Emerging research into the endocannabinoid system (ECS) over recent decades has benefitted from the advantages of advanced techniques in medicinal chemistry and structural analysis to elucidate a practical illustration of its role in lipid-mediated neurotransmission and eicosanoid regulation. Eicosanoids are derivatives of arachidonic acid (AA) supplied by the action of phospholipase A2 cleavage from glycerophospholipids of the plasma membrane that further undergo intracellular biosynthetic processing involving oxygenation by enzymes cytochrome P450 (CYP), cyclooxygenase 1 and 2 (COX-1/2), or lipoxygenase (LOX). Subsequent regionally specific enzymatic redox alterations occur to form lipid autacoids prostaglandins (PG), prostacyclins (PGI2), thromboxane (TX2), leukotrienes (LT), and lipoxins (LX). Eicosanoids target specific G-protein coupled receptors responsible for a diverse range of homeostatic functioning including smooth muscle control of cardiovascular and pulmonary systems, as well as immune system regulation during inflammation (Hilal-Dandan 2014). Similarly, endocannabinoids (eCBs) are derived from AA containing membrane phospholipids, yet retain unique moieties to the acyl group that postpone the immediate entrance into eicosanoid synthesis and provide them with an affinity for a specific class of inhibitory Gi/o-protein coupled receptors known as cannabinoid receptor type-1 and type-2 (CB1/CB2), so named for its affinity for exogenous ligand (-)-trans-9-tetrahrdocannabinol (THC). Other eCB target receptors include GPR119, GPR55, transient receptor potential vanilloid type-1 (TRPV1) and nuclear receptor peroxisomal proliferator-activated receptor α (PPARα) (Pistis 2017). The discovery of two principle eCBs N-arachidonoylethanolamide (anandamide, AEA), a partial CB1/CB2 agonist, and 2-arachidonoylglycerol (2-AG), a full CB1/CB2 agonist, has generated an immense interest in defining the role of the ECS in pathophysiological disorders. The Center for Drug Discovery (CDD) at Northeastern University has contributed a wide range of publications concerning biophysical properties of endocannabinoids, including numerous synthetic analogues catalogued as AM-compounds, which have served as invaluable investigative tools in preclinical studies (Makriyannis 2014).

Unlike other neurotransmitters that are stored in vesicles after synthesis (i.e. catecholamines), endocannabinoids are rapidly synthesized “on-demand” by stimulated intracellular calcium release or depolarization and quickly degraded to free their AA component for downstream eicosanoid synthesis (Figure 1-a). While the biosynthesis for 2-AG appears to be the direct action of phospholipase-C β (PLCβ) cleavage of inositol-tri-phosphate (IP3) from arachidonoyl-containing phosphatidylinositol 4,5-bisphosphate (PIP2) and subsequent hydrolysis of the diacylglycerol by diacylglycerol lipase (DAGLα/β), the biosynthesis pathway of AEA is shared among N-acyl ethanolamines (NAEs), whereby Ca²⁺-dependent N-acyltransferase (Ca-NAT) transfers a fatty acid (i.e. palmitate, stearate, oleate, linoleate, arachidonate; Figure 1-b) from another glycerophospholipid to the primary amine of a membrane phosphatidylethanolamine to produce a N-acyl phosphatidylethanolamine (NAPE) precursor, which then undergoes subsequent hydrolysis of glycerol substituents by multiple enzymes, the most well studied of which is N-acyl phosphatidylethanolamine-phospholipase D (NAPE-PLD) that directly liberates the NAE from the phosphate group (Rahman 2014). Degradation of endocannabinoids involves the hydrolysis of the acyl moiety to free the fatty acid from the substituent group as 2-AG is primarily degraded by monoacylglycerol lipase (MGL) or αβ-domain hydrolases 6 and 12 (ABHD6 and 12) with a glycerol by-product and NAEs are degraded primarily by fatty acid amide hydrolase (FAAH) or lysosomal NAE-hydrolyzing acid amidase (NAAA) with a by-product of ethanolamine. Consequently, the AA of endocannabinoids are subject to similar oxygenase activity of eicosanoid synthesis whereby 2-AG may be converted to prostaglandin PGE2-glycerol ester by COX-2, while AEA may be converted to prostamides by COX-2, hydroperoxy-eicosatetraenolyethanolamides by LOX, or hydroxy-eicosatetraenolyethanolamides by CYP-450 (Rahman 2014). A focus of pharmacological research of the ECS has been the inhibition of degrative enzymes to raise the levels of endocannabinoids and prolong their mechanisms of action.

Figure 1: a. Synthetic and Degradative Fates for Arachidonic Acid and Endocannabinoids AEA and 2-AG for Select Enzymatic Pathways as described in the text. b. Structures of possible N-acyl Ethanolamides (Adapted from Hilal-Dandan 2014, Rashman 2014)

The intrinsic pharmacokinetic (PK) and pharmacodynamic (PD) profile for eCBs, including high lipophilicity (clogP=6.3) and short half-life (t_1/2<5min), present a series of challenges in developing a reliable schematic outlining the neurotransmitter’s cycle, however, in the CNS, eCBs are determined to operate as a retrograde signal with post-synaptic release targeting presynaptic CB1 receptors to repolarize the pre-synaptic neuron and decrease excitatory neurotransmission as evidenced by post-synaptic localization of synthetic enzymes and correlation of post-synaptic Gq/11-linked GPCRs that mediate intracellular Ca²⁺ release (Figure 2) (Ahn 2008, Lu 2017). Several mechanisms have been proposed for eCB transport across the membrane including facilitated diffusion dependent on an inward concentration gradient generated by increased enzymatic degradation and caveolae lipid rafts for endocytosis as well as carrier proteins such as albumin, heat shock protein (Hsp70), fatty acid binding proteins (FABPs), and FAAH-like anandamide transporter protein (FLAT) required for intracellular trafficking to membrane bound enzymes and receptors (MacFarland 2004, Seillier 2017). Studies have concurred membrane transport as a saturable process and the use of synthetic probes that show weak inhibition of degradative enzymes but inhibit transport suggest antagonism of a putative membrane transport protein that operates bi-directionally, regulating both eCB uptake and release. Chicca et al. performed a battery of assays on U937 cells (myeloid cells with active eCB uptake) using radiolabeled [H³]AEA to measure transport in the presence of proposed putative eCB-membrane transporter (EMT) inhibitors UCM707 and OMDM-2 to demonstrate increased levels of extracellular AEA comparable to the use of FAAH inhibitors URB597 or PMSF (phenylmethylsulphonyl fluoride) alone as well as showing a synergistic result when used in combination; further investigation included the role of FABPs, which had been a suspected target of EMT inhibitors, using U937 macrophages that show increased FABP phenotype compared to U937 monocytes as both cell lineages demonstrated similar AEA uptake inhibition in presence of UCM707 and OMDM-2 as well as an efflux function of the EMT from radioligand loaded U937 cells as EMT-inhibitors blocked release (Chicca et al. 2012). Interest in the biophysical configuration of the AA component of eCBs has been a parameter of distinction when considering free-ligand membrane kinetics and bound-ligand conformation to receptors or enzymes. Tian et al. designed an NMR study recording coupling interactions between specific radiolabeled atoms of di-palmitoylphosphatidlycholine (DPPC) multilamellar bilayers to radiolabeled atoms on AEA to establish among the three hypothetical ligand conformations (U-shaped, J-shaped, or extended; Figure 3-d.) that AEA maintains an extended conformation within the membrane bilayer with ethanolamine head group level with polar phosphate groups and terminal n-pentyl chain stabilized toward the bi-layer center, which further purports a mechanism of activation for CB1 receptors via lateral diffusion to an entry port within transmembrane helices 3 and 6 (Tian 2005).

Figure 2. General Schematic of Endocannabinoid Retro-signaling (at glutamatergic synapse) as described in the text. (Adapted from Ahn 2008, Lu 2017, and Melis 2017)

As stated, characterization of eCB receptors and preeminent degradative enzymes of the ECS has demanded an extensive library of synthetic analogues and, seeing to that objective, the CDD at Northeastern University has produced many structure activity relationship studies. Novel AEA analogues had been synthesized to stress the importance of the hydrophobic tail in CB1 affinity as a Wittig olefination of the final AA cis double bond by phosphonium salts of phenyl, aryl and furyl groups with varying methylene chains concluded lengths of 4 carbons showed the greatest affinity when compared with 2, 3, and 5 carbon chains (Figure 3-c) (Yao 2008). Likewise, preparation of hypothesized metabolically stable analogues of AEA have involved methylation of the C7, C10 and C13 positions of the tetra-olefinic chain to anticipate a hydrogen abstraction step in the enzymatic oxygenation process of COX-2 (Papahatjis 2010). COX-2 is an intracellular membrane localized homodimer with two active sites, Oxidation of a heme prosthetic group by hydroperoxide in one monomer oxidizes a tyrosine (Y385) to form a tyrosyl radical that abstracts the (C)13-pro-(S)-hydrogen from the AA olefinic chain. This gives a carbon-centered radical that gets trapped at C11 by molecular oxygen to form an 11-(R)- peroxyl radical. Production of a bicyclic endoperoxide relocates the radical towards the terminal alkyl chain to enable the formation of a peroxyl radical at C15 by incorporation of another molecular oxygen. The peroxyl radical re-abstracts the hydrogen from Y385 to reform the tyrosyl radical and produces a hydroperoxyl endoperoxide prostaglandin that enters the second active site where the 15-hydroperoxide group is reduced to an alcohol (Figure 3-b) (Hermanson 2014). In a follow up experiment testing the metabolic stability of 13(S)-methyl anandamide (AM-313), Kudalkar et al. found that, while AM-313 demonstrated significantly reduced oxygenation, 13(S)-methyl arachidonic acid (AM-8138) proved complete metabolic stability and instead demonstrated affinity for an allosteric site on the companion monomer to potentiate oxygenation of the poor substrate 2-AG (Kudalkar 2015). Solving the crystal structure for AM-8138 bound COX-2, the catalytic site was further characterized in mutagenesis assays noting the restoration of 2-AG oxygenation for Y355A, R120A, R120Q, and R120a/Y135A mutants in the presence of AM-8138; suggesting, according to steady-state kinetic assays, that 2-AG participates in a substrate dependent inhibition that AM-8138 relieves via a higher affinity for the allosteric site (Kudalkar 2015).

The serine protease enzyme FAAH has also garnered interest as a potential target to modulate levels of eCBs as analgesic, anxiolytic, antidepressant, and anti-inflammatory effects have been observed in FAAH knock-out mice and possible therapeutic intervention might provide greater specificity without side effects of hypothermia or catalepsy as observed with direct CB1 agonism (Ahn 2008). Structural analysis suggests FAAH is an intracellular bound homodimer that possesses the unique amidase signature domain (AS; conserved among serine proteases) of a Serine-Serine-Lysine (S241-S217-K142) catalytic triad in a pocket of hydrophobic residues termed the “acyl-chain binding” channel, whereby S241 becomes activated for nucleophilic attack to the substrate acyl group by K142 via S217 and, as the resulting tetrahedral intermediate proceeds to eliminate the leaving group that exits via a cytosolic port channel, the acyl-enzyme intermediate undergoes hydrolysis to restore the original formal charges on both the amino acids and the acyl moiety of the fatty acid (Figure 3-a) (Ahn 2008, Mileni 2010). Surmounting evidence on the efficacy of FAAH inhibitors in preclinical trials enforce the importance of eCBs in mediating tonic and phasic signaling in the CNS, with AEA operating as a tonic mediator while 2-AG operates in phasic signaling, as pathologically induced expression of postsynaptic FAAH relates to overexcitation of sympathetic neural pathways. For instance, AM-3506, an analog of PMSF, with a selective action on CNS over peripheral FAAH, has demonstrated the effect of reduced norepinephrine in hypertensive rats leading to a decrease in mean arterial pressure and heart rate where it’s effect could be reversed by CNS-penetrant CB1 antagonists but not peripherally restricted CB1 antagonists (Godlewski 2010, Gunduz-Cinar 2013). Consideration that access to the catalytic site requires hydrophobic characteristics of the substrate presents a credible obstacle in drug design concerning lipophilicity. FAAH inhibitor mimicry of substrate moieties have included long chain unsaturated fatty acids as well as α-keto-heterocycles and carbamates with lipophilic tails to produce reversible and irreversible inhibitors. Unfortunately, as investigated by Zhuang et al., trifluoromethyl ketone FAAH inhibitors AM5206 and AM5207 demonstrate albumin binding behavior, which indicates a preference for compartmentalization (Zhuang 2013). As FAAH inhibitors enter clinical trials, the PK and PD profile in humans may prove different than in preclinical organisms as it may have for BIA 10-2474, which experienced serious adverse events including one death during phase I clinical trials due to suspected toxic accumulation of non-excretable metabolites (Mallet 2016).

Figure 3. a. Mechanism of FAAH Ser-Ser-Lys Catalytic Site (Adapted from Ahn 2008, Mileni 2010) b. Mechanism for Cyclooxygenase Catalytic Site (Adapted from Hermanson 2014) c. Synthetic Approach to Endocannabinoid Structure including Methylation sites 7,10 & 13 and Novel Tail Chains (Adapted from Papahatjis 2010, Yao 2008) d. In vivo Endocannabinnoid Conformations (Adapted from Tian 2005)

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