in the forced swim test. Finally, we measured blood corticosterone and prefrontal cortex AEA concentrations following the forced swim test and found that all tested compounds decreased corticosterone and increased AEA, demonstrating that isoflavonoids are promising therapeutic targets as FAAH inhibitors.


| INTRODUC TI ON
Modulation of the endocannabinoid system (ECS) for therapeutic benefits has been a focus since the discovery of cannabinoid receptors and their endogenous ligands. 1,2 The two principal cannabinoid receptors, CB1R and CB2R, are G-protein coupled receptors located in the central nervous system as well as in the periphery. 3 The two primary endogenous ligands, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are produced on-demand in the brain and activate the CB receptors. 4 The diversity in location of the receptors, and complexity of their downstream activity in neural circuits, leads to modulation of numerous aspects of synaptic activity. 5 Ultimately, the ECS regulates several physiological and pathological conditions including immunomodulation, pain, addictive behaviors, cognition, sociability, and stress responses. 6,7 Substantial preclinical and clinical evidence implicate ECS in depressive disorders. 3,8 CB1 receptors and the enzymes involved in AEA synthesis are highly expressed in the limbic system of the brain, including prefrontal cortex, hippocampus, amygdala, and thalamus. 9 Moreover, the functional activity of these regions is modulated by endocannabinoids, ultimately effecting mood and emotional behavior. 10 The ECS not only modulates neuronal circuits, but also alters several hormones that play a vital role in mood disorders 11 and endocannabinoids can act as anti-inflammatory agents by cyclooxygenase inhibition. 12,13 As such, modulation of the ECS has the promise to help treat and prevent depressive disorders by several mechanisms.

Several medicinal plants and their extracts have been used for
generations as natural remedies to treat depression 14 and some (Ginkgo biloba, St. John's wort, Valerian) have been validated in preclinical in vivo and in vitro studies. [15][16][17] Isoflavonoids are one such example, being identified as plant-derived compounds that target the ECS by modulating the metabolism of endocannabinoids. In particular, AEA bioavailability in the brain is controlled by the metabolic activity of the enzyme fatty acid amide hydrolase (FAAH). FAAH rapidly breaks down AEA into arachidonic acid and ethanolamide to limit AEA's ability to regulate neural transmission. 18,19 Therefore, enhancing ECS signaling can be achieved by FAAH inhibition.
We conducted this study to explore the therapeutic potential of three specific isoflavonoid compounds-7-hydroxyflavone, biochanin-A, and genistein-identified using an in-silico drug discovery platform to detect putative FAAH inhibitors. Using a mouse model, we find that each of these compounds not only inhibits FAAH activity in vitro, but also reduces immobility time in the forced swim test, a common test for antidepressant efficacy. Furthermore, isoflavonoid-treated animals had elevated AEA and decreased corticosterone following the forced swim test, demonstrating the efficacy of these compounds to regulate brain and behavior. Our data highlight the utility of in silico drug screening methods and identify isoflavonoids as potential novel therapeutics.

| In silico docking analysis
Molecular docking analysis was performed to predict the probable binding affinity between ligand and receptor and to portray distinct binding modes. The Molecular Operating Environment (MOE) software (http://www.chemc omp.com), (version 2015.10) was used to describe and predict compound binding interaction with fatty acid amide hydrolase (FAAH). The crystallographic X-ray structure of FAAH coupled with inhibitor PF-750 (PDB ID: 2VYA) 20 ; was downloaded from the Protein Data Bank (http://www.rcsb.org). The unwanted/extra chain and water molecules were removed. Using default parameters, energy was minimized, and hydrogen was added. 21 Genistein, biochanin-A, and 7-hydroxyflavone were screened through molecular docking. The molecular structures for these compounds were obtained from the PubChem database 22 and MAPS database. 23 The docking algorithm was authenticated by redocking the co-crystallized ligand PF-750 in the FAAH active site. Subsequently, a refinement induced-fit method was performed, allowing both ligand and receptor to move freely. The positions were rescored by GBVI/ WSA dG scoring function. The cognate redocking was performed to validate the docking protocol and RMSD value of co-crystallized ligand was calculated. 24 Ultimately, docking scores, best poses, and two-and three-dimensional structures were recorded.

| FAAH inhibitor screening assay
Analysis of FAAH inhibition was performed using the FAAH Inhibitor Screening Assay Kit (Cayman Chemicals, Cat No. #10005196) according to manufacturer instructions. This kit is a fluorescence-based method for screening FAAH inhibitors and has been successfully used by others. 25,26 Agents were tested in triplicate and the average fluorescence of each was calculated. The percentage inhibition for each agent was calculated using the following formula: swim test and found that all tested compounds decreased corticosterone and in-   33 The effect was measured after chronic administration of drugs, that is, 14 days of compounds/drug treatment followed by forced swim test. The minimum, maximally effective dose based on immobility time in forced swim test was selected.

| Animal behavior testing
Once assigned to an experimental group, mice underwent 14 con- and testing, mice were immediately euthanized by cervical dislocation to obtain blood and brain samples for biochemical analysis.

| Open field test
The open field test was performed as described previously 36

| Forced swim test (FST)
The FST was performed as described previously. 37 The clear polycarbonate cylinder (20 cm diameter, 30 cm high) was filled with water to a height of 15 cm and water temperature was maintained between 23 and 25°C. Mice were held by the tail and slowly lowered into the water to prevent the animal's head from submerging. Mice were video recorded by a side-facing camera for a period of 6 min, at which point animals were removed from the water, dried, and placed back into their home cage. 38 Time spent immobile during the last 4 min of the test was manually quantified by an experimenter blind to experimental group as described in. 37

| Corticosterone measurement
Blood samples were collected from mice immediately following the #K014-H5) was used according to manufacturer instructions.

| Anandamide quantification
Brain samples were collected from mice immediately following the FST on the 14th day and the prefrontal cortex was excised and flash frozen.
Samples were stored at −80°C until analyzed. Anandamide measurement from brain samples was performed using liquid chromatography/ tandem mass spectrometry as previously described. 39 Frozen brain samples were homogenized in glass tubes containing 2 ml of acetoni- Anandamide quantification was conducted by liquid chromatography/ tandem mass spectrometry on a Eksigent Ekspert micro liquid chromatographer 200 coupled to an AB SciexQtrap5500 mass spectrometer, which was outfitted with a Turbo V Spray ion source at the Southern Alberta Mass Spectrometry Centre at the University of Calgary, as previously described. 39 AEA concentration (in pmol/μl) was normalized to brain sample weight for statistical analysis and graphing.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism (version 8.0.1) software and results were considered significant if p < .05. In vivo and ex vivo data were analyzed by two-way ANOVA with sex and treatment as factors. Specific group comparisons were tested using Tukey's HSD to compare the effect of treatment, as there was no effect of sex, or interaction, for any measure. Data are represented as the mean ± standard error of the mean.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guide topha rmaco logy.
org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, 40 and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20. 41

| In silico analysis of FAAH inhibitors
We

| In vitro analysis of FAAH inhibitory activity
To determine the extent to which 7-Hydroxyflavone, Biochanin-A, and Genistein were able to inhibit FAAH activity, we performed a FAAH inhibition assay using a commercially available ELISA-based kit. All three compounds were tested and compared to the standard   (Figure 2A-D).

| In vivo analysis of locomotion and depressive-like behaviors
To assess the therapeutic potential of biochanin-A, genistein, and  Figure 4C).

| Ex vivo analysis of anandamide and corticosterone concentrations
Finally, to determine the efficacy of each isoflavonoid compound in modulating endogenous AEA and corticosterone, we collected the brains and blood from male and female mice immediately after the forced swim test. We isolated the prefrontal cortex and used mass spectrometry to determine the AEA concentrations and used a commercially available ELISA kit to measure corticosterone concentrations in the serum of blood samples.

| DISCUSS ION
The current results demonstrate that selected isoflavonoid compounds-7-hydroxyflavone, biochanin-A, and genistein-have which is predicted to block the conformational changes necessary for FAAH activity upon ligand binding. Thus, all three compounds were predicted to be efficacious FAAH inhibitors.
Our data confirm the in silico predictions and demonstrate that all three compounds are able to inhibit FAAH activity in vitro in a dose-dependent manner. We found that genistein was the most potent inhibitor, followed by biochanin-A and 7-hydroxyflavone. The data for genistein are in agreement with another study showing that genistein is a competitive FAAH inhibitor in vitro, 48   As such, our current findings mirror this established relationship between prefrontal AEA signaling and stress reactivity and further highlight the link between the ECS and the biochemical/behavioral response to stressors, while also demonstrating the efficacy of isoflavonoids to regulate these processes. Similarly, isoflavonoid treatment significantly reduced blood corticosterone concentrations in all groups. While an earlier study reported that fluoxetine treatment did not alter corticosterone concentrations following the forced swim test, 64 we did find a decrease in corticosterone in fluoxetinetreated animals in this study. Furthermore, the decrease observed in isoflavonoid-treated animals is corroborated by previous studies suggesting that isoflavonoid-modulation of FAAH activity facilitates fear extinction learning 65 and decreases circulating corticosterone 66 Similarly, arch-5HT was reported to be involved in normalization of HPA axis and regulation of plasma corticosterone levels. 67 It is important to note that we did not detect sex differences in any of the measures reported here. While depressive disorders are far more prevalent in women than in men, 68 we found that the effects of isoflavonoid treatment on behavior, AEA and corticosterone concentrations were equally effective in non-stressed male and female mice. While no quantitative differences in immobility time between male and female mice have been previously reported, it is possible that there may be qualitative differences in the expression of the behavior, such as head swinging behavior, that we did not quantify here. 69 Together our data highlight the utility of using in silico techniques to identify novel protein-ligand interactions by screening natural compounds for putative therapeutic utility. Our in vitro screening and in vivo rodent behavioral assessments demonstrate that isofla-

ACK N OWLED G M ENTS
The authors thank Dr. Todd Gould for providing technical expertise and equipment to perform the behavioral analyses. The authors also acknowledge the Southern Alberta Mass Spectrometry Centre,

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data that support the findings of this study are available within the article andits supplementary materials, at https://doi.org/10.1002/prp2.999

D I SCLOS U R E
The authors declare no conflict of interest.

E TH I C A L S TATEM ENT
All experiments were performed in accordance with the Institutional