Phytocannabinoid Biochemistry

Phytocannabinoids are terpenophenolic metabolites produced in the glandular trichomes of Cannabis sativa L. In lieu of ongoing taxonomic debate, subspecies are classified as varieties: var. sativa, var. indica, and var. ruderalis (Kinghorn et al. 2017, ElSohly et al. 2017). Of the 565 natural products identified from C. sativa L., 120 are considered phytocannabinoids, which utilize the prefix phyto- to distinguish their origin from endogenous (endocannabinoids; eCB) and synthetic cannabinoids and share the characteristic biosynthetic step of polyketide prenylation by geranyl pyrophosphate (GPP) to olivetolic acid (OA) that produces a 21-carbon scaffold (Kinghorn et al. 2017, ElSohly et al. 2017). Geranyl pyrophosphate is a resulting monoterpene from the condensation of isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are isomers produced via either the mevalonic acid (MVA) or methylerythritol 4-phosphate (MEP) pathways (Gagne et al. 2012, Rodriguez-Concepcion et al. 2002). The MVA pathway occurs in the cytoplasm beginning with the condensation of two acetyl-CoA precursors, whereas the MEP pathway occurs in plastid organelles and involves the initial condensation of pyruvate and glyceraldehyde 3-phosphate precursors; while the MEP pathway is believed to account for the majority of GPP production, both pathways are exhibited in C. sativa L. (Gagne et al. 2012, Rodriguez-Concepcion et al. 2002; see Figure 1). Olivetolic acid is a polyketide moiety resulting from the type III polyketide synthase (PKS) catalyzed condensation of one hexanoyl-CoA with three malonyl-CoA units that also undergoes cyclization via olivetolic acid cyclase (OAC), a unique dimeric α/β-barrel protein (Gagne et al. 2012). The ultimate condensation of GPP and OA is catalyzed by the aromatic prenyltransferase known as geranyl pyrophosphate: olivetolate geranyltransferase to produce the phytocannabinoid precursor cannabigerolic acid (CBGA), which retains the carboxylic acid function (see Figure 2).

Figure 1: Biosynthetic Pathways Mevalonate (MAP) and Methylerythritol 4-Phosphate (MEP) for Cannabinoid Terpene Precursors Isopentyl Pyrophosphate (IPP) and Dimethylallyl Pyrophosphate (DMAPP)

Figure 2: Biosynthetic Pathways for Geranyl Pyrophosphate and Olivetolic Acid with resulting Condensation to Cannabigerolic Acid.

From the CBGA precursor, cannabinoids may transform into several general class types including (-)-Δ⁹-trans-tetrahydrocannabinol (Δ⁹-THC), (-)-Δ⁸-trans-tetrahydrocannabinol (Δ⁸-THC), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), cannabidiol (CBD), and cannabinodiol (CBND) among others, via enzymatic cyclization and non-enzymatic decarboxylation (Kinghorn et al. 2017, ElSohly et al. 2017; see Figure 4A). Crystallographic and mutagenic studies have confirmed residues of an enzyme called THCA synthase to participate in stabilizing the CBGA precursor while abstracting the phenolic hydrogen and allowing a covalently linked molecule of flavin adenine dinucleotide (FAD) to abstract a hydride from the C-1 carbon to catalyze cyclisation of THCA (ElSohly et al. 2017; see Figure 3A). Interestingly, both the product, THCA, and byproduct, hydrogen peroxide (H₂O₂) from the oxidation of FADH₂ to reform FAD, are toxic and some suggest the selective benefit may be a defense mechanism, especially as reactions are localized to the glandular trichomes that form superficial to the epidermis. Nevertheless, the enzyme CBDA synthase was cloned to reveal similar flavinylated residues as THCA synthase but is suggested to involve stereoselective hydrogen abstraction prior to ring closure that results in the retention of the aromatic diol (ElSohly et al. 2017; see Figure 3B). While cannabichromenic acid (CBCA) is also the product of a specific CBCA synthase reaction from CBGA, other class types, such as CBN and CBND, are derivatives of pre-synthesized types, THC and CBD respectively, whereas cannabivarin type homologues, which are cannabinoids with n-propyl chains in place of n-pentyl chains, are suggested to result from the prenylation of divarinolic acid to form precursor cannabigerovarinic acid (CBGVA), a 19-carbon terpenophenolic compound (ElSohly et al. 2017).

Figure 3: A) THCA Synthase Mechanism to transform CBGA to THCA. B) Comparison of Regioselection between THCA Synthase and CBDA Synthase

Phytocannabinoid Pharmacology

While the exploration of the medicinal properties of cannabis pre-date recorded history, the isolation and characterization of THC by R. Mechoulam in 1964 led to the discovery of a G_i/o sub-class G-protein coupled receptor (GPCR) expressed primarily on synaptic terminals of the central nervous system (CNS), thereafter named cannabinoid receptor 1 (CB1; Maayah et al. 2020, Maccarrone et al 2015). The discovery of CB1 supported evidence of endogenous ligands to interact with the same receptor, thereafter, known as endocannabinoids (Maayah et al. 2020, Maccarrone et al 2015). Endocannabinoids (eCBs) are lipid mediators, primarily derivatives of arachidonic acid (AA), that operate in retrograde neurotransmission and autocrine/paracrine inflammatory processes by two predominant endogenous ligands known as arachidonoylethanolamide (AEA) and 2-arachidonoyl-glycerol (2-AG) (Maccarrone et al 2015; see Figure 4B). Other saturated, mono-, and polyunsaturated lipids may also undergo conjugation to ethanolamine, collectively known as N-acyl ethanolamines (NAEs), whose dynamics are consistent with eCB activity, and a subject incorporated into much eCB research (Maccarrone et al 2015). Cannabinoid receptor 2 (CB2) was discovered via cloning experiments to show homologous amino acid sequence and similar ligand affinity as CB1 but later showing to maintain a unique expression profile primarily to reproductive tissue of both genders, immune tissue, including the tonsils, spleen, and thymus, and immune cells, such as leukocytes and macrophages. Generalizations aside, CB1 has been identified in the periphery (i.e. cardiac, gastrointestinal, and reproductive tissues), while splice variants of CB2 have been identified in neural tissue of the CNS, yet the majority of inducible CNS CB2 remains the product of microglia and other CNS immune cells (Turcotte et al. 2016, Atwood et al. 2012, Jordan et al. 2018, Svizenka et al. 2008, Liu et al. 2009). Class A G_i/o GPCRs, such as CB1 and CB2, are seven transmembrane proteins with extracellular N-terminal, intracellular C-terminal, extracellular loops for ligand interactions, and intracellular loops for interactions with a heterotrimer G-protein as well as participating in other signaling events, such as G-protein receptor kinase (GRK) phosphorylation of intracellular residues for the recruitment of β-arrestin mediated receptor termination and endocytosis (Morales et al. 2017, Goodman et al. 2013, Porter-Stransky et al. 2017). Conformational change allows exchange of GDP to GTP to dissociate the heterotrimer from the GPCR, whereby the G_i/oα subunit proceeds to inhibit adenylyl cyclase production of cAMP while the G_i/oβγ subunits proceed to modulate G-protein inwardly rectifying potassium channels (GIRKs), activate mitogen-activating protein kinases (MAPK), and mobilizes phospholipase C (PLC) to produce diacylglycerol (DAG) and inositol triphosphate (IP₃) to stimulate phospholipase A2 (PLA2) activity and intracellular calcium release (Goodman et al. 2013; see Figure 5C). In sum, G_i/o GPCRs function to attenuate cellular activity and hyperpolarize resting potential, yet, interestingly, while CB2 does not show GIRK activity, GPCRs are known to form heterodimers that increase variety of effector events (Atwood et al. 2012, Mackie et al. 2005).

Figure 4: A) Chemical Structures for Phytocannabinoids - iterative of the olivetolic and divarinolic derivatives. B) Primary Endocannabinoids.

Parallel the interactions with CB1 and CB2, cannabinoids demonstrate a dynamic polypharmacology with a series of effector targets that comprise a ‘non-cannabinoid’, or ‘CB1/CB2-independent’, system responsible for many of their therapeutic qualities (Soderstrom et al. 2017, Morales et al. 2017, Maayah et al. 2020). Deorphanized GPCRs GPR55, GPR18, GPR119 and GPR35, have demonstrated affinity for phytogenic, endogenous, and synthetic cannabinoids to offer unique mechanisms to treat disease; for instance, cholangiocarcinoma cells expressing CB1 and GPR55 show antiproliferative effects of AEA administration with selective CB1 antagonist (Soderstrom et al. 2017). Other families of GPCRs, such as opioid (µ, δ, and κ), serotonin (5HT_1A), and adenosine (A_2A) receptors have demonstrated analgesic, anxiolytic, and anti-inflammatory effects under phytocannabinoid administration, while research suggests ligand-gated ion channels such as N-methyl-D-aspartate (NMDA), α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic (AMPA), glycine, γ-aminobutyric acid (GABA_α), and nicotinic-cholinergic receptors primarily interact with endocannabinoids (Soderstrom et al. 2017; see Figure 5A,5B). A superfamily of transmembrane ion-channels, known as transient receptor potential (TRP) channels, consists of six subfamilies: vanilloid (TRPV), ankyrin (TRPA), melastatin (TRPM), canonical (TRPC), polycystin (TRPP), and mucolipin (TRPM); three of which afford six channels (TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8) known to interact with cannabinoids and have thence been labeled ‘ionotropic cannabinoid receptors’ (Muller et al. 2018; see Figure 5B). Structural distinctions aside, including varying N-terminal ankyrin repeat domain (ARD), inner channel coiled-coil motif, and C-terminal ‘TRP box’ domain, all TRPs transduce mono- and/or divalent cations across plasma membranes of either cell membrane or organelles, such as the endoplasmic reticulum, in response to physical, chemical, and/or pathological stimulus. They are primarily expressed in neural tissue of the CNS and peripheral nervous system (PNS), but have been identified in epithelial cell, osteoblasts, blood vessels, as well as organ such as heart, liver, and reproductive tissue (Morales et al. 2017, Muller et al. 2019). Nuclear receptors called peroxisome proliferator-activated receptor (PPAR) that are transcription factors for metabolism and cell differentiation exist as α-, δ-, and ϒ-subtypes and studies have shown anti-proliferative effects via agonism by cannabinoids (Soderstrom et al. 2017; see Figure 5D). Other interactions of cannabinoids involve indirect agonism/antagonism via modulation of membrane neurotransmitter transporters, metabolic enzymes, and intracellular mediators such as antioxidants, which may be of increased consideration to full-spectrum cannabis extracts that include multiple phytocannabinoids and terpenes above a threshold of 0.5%v/w to enhance therapeutic efficacy by a so-called ‘entourage effect’ (Soderstrom et al. 2017, Maayah et al. 2020).

Figure 5: Diagrammatical Depiction of Phytocannabinoid Targets. A) Ligand-gated Ion Channels with Inhibitory Effect, B) Ligand-gated Ion Channels with Excitatory Effect, C) G_i/o-coupled GPCR (i.e. CB1, MOR), D) Nuclear Receptor Pathway (Fatty Acid Binding Protein; FABP), E) Active Transport Channel.