# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 5 CB Receptors and Their Ligands
### Classical Cannabinoid Receptors and Their Ligands  
Class A GPCRs CB1 and CB2 have been extensively studied in recent years and confirmed as cannabinoid targets. CB2 shares 44% sequence identity with CB1 [91]. CB1 and CB2 have been implicated in many pathological processes, such as pain, epilepsy, anxiety, depression, Parkinson's and Huntington's diseases, amyotrophic lateral sclerosis, stroke, cancer, drug dependence, glaucoma, autoimmune uveitis, osteoporosis, sepsis, and hepatic, renal, intestinal and cardiovascular disorders [92]. Although phylogenetically related, they are distributed differently. CB1 is expressed all over the body and to a large extent in the central nervous system (CNS), while CB2 is expressed predominantly in the immune system, and less so in the CNS [93; 94; 95; 96; 97]. Due to this difference, it is generally accepted that many side effects of agonist cannabinoid come from CB1 activation [92]; thus there is value in screening a derivatized cannabinoid library to discover cannabinoids with specific binding to CB2 or other candidates, in order to avoid off-target effects. In terms of structural data, CB1 and CB2 structures have only been resolved in the recent decade. CB1 X-ray crystal structures came earlier (PDB 5 TGZ [98], 5 XRA, 5 XR8 [99], 5U09 [100], 6 KQI [101]), followed by those of CB2 (PDBs 5 ZTY [102], 6 KPC [103]). The cryo-EM structures of both receptors in complex with G proteins were obtained in the past three years: CB1 (PDBs 6N4B [104], 6 KPG [103]); CB2 (PDBs 6 KPF [103], 6 PT0 [105]). GPCRs have seven transmembrane (TM) helices connected by extracellular and intracellular loops. Activation by agonist binding typically involves an agonist binding at an extracellular site formed by gaps between TM bundles, which induces conformational changes facilitating G-protein binding, and leading to signal transduction. Differences in the amino acid sequences of CB1 and CB2 are found in the N-terminal extracellular loop II (ECL2), near the orthosteric ligand binding site, C-terminal of TM7, and C-terminus. Their activation involves key residues termed toggle switches: Phe200 and Trp35 in CB1; Trp258 in CB2. Large relative movements of TM3 and TM6 occur, which opens up the G-protein binding site. Known ligands of CB1 and CB2 are generally highly lipophilic in nature. There are indications suggesting that these ligands diffuse laterally from the lipid bilayer, instead of the aqueous extracellular phase [106]. Compared to synthetic cannabinoids, which are full agonists of CB1/2, the classical phytocannabinoid (\(-\))-trans-\(\Delta^{9}\)- THC is a partial agonist of both CB1 and CB2 (K\({}_{\text{i}}\) = 10 nM, 24 nM, respectively [107]). The THC chemical structure consists of a tricyclic core (aromatic A-ring, pyran B-ring, and cyclohexenyl C-ring) and various functional groups (Figure 15). The C3\({}^{\prime}\) pentyl side chain on the A-ring has been the subject of many modifications by different research groups. Increasing its linear length increases THC binding affinity to CB1/2 [75; 80], while restricting its flexibility increases affinity when certain conformations are favored. In addition, the substitution of C1\({}^{\prime}\) to bulky ring groups increases affinity and indifference to various heteroatom substitutions, suggesting a CB1 hydrophobic binding subsite near the A-ring, whereas CB2 prefer smaller rings. Removal of the A-ring hydroxyl significantly increases selectivity against CB2. Substitutions of the C-ring C11 methyl group do not affect selectivity between the two receptors but can modulate affinity: Nabilone is a ketone substitution of C11 methyl with high affinity to both CB1/2. The pyran B-ring is not required for CB1/2 activity: CP-55,940, a potent full agonist used as an assay reference, and notably cannabinoid (CBD), has no C-ring pyran core. [PERSON] and [PERSON] provided an excellent comprehensive review regarding various THC analogues and their interactions with receptors [75].