International Journal of Molecular Sciences

Review Targeting Cannabinoid Receptors: Current Status and Prospects of Natural Products

Dongchen An, Steve Peigneur , Louise Antonia Hendrickx and Jan Tytgat * Toxicology and Pharmacology, KU Leuven, Campus Gasthuisberg, O&N 2, Herestraat 49, P.O. Box 922, 3000 Leuven, Belgium; [email protected] (D.A.); [email protected] (S.P.); [email protected] (L.A.H.) * Correspondence: [email protected]



Received: 12 June 2020; Accepted: 15 July 2020; Published: 17 July 2020


Abstract: Cannabinoid receptors (CB1 and CB2), as part of the endocannabinoid system, play a critical role in numerous human physiological and pathological conditions. Thus, considerable efforts have been made to develop ligands for CB1 and CB2, resulting in hundreds of phyto- and synthetic cannabinoids which have shown varying affinities relevant for the treatment of various diseases. However, only a few of these ligands are clinically used. Recently, more detailed structural information for cannabinoid receptors was revealed thanks to the powerfulness of cryo-electron microscopy, which now can accelerate structure-based drug discovery. At the same time, novel peptide-type cannabinoids from animal sources have arrived at the scene, with their potential in vivo therapeutic effects in relation to cannabinoid receptors. From a natural products perspective, it is expected that more novel cannabinoids will be discovered and forecasted as promising drug leads from diverse natural sources and species, such as animal venoms which constitute a true pharmacopeia of toxins modulating diverse targets, including voltage- and ligand-gated ion channels, G protein-coupled receptors such as CB1 and CB2, with astonishing affinity and selectivity. Therefore, it is believed that discovering novel cannabinoids starting from studying the biodiversity of the species living on planet earth is an uncharted territory.

Keywords: cannabinoid receptor type 1 (CB1) and type 2 (CB2); phytocannabinoids; synthetic cannabinoids; structural analysis; animal venoms

1. Introduction Cannabis sativa, commonly known as marijuana, is a plant which has been used throughout human history to treat a wide variety of ailments, such as pain and anxiety, and for recreational purposes. The first natural product isolated from the cannabis plant and then characterized was the phytocannabinoid cannabinol (CBN) [1], followed by the isolation and pharmacological elucidation of the psychoactive ∆9-tetrahydrocannabinol (∆9-THC) and the non-euphoric cannabidiol (CBD) [1,2]. The knowledge about the structure and pharmacology of CBN, ∆9-THC and CBD has led to major breakthroughs in our understanding of the effects of this plant. Insights into the mechanism of the action of phytocannabinoids led to the identification of two G protein-coupled receptors (GPCRs), cannabinoid receptors type 1 (CB1) and type 2 (CB2) [3,4]. Consequently, endogenous ligands of cannabinoid receptors, also known as endogenous cannabinoids or endocannabinoids, were identified [2,5]. Amid the recognized endocannabinoids, anandamide (synonym for N-arachidonoylethanolamine or AEA) and 2-arachidonoyl glycerol (2-AG) were discovered first [6–8]. Thereafter, it became clear that endocannabinoids and cannabinoid receptors are pleiotropic signaling molecules belonging to the endocannabinoid system, which also involves the enzymes that catabolize these compounds [9–12]. This signaling system has been shown to contribute to re-establishing homeostasis after insults,

Int. J. Mol. Sci. 2020, 21, 5064; doi:10.3390/ijms21145064 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5064 2 of 32 which highlights the therapeutic opportunities for multiple pathologies, such as pain, inflammation, cardiovascular regulation, metabolic disorders, cancer and neurodegenerative disorders [2,13]. In addition, CB1 and CB2 have been proven to play a crucial role in various bioactivities of phytocannabinoids [14], indicating the significance of cannabinoid receptors for the therapeutic effects of the cannabis plant. These discoveries subsequently inspired the constant generation of a wide variety of synthetic cannabinoids with similar or distinct structures as compared with endo- and phyto-cannabinoids. Simultaneously with the progress made in the medical field to develop selective CB1 or CB2 ligands that can modulate biological functions and treat associated diseases, some synthetic cannabinoids have become problematic in the field of recreational use, such as SPICE and K2 [15]. As a therapeutic target, CB2 has a remarkable advantage over CB1 regarding its expression pattern. CB1 is mainly expressed in the human central nervous system (CNS) (Figure1), and is the main receptor responsible for the psychotropic effects of ∆9-THC as well as the deleterious psychiatric side effects of drugs targeting CB1 [2,16]. The CB1 inverse agonists rimonabant (SR141716) and taranabant (MK-0364) were developed as anti-obesity drugs, but both produce crippling CNS side effects, such as anxiety, depression, and suicidal ideation [17–19]. As a consequence, they were either withdrawn from the market or dropped in clinical trials. In contrast, CB2 is predominantly expressed in peripheral tissues, such as the immune system, where it modulates immunological function, cell migration and cytokine release [16,20] (Figure1). CB2 expression has also been detected in the brain, albeit to a much lower extent in comparison to the immune system or the level of CB1 expression [16] (Figure1). Notwithstanding a rather limited expression of CB2 in the peripheral nervous system and the CNS, it is undeniable that the CB2 plays an active role in neurological activities, including nociception and neuroinflammation [21,22]. Some CB2-selective agonists have been developed, showing significant efficacy in in vitro assays and in animal models without displaying unwanted psychoactive effects. Examples of such CB2-selective agonists are JWH-015, HU-308 and GW-405833 [23–27]. So far, besides a few phytocannabinoids and their analogs, no other CB targeting drugs have reached the market yet for clinical use. Thus, it is believed that selectively targeting CB2 provides a promising pathway of new drug discovery in the area of natural products for the treatment of a number of disorders while avoiding the severe psychiatric side effects associated with CB1. Recently, structural determination of CB1/CB2 coupled to the Gi protein has revolutionized our understanding of their structures and functions [28,29], alongside a parallel revolution in the methods for determining structures of cannabinoid receptors [30]. For the past few years, X-ray crystallography has been the method of choice for elucidating CB1 and CB2 structures [30–33]. Thanks to the advent of higher resolution cryo-electron microscopy (cryo-EM) structures that eliminate the problem of crystal-packing artifacts and generate an ensemble of structures [34], cryo-EM has now become the primary method in order to obtain ligand-bound CB1 or CB2 in the active state coupled to the heterotrimeric G protein complex [28,29]. Therefore, the activation mechanisms of CB1 and CB2 have been revealed [28,29]. This is expected to facilitate the rational structure-based design/discovery of drugs selectively targeting cannabinoid receptors. In the meantime, some efforts have been made to discover novel cannabinoids over the past few years, leading to the emergence of peptide-type ligands of CB1 and/or CB2 from natural sources, other than the cannabis plant. Examples hereof are hemopressin (Hp) and the related peptides VD-Hpα and RVD-Hpα found in mice, rats or humans, as well as Pep19 derived from peptidyl-prolyl cis-trans isomerase A in humans, showing a variety of in vivo pharmacological effects depending on CB1, e.g., antinociception and neuromodulation [35,36]. Moreover, Pep19 did not exhibit CNS side effects in rats [35]. These peptides represent valuable starting points for the development of peptide drugs targeting cannabinoid receptors. Based on the available literature on cannabinoids, it is evident that natural products have been an important source of CB1 and CB2 ligands. Cannabinoid receptors are one of the primary targets of natural products, since over 600 natural GPCR ligands have been isolated from plants, animals, fungi, and bacteria; they predominantly target aminergic, opioid, cannabinoid, and taste 2 receptors [37]. Int. J. Mol. Sci. 2020, 21, 5064 3 of 32 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 34

Among[37]. Among the diverse the diverse natural natural GPCR GPCR ligands, ligands, nature-derived nature-derived peptides peptides isolated isolated from from bacteria, bacteria, fungi, fungi, plants, andplants, venomous and venomous animals areanimals an emerging are an compoundemerging compound class for GPCRclass for ligand GPCR discovery ligand accordingdiscovery to publishedaccording data to published [37]. Over data 50% [37]. of nature-derived Over 50% of na peptidesture-derived targeting peptides GPCRs targeting discovered GPCRs so discovered far originate fromso far animal originate venoms from [37 animal]. Animal venoms venoms [37]. contain Animal a venoms true pharmacopeia contain a true of peptidespharmacopeia acting of on peptides molecular targets,acting e.g.,on molecular GPCRs, targets, often with e.g., GPCRs, impressive often a ffiwithnity impressive and selectivity affinity [and38]. selectivity Examples [38]. of theExamples value of venomof the peptidesvalue of venom in guiding peptides the in development guiding the de ofvelopment human therapeutics of human therapeutics targeting GPCRstargeting include GPCRs the antidiabeticinclude the exenatide antidiabetic (Byetta exenatide®) from (Byetta the venomous®) from the Gilavenomous monster Gila (Heloderma monster (Heloderma suspectum )[suspectum38] and) the analgesic[38] and cobratide the analgesic (also cobratide known as(also cobratoxin) known as from cobratoxin) the Chinese from cobrathe Chinese (Naja atracobra). None(Naja atra of the). None known venomof the peptides known venom have been peptides described have been as ligands described of cannabinoid as ligands of receptors cannabinoid to the receptors best of ourto the knowledge. best of Therefore,our knowledge. for future Therefore, perspective, for future animal perspective, venoms can animal be seen venoms as a can promising be seen andas a promising yet untapped and sourceyet tountapped find selective source and to potentfind selective ligands and of CB1potent and ligands/or CB2. of CB1 and/or CB2.

FigureFigure 1. 1.Major Major localization localization ofof CB1CB1 and CB2 and and their their asso associatedciated physiology physiology in inthe the human human body. body. CB1: CB1: thethe majority majority of of CB1 CB1 was was found found toto bebe expressedexpressed in in the the brain, brain, where where it it modula modulatestes various various neurological neurological activities.activities. CB1 CB1 is is located locatedin in thethe peripheralperipheral tissues, and, and, although although to to a alesser lesser extent, extent, also also participates participates in in thethe modulations modulations of of local local tissue tissue functions. functions. CB2: the the predominant predominant expre expressionssion of of CB2 CB2 was was revealed revealed to be to be inin the the immune immune system system (such (such asas thethe spleen),spleen), where it it exhibits exhibits the the effects effects of of immune immune modulation, modulation, and and otherother peripheral peripheral tissues. tissues.

InIn this this review, review, we we first first brieflybriefly introduceintroduce the cannabinoid cannabinoid receptors receptors CB1 CB1 and and CB2, CB2, and and then then discussdiscuss the the key key similarities similarities andand diversitiesdiversities of thei theirr activation activation mechanisms mechanisms based based on on the the structural structural informationinformation obtained obtained byby cryo-EM.cryo-EM. Furthermore,Furthermore, we we provide provide an an overview overview ofof the the current current research research statusstatus of of phytocannabinoids phytocannabinoids andand syntheticsynthetic cannabinoids that that have have been been shown shown to tobe beligands ligands of CB1 of CB1 andand/or/or CB2. CB2. InIn the light light of of a anatu naturalral products products perspective, perspective, recently recently emerged emerged novel novelcannabinoids, cannabinoids, i.e., i.e.,peptide-type peptide-type ligands ligands of ofcannabinoid cannabinoid receptors receptors from from animal animal sources, sources, are are summarized summarized and and the the potentialpotential of of animal animal venoms venoms as as a sourcea source of of novel novel cannabinoids cannabinoids is demonstrated.is demonstrated. In In addition, addition, CB1 CB1 or or CB2

Int. J. Mol. Sci. 2020, 21, 5064 4 of 32 expression systems that can be used to rapidly screen unlabeled natural products in vitro are described in the final section.

2. Cannabinoid Receptors CB1 is encoded by the gene CNR1 and consists of 472 amino acids in humans (473 amino acids in rats and mice) [16]. The amino acid sequence identity among these species is 97–99% [16]. CB2 is encoded by the gene CNR2, which consists of 360 amino acid in humans. It shares only 44% sequence homology with CB1 at the protein level [16]. Additionally, CB2 has greater species differences between humans and rodents, compared with CB1, as its amino acid sequence identity among humans and rodents is slightly higher than 80% [16]. CB1 and CB2 are both class A (rhodopsin-like) GPCRs. Generally, the structure of cannabinoid receptors contains seven transmembrane alpha helices (TMHs) arranged to form a closed bundle and loops connecting TMHs that extend intra- and extracellularly [34]. In addition, it contains an extracellular N terminus and an intracellular C terminus that begins with a short helical segment (Helix 8)-oriented parallel to the membrane surface [34]. CB1 primarily couples to Gi/o protein and, under certain conditions, couples to Gs and Gq, while CB2 only couples to Gi/o [34], to trigger the further activation and downstream signaling.

3. Ligand-Bound CB1/CB2-Gi Complex

3.1. Activation Mechanism of CB1 and CB2 Ligand-bound cryo-EM structures of the active cannabinoid receptors in complex with Gi (Figure2A,C) have recently been built and utilized to reveal activation mechanisms of CB1 and CB2. The overall structures of the active CB1-Gi and CB2-Gi complexes are alike [29]. The binding poses of agonists in CB1 and CB2 are superimposable [28,29]. Moreover, the agonist-binding pockets and conformations of critical residues for the receptor activation are almost identical between the active conformations of CB1 and CB2 [29]. On the other hand, intriguing differences in the activation process of both cannabinoid receptors have also been revealed by structural analysis. Firstly, in the case of agonist-bound cannabinoid receptor-Gi complexes, the cytoplasmic region of transmembrane region 5 (TM5) in CB1 is simply extended and moves inward during activation, resulting in more polar and hydrophobic interactions with α5 of the Gαi protein [29]. In contrast, the cytoplasmic portion of the TM5 in CB2 extends and moves outward to form extensive interactions with the α5 helix of Gαi [29]. Secondly, in CB1, TM6 in the intracellular region moves inward to interact with α5 of the Gαi protein [29]. However, a large outward movement of the intracellular part of TM6 in CB2 occurs to accommodate the mounting of α5 from the Gαi protein [29]. In addition, the residues on the cytoplasmic ends of TM5 and TM6 in CB2 shift modestly upward, relative to those of CB1 [28]. The movements of TM5 and TM6 have a certain significance in the activation processes of cannabinoid receptors: an outward movement of TM6 is suggested as a characteristic of cannabinoid receptor activation and an extension of TM5 can result in new interactions with Gαi [29]. Notably, the critical so-called “toggle switch” residues of cannabinoid receptors show differences for both receptors. In CB1, the “twin toggle switch”, F200 and W356 (Figure2B), experiences synergistic conformation changes, while in CB2, the “single toggle switch”, W258 (corresponding to W356 in CB1) (Figure2B), triggers the activation and the downstream signaling [29]. Alternatively, when taking into consideration residue F117 in CB2 (corresponding to F200 in CB1), another way to think about the differences of the toggle switch in the cannabinoid receptors is the following: the distance between F200 and W356 in CB1 is longer than that between F117 and W258 in CB2 (Figure2D) [ 28]. This is a result of the upward position of W356 and the rotation of F200 in CB1 compared with the analogous residues in CB2 [28]. The different arrangement of the toggle switch in CB2 causes a rotation of F202 in its TM5 in comparison with the corresponding L287 in CB1 [28]. In general, CB2 only experiences minor conformational changes upon agonist binding, Int. J. Mol. Sci. 2020, 21, 5064 5 of 32

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 34 while CB1 is exceptional and displays larger conformational changes when modulated by agonists [29]. different states facilitates its inherent ability to respond to a diverse array of ligands compared to CB2 Furthermore,[29]. the high plasticity of CB1 during the transitions between different states facilitates its inherent ability to respond to a diverse array of ligands compared to CB2 [29].

Figure 2. (A) Binding pocket of human CB1-Gi complex bound to agonist AM841 and human CB2-Gi complexFigure bound 2. (A) toBinding agonist pocket AM12033. of human Orange CB1-Gi cartoon, comple CB1x bound structure; to agonist green AM841 cartoon, and human CB2 structure; CB2-Gi yellowcomplex sticks, bound AM841; to agonist orange AM12033. sticks, AM12033. Orange cartoon, (B) Comparison CB1 structure; of thegreen “toggle cartoon, switch” CB2 structure; residue conformationyellow sticks, in human AM841; CB1, orange orange sticks, cartoon; AM12033. and in human(B) Comparison CB2, green of cartoon. the “toggle (C) Binding switch” pocket residue of humanconformation CB1-Gi complex in human bound CB1, toorange agonist cart FUBoon; andand humanin human CB2-Gi CB2, green complex cartoon. bound (C) toBinding agonist pocket WIN 55,212-2.of human Marine CB1-Gi cartoon, complex CB1 bound structure; to agonist purple FUB cartoon, and human CB2 structure; CB2-Gi complex green sticks, bound FUB; to agonist cyan sticks, WIN WIN55,212-2. 55,212-2. Marine (D) Comparison cartoon, CB1 of structure; the “toggle purple switch” cartoon residue, CB2 structure; conformation green insticks, human FUB; CB1, cyan marine sticks, cartoon;WIN and 55,212-2. in human (D) Comparison CB2, purple of cartoon. the “toggle (A) andswitch” (B) areresidue derived conformation from Hua in et human al. [29], CB1, (C) andmarine (D) are derivedcartoon; from and in Xing human et al. CB2, [28]. purple cartoon. (A) and (B) are derived from Hua et al. [29], (C) and (D) are derived from Xing et al. [28]. 3.2. Implications for CB1 and CB2 Ligand Selectivity 3.2.Structure Implications determination for CB1 and ofCB2 cannabinoid Ligand Selectivity receptors coupled to Gi indicates that discovering a selective agonistStructure may determination be a huge challenge of cannabinoid due to similarreceptors binding coupled pockets to Gi in indicates both cannabinoid that discovering receptors. a Nevertheless,selective agonist there is clear-cutmay be a evidence huge challenge that some due highly to similar selective binding CB1 or pockets CB2 agonists in both can cannabinoid be obtained. For example,receptors. arachidonyl-2’-chloroethylamide Nevertheless, there is clear-cut evidence (ACEA) that and some arachidonylcyclopropylamide highly selective CB1 or CB2 (ACPA) agonists are potentcan and be selective obtained. CB1 agonists, For asexample, described furtherarachidonyl-2´-chloroethylamide in “4.2.2. CB1-selective agonists”; (ACEA) JWH-133, and JWH-015arachidonylcyclopropylamide and PM-226 are potent and (ACPA) selective are CB2potent agonists, and selective as described CB1 agonists, further inas “4.2.3.described CB2-selective further in agonists”.“4.2.2. ThisCB1-selective strongly agonists”; suggeststhat JWH-133, the assumed JWH-015 critical and PM-226 differences are potent of activation and selective processes CB2 agonists, between

Int. J. Mol. Sci. 2020, 21, 5064 6 of 32

CB1 and CB2 might be a good starting point for the design of cannabinoid receptor-selective drugs. As we emphasized earlier, the difference of agonist-binding activation modes between CB1-Gi and CB2-Gi complexes may find its basis in the “toggle switch”. Although the agonist-bound CB1-Gi and CB2-Gi overlap very well, the “toggle switch” concept in cannabinoid receptors is regarded as a crucial role in determining efficacy of a ligand [28,29]. When the notorious switch is constrained by binding of an antagonist or inverse agonist, the activation of cannabinoid receptors can be blocked or reversed [28].

4. Phytocannabinoids and Synthetic Cannabinoids

4.1. Phytocannabinoids To date, over 100 unique phytocannabinoids have been identified [14,39]. These phytocannabinoids can be classified into several subclasses, including the ∆9-THC type, the ∆8-THC type, the CBD type, the CBN type, and several others [14]. Among these, ∆9-THC and CBD are the compounds that have been investigated the most [40]. They have been shown to bind to cannabinoid receptors and elicit the characteristic beneficial or psychoactive effects associated with cannabis. The beneficial effects of the phytocannabinoids are mediated by multiple targets, thus not solely the cannabinoid receptors, CB1 and CB2, as many people believe [14]. Two kinds of novel phytocannabinoids have recently emerged (∆9-THCP and CBDP) [39] (see further). Here, we provide an overview on the recently reported novel phytocannabinoids and the well-known/typical phytocannabinoids that are the most thoroughly studied to date.

4.1.1. ∆9-Tetrahydrocannabinol (∆9-THC) The psychotropic effects of cannabis are considered to be produced essentially by ∆9-THC (Table1a); for example, acute psychotic reactions and a temporary decline in cognitive functioning in human [1,2,40,41]. The psychoactive effects of cannabis are predominantly attributed to partial agonist activity of ∆9-THC at CB1 [14,40] (Table1a). Moreover, ∆9-THC is also characterized as a partial agonist of CB2 [14,42] (Table1a). As a typical partial agonist, ∆9-THC has a mixed agonist–antagonist effect which is presumably dependent on the cell type, the expression of receptors, and the presence of endocannabinoids or other full agonists [14]. Regarding the unwanted side effects, the safety concerns raised in connection with ∆9-THC as a psychoactive agent preclude its widespread use in the clinic. ∆9-THC undoubtedly has a range of important therapeutic benefits, such as appetite stimulation, analgesia, and anti-emetic effects, mediated by either CB1 and CB2 or non-cannabinoid targets [40,43]. This drove further large-scale investigations, leading to the approval of nabiximol (Sativex®), a combination of THC and CBD, for the treatment of pain and/or spasticity in multiple sclerosis which was a milestone in cannabis research [44]. Sativex® is a preparation administered in the form of an orally mucosal spray and licensed in more than 27 countries as a formulation delivering a consistent concentration at a one-to-one ratio of ∆9-THC:CBD [45]. After the optimization of preparation and delivery methods, another product, called Cannador®, came to the market, delivering ∆9-THC:CBD within a narrow concentration range and at a two-to-one ratio, in the form of an orally administered capsule [45]. In relation to this, many of the recent studies on medical cannabis have focused on various forms of ∆9-THC + CBD administration [46–49] and co-administration of ∆9-THC with first-line neurotherapeutic drugs [50]. On the other hand, the synthesis of ∆9-THC analogs is another effective way to avoid or reduce its severe side effects. Details of synthetic cannabinoids are summarized further in the “Synthetic cannabinoids” section.

4.1.2. Cannabidiol (CBD) Unlike ∆9-THC, CBD (Table1b) is regarded as a clinically interesting compound for its therapeutic potential in several disorders, including anti-inflammatory, analgesic, anti-anxiety, and antitumor properties [40,43,51,52]. Moreover, it has low addictive, hallucinogenic, and toxic side effects [40,43,51]. A number of studies have investigated CBD to determine its activity at cannabinoid receptors and Int. J. Mol. Sci. 2020, 21, 5064 7 of 32 shown very low affinity for CB1 and CB2 [14] (Table1b). It was reported that CBD can act as an antagonist/inverse agonist at certain concentrations below which it binds to both CB1 and CB2 orthosteric sites [53]. Recently, several studies demonstrated that CBD can act as a negative allosteric modulator of CB1, which alters the potency and efficacy of the orthosteric ligands but does not activate theInt. receptor J. Mol. Sci. 2020 itself, 21, [x54 FOR–56 PEER].For REVIEW CB2, the study showed that CBD can act as a partial7 agonistof 34 [54]. These resultsInt. J. Mol. could Sci. 2020 explain, 21, x FOR thePEER reported REVIEW ability of CBD to functionally antagonize some7 of undesirable 34 effects of4.1.2.∆9-THC Cannabidiol in animal (CBD) studies and clinical studies in humans without attenuating positive effects, Int.4.1.2. J. Mol. Cannabidiol Sci. 2020, 21, x(CBD) FOR PEER REVIEW 7 of 34 thus increasingUnlike Δ9-THC, the therapeuticCBD (Table 1b) index is regarded of ∆9-THC. as a Inclinically addition, interesting CBD exertscompound analgesic for its effects in rats byInt.4.1.2.therapeutic interactingJ. Mol. 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This studie iss an demonstrated oral solution that composedCBD can act of as the a negative active ingredient CBD, actnot as activate an antagonist/inverse the receptor itself agonist [54–56]. at Forcertain CB2, concentrations the study showed below that which CBD itcan binds act as to a both partial CB1 agonist and allosteric modulator of CB1, which alters the potency and efficacy of the orthosteric ligands but does derivedCB2 orthosteric fromTable marijuana,sites 1. Structures, [53]. Recently, tobinding treat severaltype rare and and studie bioactivities severes demonstrated of forms Δ9-THC, of CBD,that epilepsy. CBDΔ9-THCP can The andact proof CBDP.as a negative of concept delivered by Epidiolexnot activateTable® thedrives receptor 1. Structures, further itself binding [54–56]. research type For and formulatingCB2, bioactivities the study of CBDshowed Δ9-THC, in that order CBD, CBD Δ to9-THCP can apply act andas CBDa CBDP. partial to agonist other various diseases allosteric modulator of CB1, which alters the potency and efficacy of ECthe50 orthosteric/IC50 ligands but does notPhytocannabinoids activate the receptor itself [54–56].Binding For Type/CB CB2, the studyKi (nM)/CB showed that CBD can actBioactivity as a partial agonist or to improveTable 1. the Structures, efficacy binding of other type and medical bioactivities drugs of Δ in9-THC, co-administration CBD,EC(nM)/CB50 Δ/IC9-THCP50 and [ CBDP.59,60 ]. Phytocannabinoids Binding Type/CB Ki (nM)/CB Bioactivity (nM)/CB Analgesic, Table 1. Structures, binding type and bioactivities of Δ9-THC, CBD,EC50 /ΔIC9-THCP50 and CBDP. PhytocannabinoidsTable 1. Structures, Binding binding Type/CB type andKibioactivities (nM)/CB of 13.0~∆9-THC, Bioactivity CBD,Analgesic,antiemetic,∆9-THCP and CBDP. 5.00 ~ (nM)/CB EC13.0~87.0/CB150/IC 50 antiemetic,orexigenic [53]; Phytocannabinoids BindingPartial Type/CB K5.00i80.0/CB1 (nM)/CB ~ EC50BioactivityAnalgesic,/IC50 Phytocannabinoids Binding Type/CB Ki (nM)/CB (nM)/CB87.0/CB141.8, orexigenicrelief from [53]; muscleBioactivity Partialagonist/CB1, CB2 80.0/CB11.70 ~ 13.0~ (nM)antiemetic,/CB 5.00 ~ 41.8,61.0/CB2 Analgesic,reliefspasms/spasticity from muscle in agonist/CB1, CB2 1.7075.0/CB2 ~ [14] 87.0/CB1 orexigenic [53]; (a) Partial 80.0/CB1 13.0~61.0/CB2[14] antiemetic,spasms/spasticitymultiple sclerosisAnalgesic, in 5.0075.0/CB2 ~ [14] 41.8, relief from muscle (a)Δ9-THC agonist/CB1, CB2 1.70 ~ 87.0/CB1[14] orexigenicmultiple[61] sclerosis [53]; antiemetic, Partial 80.0/CB1 61.0/CB2 spasms/spasticity in Δ9-THC Partial agonist/CB1, 75.0/CB25.00~80.0 [14]/CB1 41.8, 13.0~87.0relief[61]Anti-inflammatory,/ CB1 from muscleorexigenic [53]; (a) agonist/CB1,CB2 CB2 1.70 1.70~75.0~ /CB2[14] [14] 41.8,multiple 61.0/CB2 [sclerosis14] relief from muscle Antagonist/inverse 61.0/CB2 spasms/spasticityAnti-inflammatory,anti-nociceptive, in Δ9-THC 75.0/CB273.0 [14] [61] spasms/spasticity in (a)(a) Antagonist/inverseagonist, negative [14]3860/CB1 multipleanti-nociceptive,anti-oxidant, sclerosis multiple sclerosis [61] ∆9-THC 73.0~ >10000/CB1 Anti-inflammatory, Δ9-THC agonist,allosteric negative 3860/CB1503, [61]anti-oxidant,anti-ischemic, Antagonist/inverse ~370 >10000/CB1 anti-nociceptive,Anti-inflammatory, allostericmodulator/CB1 73.0 503,2270/CB2 Anti-inflammatory,anti-ischemic,neuroprotective, Antagonist/inverse 370~ >10000/CB2 anti-nociceptive, agonist,Partial negative 3860/CB1[14] anti-oxidant,immunosuppressive Antagonist/inversemodulator/CB1agonist, negative ~ >10000/CB173.0~>10,000 /CB12270/CB2 anti-nociceptive,neuroprotective,anti-oxidant, (b) allosteric 73.0~[14] >10000/CB2 503, 3860/anti-ischemic,CB1 agonist,Partialagonist/CB2allosteric negative 370 370~>10,000/CB23860/CB1[14] anti-oxidant,immunosuppressive[62]; anti-ischemic, (b)CBD modulator/CB1 ~[14] >10000/CB1 2270/CB2503, 2270neuroprotective,/CB2 [14] allostericagonist/CB2modulator /CB1 ~ >10000/CB2[14] 503, anti-ischemic,[62];anxiolytic [43,62] neuroprotective, CBD PartialPartial agonist/CB2 370 [14] immunosuppressiveimmunosuppressive [62]; (b)(b) modulator/CB1 [14] 2270/CB2 neuroprotective,anxiolytic [43,62] CBD agonist/CB2 ~ >10000/CB2 [62]; anxiolytic [43,62] CBD Partial [14] immunosuppressive (b) [14] anxiolytic [43,62] agonist/CB2 [62]; CBD 1.20/CB1 Agonist/CB1, CB2 NA anxiolyticAnalgesic [43,62] [39] 1.20/CB16.20/CB21.20/CB1 [39] Agonist/CB1,Agonist/CB1, CB2 NA NAAnalgesic [39] Analgesic [39] 6.20/CB26.20/CB2 [39] [39 ] (c) 1.20/CB1 Agonist/CB1, CB2 NA Analgesic [39] (c)(c)Δ 9-THCP 6.20/CB2 [39] ∆9-THCP 1.20/CB1 Δ9-THCP Agonist/CB1, CB2 NA Analgesic [39] (c) 6.20/CB2 [39] Δ9-THCP (c) Δ9-THCP NANA NA NA NA NANA NA NA NA NA NA (d)(d) CBDP NA NA NA NA (d)CBDP K : binding constant; EC : half-maximal effective concentration; IC : half-maximal inhibitory concentration; NA: CBDPiKi : binding constant; ECNA50 50: half-maximal effectiveNA concentration;NA 50IC 50: half-maximalNA inhibitory (d) not available. Kconcentration;i: binding constant; NA: not EC available.50: half-maximal effective concentration; IC50: half-maximal inhibitory CBDP (d) concentration; NA: not available. Ki: binding constant; EC50: half-maximal effective concentration; IC50: half-maximal inhibitory 4.1.3.CBDP4.1.3.∆ Δ9-Tetrahydrocannabiphorol9-Tetrahydrocannabiphorol (Δ9-THCP) (∆9-THCP) and Cannabidiphorol and Cannabidiphorol (CBDP) (CBDP) 4.1.3.concentration; Δ9-Tetrahydrocannabiphorol NA: not available. (Δ9-THCP) and Cannabidiphorol (CBDP) KAti:At binding the the endend constant; of of last last year,EC year,50 :tw half-maximalo two novel novel phytocannabinoids, effective phytocannabinoids, concentration; Δ9-tetrahydrocannabiphorol IC50∆: 9-tetrahydrocannabiphorolhalf-maximal inhibitory (Δ9-THCP) (∆9-THCP) andconcentration;At cannabidiphorol the end of NA: last not year,(CBDP), available. two were novel isolated phytocannabinoids, from Cannabis Δ 9-tetrahydrocannabiphorolsativa [39]. These common names(Δ9-THCP) were and4.1.3. cannabidiphorol Δ9-Tetrahydrocannabiphorol (CBDP), (Δ were9-THCP) isolated and Cannabidiphorol from Cannabis (CBDP) sativa [39]. These common names were andderived cannabidiphorol from the traditional (CBDP), namingwere isolated of phytocannabinoids from Cannabis sativa based [39]. on theThese resorcinyl common residue, names inwere this At the end of last year, two novel phytocannabinoids, Δ9-tetrahydrocannabiphorol (Δ9-THCP) derived4.1.3.derivedcase Δ corresponding9-Tetrahydrocannabiphorol fromfrom the the traditional traditional to sphaerophorol naming naming (Δ9-THCP) of [39]. phytocannabinoids of Δ phytocannabinoids 9-THCPand Cannabidiphorol is a Δ based9-THC on basedhomolog(CBDP) the resorcinyl on with the a resorcinyl residue,seven-term in residue, thisside in this case and cannabidiphorol (CBDP), were isolated from Cannabis sativa [39]. These common names were correspondingcaseAt corresponding the end of tolast sphaerophorolto year, sphaerophorol two novel phytocannabinoids,[39]. [39]. Δ∆9-THCP9-THCP is a is ΔΔ9-THC a9-tetrahydrocannabiphorol∆9-THC homolog homolog with a seven-term with (Δ9-THCP) a seven-term side side alkyl derived from the traditional naming of phytocannabinoids based on the resorcinyl residue, in this and cannabidiphorol (CBDP), were isolated from Cannabis sativa [39]. These common names were case corresponding to sphaerophorol [39]. Δ9-THCP is a Δ9-THC homolog with a seven-term side derived from the traditional naming of phytocannabinoids based on the resorcinyl residue, in this case corresponding to sphaerophorol [39]. Δ9-THCP is a Δ9-THC homolog with a seven-term side

Int. J. Mol. Sci. 2020, 21, 5064 8 of 32 chain (Table1c) which is longer than the five-term side alkyl chain of ∆9-THC (Table1a). ∆9-THCP can bind with high affinity to both CB1 and CB2 in a radioligand binding assay [39] (Table1c). Its a ffinity for CB1 is significantly higher compared to the reported data of ∆9-THC, as shown in Table1a. Further in vivo evaluation of ∆9-THCP confirmed its cannabimimetic activity of decreasing locomotor activity and rectal temperature, inducing catalepsy and producing analgesia, thereby mimicking the properties of a full CB1 receptor agonist [39]. The cannabimimetic activity of ∆9-THCP is several times higher than that of ∆9-THC [39]. As the pharmacological activity of ∆9-THC is particularly ascribed to its affinity for CB1 receptor, it is suggested that this affinity can be increased by elongating the alkyl side chain [63]. Thus, the in vivo results of ∆9-THCP show the significance of the length of the side alkyl chain on the resorcinyl moiety in modulating the ligand affinity at CB1 [39]. Another novel phytocannabinoid was named cannabidiphorol (CBDP), which is a CBD homolog with a seven-term side alkyl chain (Table1d). At present, no data on the pharmacological e ffects of CBDP are available [39].

4.2. Synthetic Cannabinoids Synthetic cannabinoids constitute the most diverse group of cannabinoids in regard to functional profile and chemical structure [40]. Originally, the synthetic cannabinoids were used as pharmacological tools to delineate the cannabinoid receptor-mediated activity [21]. Thus, their structural characteristics allow them to bind to one of the known cannabinoid receptors present in human cells, CB1 and/or CB2 [15]. After decades, some synthetic cannabinoids emerged on the market as alternatives to phytocannabinoids for recreational drug use. Several hundreds of different synthetic cannabinoids have been produced up to date, sometimes with subtle structural changes [15,22]. These synthetic cannabinoids can be divided into classical, nonclassical, amino-alkylindoles, eicosanoids and others in terms of chemical structure [53]. Many of these synthetic cannabinoids are used in pharmacological studies involving structure–activity relationships, receptor binding studies and detailed mechanisms of action of these drugs. The FDA has approved three synthetic cannabis-related drug products: Marinol® (dronabinol), Syndros® (dronabinol), and Cesamet® (nabilone) [64]. Marinol® and Syndros® include the active ingredient dronabinol, a synthetic ∆9-THC which is considered the psychoactive intoxicating component of cannabis (i.e., the component responsible for the “high” people may experience from using cannabis). Their therapeutic uses in the United States include the treatment of nausea associated with cancer chemotherapy and the treatment of anorexia associated with weight loss in AIDS patients [64]. Another FDA-approved drug, Cesamet®, contains the active ingredient nabilone, which has a chemical structure similar to THC and is synthetically derived. Cesamet®, similarly to dronabinol-containing products, is indicated for nausea associated with cancer chemotherapy and neuropathic pain [64].

4.2.1. Mixed CB1/CB2 Agonists Most known synthetic agonists of cannabinoid receptors show little selectivity between CB1 and CB2 [21], but exhibit stronger affinity for cannabinoid receptors compared to endo- and phytocannabinoids [65]. The synthetic cannabinoids that are most commonly used in the laboratory as CB1 and CB2 receptor agonists fall essentially into three chemical groups: classical, nonclassical and amino-alkylindole. Three notable examples of such compounds are 11-hydroxy-∆8-THC-dimethylheptyl (HU-210), CP-55,940 and WIN-55,212-2. HU-210 (Table2a), an example of a classical synthetic cannabinoid, is a highly potent cannabinoid receptor agonist, and its potency and affinity at cannabinoid receptors exceed those of many other cannabinoids [66]. The high potency and affinity of HU-210 are believed to result from replacing the pentyl side chain on ∆9-THC with a dimethylheptyl group [53]. Furthermore, pharmacological effects of HU-210 in vivo are exceptionally long-lasting [66]. The non-classical synthetic cannabinoid, CP-55,940 (Table2b), is a cannabinoid receptor full agonist that is considerably more potent than ∆9-THC [67,68]. Moreover, it has comparable affinity for both CB1 and CB2 receptors in the low nanomolar range and it is highly potent in vivo [69]. Like CP-55,940, the amino-alkylindole synthetic cannabinoid WIN-55,212 (Table2c) Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 34

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 34 the low nanomolar range. However, in contrast to CP-55,940, it has slightly greater affinity for CB2 Int.Int. J. J. Mol.Mol. Sci. Sci. 20202020, 21, 21, x ,FOR 5064 PEER REVIEW 9 of 34 9 of 32 the thanlow nanomolarfor CB1 [66] range. (Table However, 2c). Overall, in contrastagonists to of CP-55,940,the cannabinoid it has receptorsslightly greater are involved affinity in for cognition, CB2 memory,Int. J. Mol. Sci. anxiety, 2020, 21, x FORcontrol PEER REVIEWof appetite, emesis, motor behavior, sensory, autonomic 9 andof 34 thanthe for low CB1 nanomolar [66] (Table range. 2c). Overall, However, agonists in contrast of the cannabinoidto CP-55,940, receptors it has slightly are involved greater inaffinity cognition, for CB2 memory,neuroendocrine anxiety, controlresponses, of immuneappetite, responsesemesis, motorand inflammatorybehavior, sensory, effects, autonomic liver injury and and exhibitsthanthe low for relativelyCB1nanomolar [66] (Table highrange. 2c). potency However, Overall, for agonists in both contrast CB1of the to and CP-55,940,cannabinoid CB2, and it receptorshas possesses slightly are greater involved CB1 and affinity in CB2 cognition, for affi CB2nities in the low neuroendocrinehepatocellular responses, carcinoma [53].immune Currently, responses among and synthetic inflammatory CB1/CB2 effects,mixed agonists,liver injury only nabiloneand nanomolarmemory,than for CB1 anxiety, range. [66] (Table However,control 2c). Overall,of inappetite, contrast agonists emes of to the CP-55,940,is, cannabinoidmotor behavior, it hasreceptors slightly sensory, are involved greater autonomic in affi cognition,nity and for CB2 than for hepatocellular(Table 1d) is carcinoma in the phase [53]. III Currently, of the clinical among trial syntheticfor non-motor CB1/CB2 symptoms mixed inagonists, Parkinson’s only diseasenabilone from neuroendocrinememory, anxiety, responses, control immuneof appetite, responses emes is,and motor inflammatory behavior, effects,sensory, liver autonomic injury andand (TableCB1ClinicalTrials.gov 1d) [66 is] (Tablein the phase2c). [70]. Overall, III of the agonistsclinical trial of for the non-motor cannabinoid symptoms receptors in Parkinson’s are involved disease in from cognition, memory, hepatocellularneuroendocrine carcinoma responses, [53]. immune Currently, responses among synthetic and inflammatory CB1/CB2 mixed effects, agonists, liver only injury nabilone and ClinicalTrials.govanxiety, control [70]. of appetite, emesis, motor behavior, sensory, autonomic and neuroendocrine responses, (Tablehepatocellular 1d) is inTable thecarcinoma phase 2. Structures, III [53]. of the Currently, chemical clinical type trialamong and for bioa non-motorsyntheticctivities CB1/CB2of symptoms mixed CB1/CB2mixed in Parkinson’s agonists, agonists. only disease nabilone from immuneClinicalTrials.gov responses [70]. and inflammatory effects, liver injury and hepatocellular carcinoma [53]. Currently, (Table 1d) is in the phase III of the clinical trial for non-motor symptoms in Parkinson’s. disease from amongSynthetic syntheticTable 2. Structures, CB1/CB2Chemical chemical mixed type agonists, and bioactivities only nabilone of mixed CB1/CB2 (Table 1agonistsd) is in the phase III of the clinical ClinicalTrials.gov [70]. Ki (nM)/CB EC50 (nM)/CB Bioactivity SynthetictrialCannabinoids for non-motorTable 2. symptomsStructures,ChemicalType chemical in Parkinson’s type and bioa diseasectivities of from mixed ClinicalTrials.gov CB1/CB2 agonists. [70]. Ki (nM)/CB EC50 (nM)/CB Bioactivity CannabinoidsSynthetic Table 2. Structures,TypeChemical chemical type and bioactivities of mixed CB1/CB2 agonists. K0.0608i (nM)/CB ~ EC50 (nM)/CB Bioactivity CannabinoidsSynthetic Table 2. Structures,TypeChemical chemical type and bioactivities of mixed CB1/CB2 agonists. 0.730/CB1Ki (nM)/CB 0.0702/CB1EC50 (nM)/CB Analgesic Bioactivity [53]; Cannabinoids Type 0.0608 ~ Synthetic CannabinoidsClassical Chemical Type0.170 ~ K i (nM)/CB[71] EC50 (nM)neuroprotective/CB Bioactivity 0.730/CB10.0608 ~ 0.0702/CB1 Analgesic [53]; 0.524/CB2 NA/CB2 [72,73] Classical 0.1700.730/CB1 ~ [71]0.0702/CB1 neuroprotectiveAnalgesic [53]; [66]0.0608 ~ (a) Classical 0.524/CB20.170 ~ NA/CB2[71] [72,73]neuroprotective 0.730/CB10.0608~0.730 0.0702/CB1/CB1 Analgesic [53]; HU-210 [66] 0.0702/CB1 [71] Analgesic [53]; (a) ClassicalClassical 0.524/CB20.170 ~0.170~0.524 NA/CB2[71]/CB2 [72,73]neuroprotective NA/CB2 neuroprotective [72,73] HU-210(a) [66]0.524/CB2 [66] NA/CB2 [72,73] HU-210(a)(a) 0.500[66] ~ HU-210 0.0462 ~ HU-210 0.5005.00/CB1 ~ 0.046231.0/CB1 ~ Anti-nociceptive, anti- Nonclassical 5.00/CB10.690~ 0.500 ~ 31.0/CB1[24,71,74] Anti-nociceptive,emetic [53] anti- Nonclassical 0.690~2.80/CB2 0.0462 ~ 5.00/CB10.500 ~ [24,71,74]NA/CB2 0.0462~31.0emetic [53]/CB1 [66] 0.500~5.0031.0/CB1/CB1 Anti-nociceptive,Anti-nociceptive, anti- (b) NonclassicalNonclassical 2.80/CB20.690~ 0.0462 ~ [24,71,74] 5.00/CB10.690~2.80 NA/CB2[24,71,74]/CB2 [66] emetic [53] anti-emetic [53] CP-55,940 [66]2.80/CB2 31.0/CB1 NA/CB2Anti-nociceptive, anti- (b) Nonclassical 0.690~ NA/CB2 [66] [24,71,74] emetic [53] CP-55,940(b) 2.80/CB2 CP-55,940 NA/CB2 CP-55,940 [66] (b) 1.89 ~ 5.50 ~ CP-55,940 123/CB1 Analgesic, anti- Amino- 1.89 ~ 3000/CB1 0.280 ~ 5.50 ~ inflammatory [53]; alkylindole 123/CB11.89 ~ [71,74–78] 5.50~3000Analgesic,/CB1 anti- Analgesic, Amino- 1.89~1233000/CB1/CB1 Amino-alkylindole0.28016.2/CB2 ~ 5.50 ~ [71inflammatory,74–neuroprotective78] [53]; anti-inflammatory[79] [53]; alkylindole 123/CB11.89 ~ 0.280~16.2 [71,74–78]NA/CB2/CB2 [66 ] Analgesic, anti- Amino- 16.2/CB2[66] 3000/CB15.50 ~ NAneuroprotective/CB2 [79]neuroprotective [79] 0.280123/CB1 ~ NA/CB2 inflammatoryAnalgesic, anti- [53]; (c) alkylindoleAmino- [66] [71,74–78]3000/CB1 16.2/CB20.280 ~ neuroprotectiveinflammatory [53]; [79] (c) (c)WIN-55,212-2 alkylindole NA/CB2[71,74–78] WIN-55,212-2 [66]16.2/CB2 neuroprotective [79] WIN-55,212-2 NA/CB2 (c) [66] WIN-55,212-2(c) Analgesic, antiemetic, 1.84/CB1 Analgesic, antiemetic, WIN-55,212-2 1.84/CB1 Analgesic,anti-inflammatory antiemetic, ClassicalClassical 1.84/CB12.19/CB2 NA NA anti-inflammatory [53]; 2.19/CB2 [66] anti-inflammatory[53]; neuroprotective Classical 2.19/CB2[66] NA Analgesic, antiemetic,neuroprotective [80,81] 1.84/CB1 [53];[80,81] neuroprotective (d) [66] anti-inflammatoryAnalgesic, antiemetic, Nabilone Classical 2.19/CB21.84/CB1 NA [80,81] Nabilone [53]; neuroprotective (d) [66] anti-inflammatory K : bindingClassical constant; EC : half-maximal2.19/CB2 effectiveNA concentration; NA: not available. Ki: bindingi constant; EC50: half-maximal50 effective concentration; NA: not[80,81][53]; available. neuroprotective Nabilone(d) [66] Ki: binding constant; EC50: half-maximal effective concentration; NA: not available.[80,81] 4.2.2.Nabilone(d) CB1-Selective Agonists Nabilone Ki: binding constant; EC50: half-maximal effective concentration; NA: not available. 4.2.2.4.2.2. CB1-SelectiveThe CB1-Selective starting Agonists point Agonists for the development of a CB1-selective agonist was the AEA molecule. Ki: binding constant; EC50: half-maximal effective concentration; NA: not available. 4.2.2.ThroughTheThe CB1-Selectivestarting startingchanging point point Agoniststhe for atom forthe thedevelopmenon the development 1′, 2′ tor of 2 acarbon CB1-selective of aCB1-selective of AEA, agonistits CB1 agonist wasselectivity the was AEA can the bemolecule. AEA enhanced, molecule. Through Throughleading changing to synthesis the atomof CB1-selective on the 1′, 2 agonists,′ or 2 carbon such ofas AEA,methanandamide its CB1 selectivity (Table 3c)can and be enhanced,O-1812 (Table changing4.2.2.The CB1-Selective starting the atom point Agonists on for the the 10 ,developmen 20 or 2 carbont of a of CB1-selective AEA, its CB1 agonist selectivity was thecan AEA be molecule. enhanced, leading to leading3d) to[66]. synthesis So far, of CB1-selective the most agonists,potent CB1-selective such as methanandamide agonists developed (Table 3c) andare O-1812 arachidonyl-2´- (Table synthesisThroughThe changingstarting of CB1-selective point the atom for theon agonists, thedevelopmen 1′, 2′ suchor 2t carbonof as a methanandamide CB1-selective of AEA, its CB1agonist selectivity (Table was 3thec) can andAEA be O-1812 enhanced,molecule. (Table 3d) [ 66]. 3d) chloroethylamide[66]. So far, the (ACEA) most (Table potent 3a) andCB1-selective arachidony agonistslcyclopropylamide developed (ACPA) are arachidonyl-2´-(Table 3b), both of leadingThrough to changing synthesis theof CB1-selective atom on the agonists,1′, 2′ or 2 such carbon as methanandamide of AEA, its CB1 (Tableselectivity 3c) and can O-1812 be enhanced, (Table chloroethylamideSowhich far, theexhibit most reasonably(ACEA) potent (Table CB1-selective high 3a) CB1 and potency. arachidony agonists ACEAlcyclopropylamide developed displays nanomolar are arachidonyl-2 (ACPA) affinity (Table at CB1´3b),-chloroethylamide andboth >1000- of (ACEA) 3d)leading [66]. to synthesisSo far, ofthe CB1-selective most potent agonists, CB1-selective such as methanandamide agonists developed (Table 3c)are and arachidonyl-2´- O-1812 (Table which(Table exhibit3a) and reasonably arachidonylcyclopropylamide high CB1 potency. ACEA displays (ACPA) nanomolar (Table 3affinityb), both at CB1 of which and >1000- exhibit reasonably chloroethylamide3d) [66]. So far, (ACEA) the most(Table potent3a) and CB1-selectivearachidonylcyclopropylamide agonists developed (ACPA) are (Table arachidonyl-2´- 3b), both of high CB1 potency. ACEA displays nanomolar affinity at CB1 and >1000-fold selectivity over CB2 [21], whichchloroethylamide exhibit reasonably (ACEA) high (Table CB1 3a) potency. and arachidony ACEA displayslcyclopropylamide nanomolar (ACPA)affinity at(Table CB1 3b),and both>1000- of while which ACPA exhibit displaysreasonably> 300-foldhigh CB1 potency. selectivity ACEA over displays CB2 [74nanomolar]. In general, affinity compounds at CB1 and >1000- with an agonistic eff ect and sufficient affinity to CB1 have a potential for abuse as cannabis substitutes. However, considering the medical potency of CB1-selective agonists, there is still an attractive interest in those compounds exploring different pharmacological strategies to minimize the unwanted CNS side effects and maximize the beneficial therapeutic effects. For example, ACEA and ACPA have both been studied and have been shown to have anti-depressant [82,83] and anti-nociceptive effects [84,85]. Furthermore, ACEA has been receiving considerable attention in terms of co-administration with Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 34

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 34 fold selectivity over CB2 [21], while ACPA displays >300-fold selectivity over CB2 [74]. In general, Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 10 of 34 foldcompounds selectivity with over an CB2 agonistic [21], while effect ACPA and sufficient displays >300-foldaffinity to selectivity CB1 have over a potential CB2 [74]. for In abuse general, as cannabis substitutes. However, considering the medical potency of CB1-selective agonists, there is foldcompounds selectivity with over an CB2 agonistic [21], while effect ACPA and sufficientdisplays >300-fold affinity to selectivity CB1 have over a potential CB2 [74]. for In general,abuse as Int.still J. Mol. an Sci.attractive 2020, 21, xinterest FOR PEER in REVIEW those compounds exploring different pharmacological strategies 10 of 34to compoundscannabis substitutes. with an agonistic However, effect considering and sufficient the medical affinity potency to CB1 of haveCB1-selective a potential agonists, for abuse there as is stillminimize an attractive the unwanted interest CNSin those side compoundseffects and exploringmaximize differentthe beneficial pharmacological therapeutic strategieseffects. For to cannabisfold selectivity substitutes. over CB2However, [21], while considering ACPA displaysthe medical >300-fold potency selectivity of CB1-selective over CB2 agonists, [74]. In general,there is minimizeexample, ACEAthe unwanted and ACPA CNS have side both effects been andstudied maxi andmize have the been beneficial shown therapeuticto have anti-depressant effects. For stillcompounds an attractive with aninterest agonistic in those effect compounds and sufficient exploring affinity different to CB1 havepharmacological a potential forstrategies abuse toas example,[82,83] and ACEA anti-nociceptive and ACPA haveeffects both [84,85]. been studiedFurthermore, and have ACEA been has shown been toreceiving have anti-depressant considerable minimizecannabis substitutes.the unwanted However, CNS sideconsidering effects andthe medicalmaximize potency the beneficial of CB1-selective therapeutic agonists, effects. there For is [82,83]attention and in anti-nociceptiveterms of co-administration effects [84,85]. with Furt hermore,different ACEAantiepileptic has been drugs. receiving It potentiated considerable the example,still an attractive ACEA and interest ACPA in have those both compounds been studied exploring and have different been shown pharmacological to have anti-depressant strategies to attentionanticonvulsant in terms activity of ofco-administration antiepileptic drugs with in variousdifferent animal antiepileptic models ofdrugs. epilepsy It potentiated and stimulated the [82,83]minimize and the anti-nociceptive unwanted CNS effects side [84,85].effects Furtand hermore,maximize ACEA the beneficial has been therapeuticreceiving considerable effects. For anticonvulsantneurogenesis in activity the brain of antiepileptic of mice, showingdrugs in variousno possible animal acute models adverse of epilepsy effects and [86–90]. stimulated This attentionexample, ACEAin terms and of ACPA co-administration have both been with studied different and haveantiepileptic been shown drugs. to have It potentiated anti-depressant the neurogenesiscombinational inactivity the brain could ofbe mice,beneficial showing to avoid no thepossible severe sideacute effects adverse of CB1-selective effects [86–90]. agonists. This anticonvulsant[82,83]Int. J. Mol. and Sci.anti-nociceptive 2020activity, 21 ,of 5064 antiepileptic effects [84,85]. drugs Furt in varioushermore, animal ACEA models has been of epilepsy receiving and considerable stimulated 10 of 32 combinationalHowever, no activityCB1-selective could beagonist beneficial is currently to avoid inthe the severe stage side of effects the clinical of CB1-selective trial according agonists. to neurogenesisattention in termsin the of brain co-administration of mice, showing with nodifferent possible antiepileptic acute adverse drugs. effects It potentiated [86–90]. Thisthe However,ClinicalTrials.gov no CB1-selective [70]. agonist is currently in the stage of the clinical trial according to combinationalanticonvulsant activity activity could of antiepileptic be beneficial drugs to avoid in various the severe animal side models effects of of epilepsy CB1-selective and stimulated agonists. However,ClinicalTrials.gov no CB1-selective [70]. agonist is currently in the stage of the clinical trial according to neurogenesisdifferent antiepileptic inTable the 3.brain Structures, drugs. of mice, chemical It potentiatedshowing type and no bioactivities thepossible anticonvulsant ofacute CB1-selective adverse activity agonists.effects of[86–90]. antiepileptic This drugs in various ClinicalTrials.govcombinationalanimal models activity [70]. of epilepsy could be beneficial and stimulated to avoid the neurogenesis severe side effects in the of brain CB1-selective of mice, agonists. showing no possible acute Table 3. Structures, Chemicalchemical type and bioactivities of CB1-selectiveEC50 agonists. However,Synthetic no Cannabinoids CB1-selective agonist is currentlyKi (nM)/CBin the stage of the clinicalBioactivity trial according to adverse effectsTable [86 3. –Structures,90]. This chemicalType combinational type and bioactivities activity of CB1-selective could(nM)/CB1 be beneficial agonists. to avoid the severe side effects ClinicalTrials.gov [70]. Chemical EC50 ofSynthetic CB1-selective Cannabinoids agonists. However, noKi CB1-selective(nM)/CB agonist isBioactivityAnti-depressant currently in [86]; the stage of the clinical ChemicalType EC(nM)/CB150 Synthetic Cannabinoids Ki (nM)/CB Bioactivityanti-nociceptive [53]; trial accordingTable to 3. ClinicalTrials.gov Structures, Typechemical type[70 and]. bioactivities of CB1-selective(nM)/CB1 agonists.Anti-depressant [86]; anti-ulcer [84]; Anti-depressantanti-nociceptive [86]; [53]; Chemical EC50 neuroprotective Synthetic CannabinoidsTable 3. Structures, chemicalK1.40,i (nM)/CB type5.29/CB1and bioactivities ofanti-nociceptiveBioactivityanti-ulcer CB1-selective [84]; [53]; agonists. Type (nM)/CB10.0317, [91,92]; Eicosanoid 195, >2000/CB2 anti-ulcerneuroprotective [84]; 1.40, 5.29/CB1 51.0 [74] Anti-depressantpotentiating activity [86]; Synthetic Cannabinoids Chemical Type[66] Ki (nM)/CB0.0317, ECneuroprotective50[91,92];(nM)/CB1 Bioactivity (a) Eicosanoid 1.40,195, 5.29/CB1>2000/CB2 anti-nociceptiveof antiepileptic [53]; 0.0317,51.0 [74] [91,92];potentiating activityAnti-depressant [86]; ACEA Eicosanoid 195,[66] >2000/CB2 anti-ulcerdrugs [87]; [84]; (a) 51.0 [74] potentiatingof antiepileptic activity anti-nociceptive [53]; [66] neuroprotectivereducing cognitive (a)ACEA 1.40, 5.29/CB1 ofdrugs antiepileptic [87]; anti-ulcer [84]; 1.40, 5.29/CB10.0317, [91,92];impairment [91] neuroprotective [91,92]; ACEA EicosanoidEicosanoid 195, >2000/CB2 0.0317,reducing 51.0 [74 cognitive] 195, >2000/CB251.0 [[74]66] drugspotentiating [87]; activitypotentiating activity of [66] impairment [91] (a)(a) reducingof antiepileptic cognitive antiepileptic drugs [87]; ACEA impairmentAnti-depressant, [91] reducing cognitive impairment ACEA 2.20/CB1 0.0551, drugs [87]; Eicosanoid anxiolytic [83]; [91] 715/CB2 [66] 37.0 [74] reducingAnti-depressant, cognitive 2.20/CB1 0.0551, anti-nociceptive [53]; (b) Eicosanoid Anti-depressant,impairmentanxiolytic [83]; [91] 2.20/CB1715/CB22.20 [66]/CB1 0.0551,37.0 [74] Anti-depressant, anxiolytic [83]; ACPA EicosanoidEicosanoid 0.0551,anxiolyticanti-nociceptive 37.0 [74 [83];] [53]; (b) 715/CB2715 [66]/CB2 [6637.0] [74] anti-nociceptive [53]; anti-nociceptive [53]; (b)(b)ACPA Anti-depressant, ACPA 2.20/CB1 0.0551, Analgesic, anti- ACPA Eicosanoid 17.9 ~ 28.3/CB1 anxiolytic [83]; 715/CB2 [66] 37.0 [74] emetic, orexigenic, Eicosanoid 815 ~ 868/CB2 1000 [77] anti-nociceptiveAnalgesic, anti- [53]; (b) 17.9 ~ 28.3/CB1 anti-proliferation,Analgesic, anti-emetic, [66] 17.9~28.3/CB1 Analgesic,emetic, orexigenic, anti- ACPA(c) EicosanoidEicosanoid 17.9815 ~~ 28.3/CB1868/CB2 1000 [77] 1000anti-migration [77] [53]orexigenic, anti-proliferation, 815~868/CB2 [66] emetic,anti-proliferation, orexigenic, (c)Methanandamide Eicosanoid 815[66] ~ 868/CB2 1000 [77] anti-migration [53] (c) anti-proliferation,anti-migration [53] Methanandamide [66] Analgesic, anti- (c)Methanandamide 17.9 ~ 28.3/CB1 Anti-nociceptive, anti-migrationemetic, orexigenic, [53] Methanandamide Eicosanoid 815 ~ 868/CB2 1000 [77] suppressing Anti-nociceptive, suppressing 3.40/CB1 anti-proliferation,Anti-nociceptive, [66] 3.40/CB1 spontaneous activity and (c) EicosanoidEicosanoid NA NA suppressingspontaneous activity 3.40/CB13870/CB23870 [66]/CB2 [66] Anti-nociceptive,anti-migration [53] catalepsy [93]; Methanandamide(d) Eicosanoid NA spontaneousand catalepsy activity [93];anti-convulsant [94] (d) 3870/CB2 [66] suppressing O-1812 3.40/CB1 andanti-convulsant catalepsy [93]; [94] (d)O-1812 Eicosanoid NA spontaneousAnti-nociceptive, activity K : binding constant; EC 3870/CB2: half-maximal [66] effective concentration; NA: not available. Ki: bindingi constant; EC50: half-maximal50 effective concentration; NA: notandanti-convulsant available. catalepsy [93]; [94] (d)O-1812 suppressing 3.40/CB1 anti-convulsant [94] O-1812 Ki: binding constant; ECEicosanoid50: half-maximal effective concentration;NA NA: notspontaneous available. activity 3870/CB2 [66] and catalepsy [93]; (d) Ki: binding constant; EC50: half-maximal effective concentration; NA: not available. 4.2.3. CB2-Selective Agonists anti-convulsant [94] O-1812

ConcerningKi: binding CB2-selective constant; EC50: half-maximal agonists, effective the concentration; most widely NA: not used available. experimental tool is the classical cannabinoid, JWH-133 (Table4a), and the less selective amino-alkylindole, JWH-015 (Table4b),

developed by Dr John Huffman [66]. Both compounds not only bind with higher affinity to CB2 than to CB1, but also behave as a potent CB2-selective agonist in functional assays [66]. Other notable CB2-selective agonists include PM-226 (Table4c), HU-308 (Table4d), the GlaxoSmithKline compound GW-405833 (Table4e), Merck Frosst (now known as Merck Canada) compounds L-759,633 (Table4f) and L-759,656 (Table4g). CB2-selective agonists have undoubtedly been the focus in the field of therapeutic uses, because modulation of the CB2 is an interesting approach avoiding CNS related side effects, to treat pain, inflammation, arthritis, addictions, neuroprotection, and cancer, among other possible therapeutic applications [24,27,95–100]. Interestingly, the use of known CB2-selective agonists (i.e., JWH-015 and L-759,656) for treating or preventing a disease associated with immune dysfunction such as HIV disease was proposed in an US patent published in 2012 [22]. Over the past decade, published patents have claimed >150 synthetic selective agonists of CB2 [22]. Nowadays, new ligands designed to interact with CB2 as selective agonists are currently the subject of research both by academia and industry. Furthermore, a number of reports dealing with in vivo and in vitro models have shown positive and very interesting results (as summarized in Table4). Nevertheless, there has still been limited success in clinical trials, partly due to the lack of translation from preclinical models and also due to the differences across species [22,101,102]. At present, at least three unique synthetic CB2 agonists have reached clinic trials, including GW842166X, S-777469 and JBT-101 from ClinicalTrials.gov[22,70,103]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 34 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 34 4.2.3. CB2-Selective Agonists Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 34 4.2.3. CB2-SelectiveConcerning Agonists CB2-selective agonists, the most widely used experimental tool is the classical 4.2.3.Concerningcannabinoid, CB2-Selective CB2-selective JWH-133 Agonists (Table agonists, 4a), theand mostthe less widely selective used amino-alkylindole,experimental tool isJWH-015 the classical (Table 4b), cannabinoid,developed JWH-133 by Dr John(Table Huffman 4a), and [66] the. Both less compounds selective amino-alkylindole, not only bind with JWH-015 higher affinity (Table to 4b), CB2 than Concerning CB2-selective agonists, the most widely used experimental tool is the classical developedto CB1, by butDr Johnalso behaveHuffman as a[66] potent. Both CB2-selective compounds agonistnot only in bind functional with higher assays affinity [66]. Other to CB2 notable than CB2- cannabinoid, JWH-133 (Table 4a), and the less selective amino-alkylindole, JWH-015 (Table 4b), to CB1,selective but also agonistsbehave as include a potent PM-226 CB2-selective (Table 4c),agonist HU in-308 functional (Table 4d), assays the [66]. GlaxoSmithKline Other notable CB2-compound developed by Dr John Huffman [66]. Both compounds not only bind with higher affinity to CB2 than selectiveGW-405833 agonists (Tableinclude 4e), PM-226 Merck (Table Frosst (now4c), HU known-308 (Tableas Merck 4d), Canada) the GlaxoSmithKline compounds L-759,633 compound (Table 4f) to CB1, but also behave as a potent CB2-selective agonist in functional assays [66]. Other notable CB2- GW-405833and L-759,656 (Table 4e), (Table Merck 4g). Frosst CB2-sele (now ctiveknown agonists as Merck have Canada) undoubtedly compounds been L-759,633 the focus (Table in the 4f) field of selective agonists include PM-226 (Table 4c), HU-308 (Table 4d), the GlaxoSmithKline compound and L-759,656therapeutic (Table uses, 4g). because CB2-sele modulationctive agonists of the have CB2 undoubtedlyis an interesting been approach the focus avoiding in the field CNS of related GW-405833 (Table 4e), Merck Frosst (now known as Merck Canada) compounds L-759,633 (Table 4f) therapeuticside effects, uses, because to treat modulationpain, inflammation, of the CB2 arthriti is ans, interesting addictions, approach neuroprote avoidingction, and CNS cancer, related among and L-759,656 (Table 4g). CB2-selective agonists have undoubtedly been the focus in the field of side effects,other possibleto treat therapeuticpain, inflammation, applications arthriti [24,27,95–10s, addictions,0]. Interestingly, neuroprote ction,the use and of knowncancer, CB2-selectiveamong therapeutic uses, because modulation of the CB2 is an interesting approach avoiding CNS related other possibleagonists therapeutic(i.e., JWH-015 applications and L-759,656) [24,27,95–10 for treating0]. Interestingly, or preventing the a use disease of known associated CB2-selective with immune side effects, to treat pain, inflammation, arthritis, addictions, neuroprotection, and cancer, among agonistsdysfunction (i.e., JWH-015 such andas HIV L-759,656) disease forwas treating proposed or inpreventing an US patent a disease published associated in 2012 with [22]. immune Over the past other possible therapeutic applications [24,27,95–100]. Interestingly, the use of known CB2-selective dysfunctiondecade, such published as HIV patents disease have was proposedclaimed >150 in an synthetic US patent selective published agonists in 2012 of CB2 [22]. [22]. Over Nowadays, the past new agonists (i.e., JWH-015 and L-759,656) for treating or preventing a disease associated with immune decade,ligands published designed patents to interacthave clai withmed CB2>150 as synthetic selective selective agonists agonists are currently of CB2 the[22]. subject Nowadays, of research new both dysfunction such as HIV disease was proposed in an US patent published in 2012 [22]. Over the past ligandsby designed academia to and interact industry. with Furthermore,CB2 as selective a number agonists of arereports currently dealing the with subject in vivo of research and in vitro both models decade, published patents have claimed >150 synthetic selective agonists of CB2 [22]. Nowadays, new by academiahave shown and industry. positive Furthermore,and very interesting a number results of reports (as summarized dealing with in Tablein vivo 4). and Nevertheless, in vitro models there has ligands designed to interact with CB2 as selective agonists are currently the subject of research both have shownstill been positive limited and success very interesting in clinical trials, results partly (as summarized due to the lack in Table of translation 4). Nevertheless, from preclinical there has models by academia and industry. Furthermore, a number of reports dealing with in vivo and in vitro models still beenand limited also due success to the in differences clinical trials, across partly species due [22,1to the01,102]. lack of At translation present, atfrom least preclinical three unique models synthetic have shown positive and very interesting results (as summarized in Table 4). Nevertheless, there has and alsoCB2 due agonists to the differences have reached across clinic species trials, [22,1 01,102].including At present,GW842166X, at least S-777469 three unique and syntheticJBT-101 from still been limited success in clinical trials, partly due to the lack of translation from preclinical models CB2 agonistsClinicalTrials.gov have reached [22,70,103]. clinic trials, including GW842166X, S-777469 and JBT-101 from and also due to the differences across species [22,101,102]. At present, at least three unique synthetic ClinicalTrials.gov [22,70,103]. CB2 agonists have reached clinic trials, including GW842166X, S-777469 and JBT-101 from Table 4. Structures, chemical type and bioactivities of CB2-selective agonists. ClinicalTrials.gov [22,70,103]. Int. J. Mol.Table Sci. 4.2020 Structures,, 21, 5064 chemical type and bioactivities of CB2-selective agonists. 11 of 32 Chemical EC50 Synthetic Cannabinoids Ki (nM)/CB Bioactivity Table 4. Structures,ChemicalType chemical type and bioactivitiesEC50 (nM)/CB2of CB2-selective agonists. Synthetic Cannabinoids Ki (nM)/CB Bioactivity Type (nM)/CB2 Attenuating TableChemical 4. Structures, chemical typeEC50 and bioactivities of CB2-selective agonists. Synthetic Cannabinoids Ki (nM)/CB AttenuatingBioactivityneurodegenerative and spatial Type (nM)/CB2 neurodegenerativememory impairment and spatial [27]; Synthetic Cannabinoids Chemical Type K (nM)/CBAttenuating EC (nM)/CB2 Bioactivity i memoryimproving impairment50 cerebral [27]; infarction neurodegenerative and spatial improving[106]; cerebral infarction Attenuating neurodegenerative memory impairment [27]; 677/CB1 [106]; anti-inflammatory, and spatial memory impairment improving cerebral infarction Classical 677/CB13.40/CB2 63.0 [105]anti-inflammatory, ameliorating sepsis [107]; [27]; improving cerebral [106]; infarction [106]; Classical 3.40/CB2[104] 63.0 [105] amelioratinganti-cancer sepsis [53]; [107]; anti-inflammatory, 677/CB1 anti-inflammatory,anti-nociceptive [108]; (a) [104] 677/CB1 anti-cancer [53]; ameliorating sepsis [107]; Classical Classical3.40/CB2 63.0 [105] amelioratingprotective63.0 [sepsis 105effects] [107]; on renal (a) JWH-133 3.40/CB2 [104anti-nociceptive] [108]; anti-cancer [53]; [104] anti-cancerischemia-reperfu [53]; sion injury JWH-133 protective effects on renal anti-nociceptive [108]; (a) anti-nociceptive [108]; (a) ischemia-reperfu[109] and againstsion injury protective effects on renal JWH-133 protective effects on renal JWH-133 [109] andcardiotoxicity against [110] ischemia-reperfusion injury ischemia-reperfusion injury cardiotoxicity [110] [109] and against cardiotoxicity [109]Attenuating and against [110] cardiotoxicity [110] Attenuatingneurodegenerative, neuroprotective [106]; 383/CB1 neurodegenerative, Attenuating neurodegenerative, Amino- Attenuatingimmunomodulatory, neuroprotective [106]; 13.8/CB2 NA neuroprotective [106]; alkylindole383/CB1 neurodegenerative,anti-inflammatory [53]; immunomodulatory, Amino- 383/CB1 immunomodulatory, Amino-alkylindole13.8/CB2[104] NA neuroprotectiveNA [106]; anti-inflammatory [53]; alkylindole 383/CB1 13.8/CB2 [104anti-inflammatory] anti-nociceptive [53]; [111]; Amino- [104] immunomodulatory, anti-nociceptive [111]; 13.8/CB2 NA anti-nociceptiveanti-cancer [111]; [112]; alkylindole anti-inflammatory [53]; anti-cancer [112]; (b) [104] anti-canceranti-obesity [112]; [25] anti-nociceptive [111]; anti-obesity [25] (b) JWH-015(b) anti-obesity [25] anti-cancer [112]; JWH-015JWH-015 (b)Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW anti-obesity [25] 12 of 34 JWH-015Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 34 >40000/CB1 Neuroprotective [95]; Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEWClassical >40,00038.7/ CB1[95] Anti-convulsant, 12 ofNeuroprotective 34 [95]; Classical 12.8/CB2 [95] anti-neuroinflammatory38.7 [95] [78] >40000/CB1 12.8/CB2 [95Neuroprotective] Anti-convulsant,neuroprotective [95]; [106]; anti-anti-neuroinflammatory [78] Int. J. Mol. Sci. 2020, 21, x FORClassical PEER REVIEW 38.7 [95] 12 of 34 (c)(c) 12.8/CB2 [95] anti-neuroinflammatoryAnti-convulsant,neuroprotectiveinflammatory [96,97]; [106]; [78] anti- PM-226 >40000/CB1 Neuroprotective [95]; (c) PM-226 Classical >10000/CB1 38.7 [95] neuroprotectiveinflammatoryanti-nociceptive [106];[96,97]; [96]; anti- Nonclassical12.8/CB2 [95] 5.57 [23] anti-neuroinflammatoryAnti-convulsant, [78] Anti-convulsant, PM-226 >10000/CB122.7/CB2 [66] inflammatoryanti-nociceptiveanti-dyskinesia [96,97]; [98]; [96]; Nonclassical 5.57 [23] neuroprotective [106]; anti-neuroprotective [106]; (c) osteoprotective [97]; >10000/CB122.7/CB2 [66] anti-nociceptiveanti-dyskinesia [96]; [98]; anti-inflammatory [96,97]; PM-226 Nonclassical 5.57 [23] inflammatory [96,97]; (d) 22.7/CB2 [66] >10,000/CB1 anti-dyskinesiaosteoprotectivealleviating septic [98]; [97]; lung injuryanti-nociceptive [96]; Nonclassical>10000/CB1 anti-nociceptive5.57 [23] [96]; (d)HU-308 Nonclassical 5.57 [23] osteoprotectivealleviating[113] septic [97]; lung injury 22.7/CB2 [66]22.7 /CB2 [66] anti-dyskinesia [98]; anti-dyskinesia [98]; (d)HU-308 alleviating[113] septic lung injury osteoprotective [97]; (d) osteoprotective [97]; alleviating septic lung injury HU-308HU-308 [113] (d) alleviating septic lung injury[113 ] HU-308 [113] Anti-nociceptive,

Amino- Anti-nociceptive,anti-inflammatory [99]; 4772/CB1 0.650 [24] Amino-alkylindole Anti-nociceptive,anti-inflammatoryprotective effects on [99]; acute liver 4772/CB13.92/CB2 [24] 0.650 [24] Anti-nociceptive, Amino-alkylindole anti-inflammatoryprotectiveinjury [114] effects [99]; on acute liver 4772/CB13.92/CB2 [24]4772 0.650 /CB1 [24] Anti-nociceptive, anti-inflammatory [99]; alkylindoleAmino-alkylindole protectiveinjury0.650 [114] effects [24 ] on acute liver Amino- 3.92/CB2 [24] 3.92/CB2 [24] anti-inflammatory [99]; protective effects on acute liver (e) 4772/CB1 0.650 [24]injury [114] injury [114] alkylindole protective effects on acute liver GW-405833 3.92/CB2 [24] (e) injury [114] (e) GW-405833(e) GW-405833GW-405833 (e) 1043, GW-405833 1043,15850/CB1 Classical 8.10 [115] Analgesic [53] 1043,15850/CB16.40, 20.0/CB2 1043, 15850/CB1 ClassicalClassical 8.10 [115] Analgesic8.10 [ 115[53]] Analgesic [53] 15850/CB16.40,[66] 20.0/CB2 6.40, 20.0/CB2 [66] Classical 1043, 8.10 [115] Analgesic [53] (f) 6.40,[66] 20.0/CB2 (f) 15850/CB1 (f)L-759,633 Classical [66] 8.10 [115] Analgesic [53] L-759,633 6.40, 20.0/CB2 (f) L-759,633 [66] L-759,633 (f) 529 L-759,633 529~ >20000/CB1 529~ >20000/CB1 ClassicalClassical 3.10 [115] Analgesic3.10 [ 115[53]] Analgesic [53] 529~11.8 >20000/CB1 ~ 57.0 11.8~57.0 [66] Classical 3.10 [115] Analgesic [53] ~ >20000/CB111.8[66] ~ 57.0 (g) Classical 529 3.10 [115] Analgesic [53] (g) 11.8[66] ~ 57.0 L-759,656 ~ >20000/CB1 (g)L-759,656 Classical [66] 3.10 [115] Analgesic [53] 11.8 ~ 57.0 (g)L-759,656 Ki: bindingKi :constant; binding EC constant;50: half-maximal EC50: half-maximal effective concentration; effective NA: concentration; not available. NA: not available. [66] L-759,656 (g) Ki: binding constant; EC50: half-maximal effective concentration; NA: not available. 4.2.4.L-759,656 CB1-SelectiveK i: binding Antagonists/Inverse constant; EC50: half-maximal Agonists effective concentration; NA: not available. 4.2.4. CB1-Selective Antagonists/Inverse Agonists 4.2.4.Since CB1-Selective the Kdiscoveryi: binding constant; Antagonistsof CB1 andEC50 : thehalf-maximal/ Inversesubsequent effective Agonists development concentration; of the NA: CB1-selective not available. and potent 4.2.4. CB1-Selective Antagonists/Inverse Agonists antagonistSince theSR141716 discovery (also of called CB1 andRimonabant) the subsequent (Table development5a) by Sanofi-Aventis of the CB1-selective [19,116], there and has potent also antagonistbeen4.2.4.Since considerableSince CB1-Selective the SR141716 discovery the discoveryinterest Antagonists/Inverse(also of CB1 called in andthe of Rimonabant) therapeuticthe CB1 subsequent andAgonists the(Tablepotential development subsequent 5a) ofby CB1-selectiveSanofi-Aventis of developmentthe CB1-selective antagonists. [19,116],of thereand Researchers the potenthas CB1-selective also and potent antagonistbeenfoundantagonist considerable Sincetheir SR141716 therapeuticthe SR141716 discovery (alsointerest potentialcalled of (alsoin CB1 theRimonabant) in calledandtherapeutic the thetreatment Rimonabant)subsequent (Table potential of 5a)disorders developmentby of Sanofi-Aventis (TableCB1-selective in which5 a)of thethe by [19,116], antagonists.endocannabinoid CB1-selective Sanofi-Aventis there Researchers hasand systemalso potent [ 19 ,116 ], there has also beenfoundappearsbeenantagonist considerable their considerable to induce therapeutic SR141716 interest undesirable (also potential interestin calledthe symptoms therapeuticin inRimonabant) the the treatment following therapeutic potential (Table of itsdisorders upregulationof5a) CB1-selective potentialby inSanofi-Aventis which [1]. ofthe Otherantagonists. CB1-selectiveendocannabinoid [19,116],notable Researchers CB1there selective antagonists.systemhas also Researchers foundappearsantagonists their to therapeutic induce include undesirable potentialanalogs ofsymptomsin therimonabant, treatment following ofAM-251 disorders its upregulation (Table in which 5b) and[1].the OtherendocannabinoidAM-281 notable (Table CB1 5c)system selective [117]. foundbeen considerable their therapeutic interest in potential the therapeutic in the potential treatment of CB1-selective of disorders antagonists. in which Researchers the endocannabinoid system appearsantagonistsRimonabant,found to their induce include therapeuticAM-251 undesirable analogs and potential AM-281 symptoms of rimonabant, innot the only following treatment act asAM-251 antagonistsits of upregulation disorders (Table attenuating in5b) which[1]. and Other the effectsAM-281 notableendocannabinoid of CB1(Table CB1 agonists, selective 5c) system[117]. but antagonistsRimonabant,actappears as inverse toinclude induce AM-251 agonists analogs undesirable and which AM-281 of can rimonabant, symptoms by not themselves only followingact AM-251 as elic antagonistsit itsresponses(Table upregulation attenuating5b) in and some [1].AM-281 CB1-containing effectsOther notableof(Table CB1 agonists, 5c)CB1 tissues [117]. selective thatbut Rimonabant,actareantagonists asopposite inverse AM-251 in agonistsinclude direction and whichanalogs AM-281 from can thoseof not by rimonabant, onlyelicitedthemselves act byas antagonists CB elicAM-2511 itagonists responses (Table attenuating [117]. in5b) someMore and effects CB1-containingspecifically, AM-281 of CB1 (Table agonists, they tissues appear5c) but [117].that to actareproduce asRimonabant, oppositeinverse inverse agonists in AM-251 direction cannabimimetic which and from canAM-281 those by effects themselves notelicited inonly at by leastact elic CB as soit 1antagonists responses meagonists tissues [117]. inby attenuating some somehow More CB1-containing specifically, effects reducing of CB1 the they tissues constitutiveagonists, appear that butto areproduceactivity oppositeact as inverse inverseof in CB1. direction agonists cannabimimetic The fromconstitutive which those can effects elicited byactivity themselves in byat isleast CB un1 soderstoodagonistselicmeit tissuesresponses [117].as bythe More somehowin coupling some specifically, CB1-containing reducing of CB1 they the to appearconstitutiveits tissues effector to that produceactivitymechanismsare opposite inverse of CB1. that, cannabimimeticin directionThe it is constitutive thought, from effects those can activity in occurelicited at least inis by theunso CBmederstood absence1 tissues agonists of asby exogenously [117].somehowthe coupling More reducing specifically, added of CB1 theor toconstitutiveendogenously they its appeareffector to activitymechanismsreleasedproduce of CB1CB1. inverse that,agonists The cannabimimeticit constitutiveis [117].thought, Since canactivity effectsthe occur withdrawal inis at inun leastthederstood absenceofso merimonabant tissuesas of the exogenously by couplingfrom somehow the market ofadded reducing CB1 inorto 2008, endogenouslytheits constitutiveeffector due to its mechanismsreleasedsevereactivity psychiatric CB1of that, CB1.agonists it isTheside thought, [117]. constitutiveeffects, Since can research theoccur activity withdrawal inon the isCB absenceun1-selective ofderstood rimonabant of exogenously antagonists’as the from coupling the addedpotential market of or CB1in pharmacologicalendogenously 2008, to itsdue effector to its releasedsevereeffectsmechanisms CB1haspsychiatric agonistscontinued. that, sideit[117]. isFor effects,thought, Since instance, theresearch can withdrawal a occurrecent on instudCB theof1-selectivey rimonabant absenceshowed antagonists’ofthat fromexogenously rimonabant the market potential added protects in 2008, pharmacologicalor againstendogenously due to light-its severeeffectsinducedreleased psychiatric has retinal CB1 continued. agonists degeneration side effects,For [117]. instance, inSinceresearch vitro the anda recent withdrawalon in vivoCB stud1-selective viay regulating ofshowed rimonabant antagonists’ that CB1 rimonabant [118].from potential theMore market protectsrecently, pharmacological in 2008,against it was due shown light- to its effectsinducedtosevere exhibit has retinalpsychiatriccontinued. neuroprotective degeneration Forside instance, effects,effects in vitro inresearcha arecentand retinal in vivostudon degene CBviay showed1-selective regulatingration thatmodel antagonists’CB1 rimonabant by [118]. blocking More potentialprotects CB1recently, [119]. againstpharmacological it At was the light- shown same inducedtotime,effects exhibit as retinal ahas selectiveneuroprotective continued.degeneration antagonist/inverse For effectsin instance, vitro in and a retinal agonistain recent vivo degenevia of stud CB1,regulatingyration implicationsshowed model CB1 that [118]. by ofrimonabant blockingrimonabant More recently, CB1 protects in [119]. weight it was againstAt loss,shownthe same anti-light- to time,diabetesexhibitinduced as neuroprotective a and selectiveretinal reduced degeneration antagonist/inverse drug effects dependency inin vitro a retinal agonistand have indegene vivo of been CB1, viaration established regulating implications model byCB1[53]. blockingof [118]. However,rimonabant More CB1 although recently,[119]. in weight At itresearchthe wasloss, same shown anti- on time,diabetes to as exhibit a selective and neuroprotective reduced antagonist/inverse drug dependencyeffects in agonist a retinal have of CB1,degenebeen implications establishedration model [53].of rimonabantby However, blocking inalthoughCB1 weight [119]. loss,research At the anti- same on diabetes time, andas a reducedselective drugantagonist/inverse dependency have agonist been of establishedCB1, implications [53]. However, of rimonabant although in weight research loss, on anti- diabetes and reduced drug dependency have been established [53]. However, although research on

Int. J. Mol. Sci. 2020, 21, 5064 12 of 32

appears to induce undesirable symptoms following its upregulation [1]. Other notable CB1 selective antagonists include analogs of rimonabant, AM-251 (Table5b) and AM-281 (Table5c) [ 117]. Rimonabant, AM-251 and AM-281 not only act as antagonists attenuating effects of CB1 agonists, but act as inverse agonists which can by themselves elicit responses in some CB1-containing tissues that are opposite in direction from those elicited by CB1 agonists [117]. More specifically, they appear to produce inverse cannabimimetic effects in at least some tissues by somehow reducing the constitutive activity of CB1. The constitutive activity is understood as the coupling of CB1 to its effector mechanisms that, it is thought, can occur in the absence of exogenously added or endogenously released CB1 agonists [117]. Since the withdrawal of rimonabant from the market in 2008, due to its severe psychiatric side effects, research on CB1-selective antagonists’ potential pharmacological effects has continued. For instance, a recent study showed that rimonabant protects against light-induced retinal degeneration in vitro and in vivo via regulating CB1 [118]. More recently, it was shown to exhibit neuroprotective effects in a retinal degeneration model by blocking CB1 [119]. At the same time, as a selective antagonist/inverse Int. J.agonist Mol. Sci. 2020 of, CB1,21, x FOR implications PEER REVIEW of rimonabant in weight loss, anti-diabetes and 13 reducedof 34 drug dependency Int. J.have Mol. Sci. been 2020, 21 established, x FOR PEER REVIEW [53]. However, although research on the development 13 of of 34 synthetic CB1-selective theInt. J.development Mol. Sci. 2020, 21 ,of x FOR synthetic PEER REVIEW CB1-selective antagonists sounded very promising, it remains 13 of 34 theassociated antagonistsdevelopment with unideal soundedof synthetic convoys. very CB1-selective promising,Several compounds antagonists it remains have sounded associatedbeen withdrawnvery withpromising, unidealfrom commercialit remains convoys. Several compounds associatedmarketsthehave development and been with clinical withdrawnunideal of trials synthetic convoys.[120]. from Besides CB1-selective Several commercial rimonabant, compounds antagonists markets taranabant have andsounded been(MK-0364) clinical withdrawn very (Table trials promising, 5d)from [120 and ].commercial it Besidesotenabant remains rimonabant, taranabant markets(CP-945,598)associated(MK-0364) and with clinical(Table (Tableunideal 5e)trials were5d) [120].convoys. andboth Besides otenabantdiscontinued Several rimonabant, compounds (CP-945,598) in ph taranabantase III have clinical (Table (MK-0364)been trials 5withdrawne) for were(Table treating both 5d)from andobesity discontinued commercial otenabant due to in phase III clinical (CP-945,598)themarkets trialsrisk/reward and for (Table clinical treating ratio 5e) trials[17,121] were obesity [120]. both and Besides discontinuedsurinabant due rimonabant, to the (SR147778) in risk ph asetaranabant/reward III (Table clinical ratio(MK-0364)5f) trialswas [17 discontinuedfor, 121(Table treating] and 5d) obesity andfrom surinabant otenabant clinicaldue to (SR147778) (Table5f) thetrials(CP-945,598) risk/reward for smoking (Table ratio cessation 5e) [17,121] were [53]. bothand Moreover, surinabantdiscontinued there (SR147778) in is phnoase CB1-selective (TableIII clinical 5f) wastrialsantagonist discontinued for treating which obesityisfrom now clinical indue the to was discontinued from clinical trials for smoking cessation [53]. Moreover, there is no CB1-selective trialsstagethe risk/reward forof the smoking clinical ratio cessation trial [17,121] according [53]. and Moreover, to surinabant ClinicalTria there (SR147778) ls.govis no CB1-selective [70]. (Table Therefore, 5f) antagonistwas th ediscontinued current which strategy is from now towards clinicalin the stagetacklingtrialsantagonist of for thethese smoking clinical adverse which cessationtrial effects according is now [53]. may Moreover, in beto thetoClinicalTria restrict stage there bi ofls.govnding is the no of[70].CB1-selective clinical CB1 Therefore, antagonists trial antagonist th accordinge tocurrent CB1 which in strategy CNS, to ClinicalTrials.govis nowlimit towards intheir the [ 70]. Therefore, tacklingcrossingstagethe of current these ofthe the clinical adverse blood strategy trial brain effects according barrier, towards may beor to toco-administer tacklingClinicalTria restrict bi thesendingls.gov CB1 of adverse antagonists[70]. CB1 Therefore, antagonists eff withects th drugs mayeto current CB1 blocking be in to strategyCNS, restrict side limit towards effects. their binding of CB1 antagonists crossingtacklingto CB1 of these the in bloodadverse CNS, brain limiteffects barrier, their may or be crossing co-administer to restrict of binding the CB1 blood antagonistsof CB1 brainantagonists with barrier, drugs to CB1 blocking or in co-administer CNS, side limit effects. their CB1 antagonists with crossingTable of the5. Structures, blood brain chemical barrier, type or and co-administer bioactivities CB1of CB1-selective antagonists antagonists/inverse with drugs blocking agonists. side effects. drugsTable blocking 5. Structures, side chemical effects. type and bioactivities of CB1-selective antagonists/inverse agonists. Chemical IC50 SyntheticTable Cannabinoids 5. Structures, chemical type and Kbioactivitiesi (nM)/CB of CB1-selective antagonists/inverseBioactivity agonists. ChemicalType IC(nM)/CB150 Synthetic CannabinoidsTable 5. Structures, chemicalKi (nM)/CB type and bioactivities ofBioactivity CB1-selective antagonists/inverse agonists. TypeChemical (nM)/CB1IC50 Synthetic Cannabinoids Ki (nM)/CB Bioactivity Type (nM)/CB1 Synthetic Cannabinoids Chemical Type Ki (nM)/CB IC50 (nM)/CB1 Bioactivity 1.80 ~ Anti-obesity, 1.8012.3/CB1 ~ Anti-obesity,smoking cessation [53]; 5.60 ~ 48.0 Others 12.3/CB17021.80 ~ ~ smokingprotectiveAnti-obesity, cessation effects of [53]; retinal 5.60[116,122] ~ 48.0 Anti-obesity, Others 70213200/CB2 ~ protectivedegeneration effects [118]; of retinal 12.3/CB1 [116,122] smoking cessation [53]; smoking cessation [53]; [66] 5.601.80~12.3 ~ 48.0/ CB1neuroprotective [119] Others Others13200/CB2702 ~ degenerationprotective5.60~48.0 effects [118]; of [116 retinal,122] protective effects of retinal [116,122]702~13200 /CB2 [66] [66]13200/CB2 neuroprotectivedegeneration [118]; [119] degeneration [118]; (a) [66] neuroprotective [119] neuroprotective [119] (a)Rimonabant (SR141716) Rimonabant(a) (a) (SR141716) Rimonabant (SR141716) Rimonabant (SR141716) Anti-obesity; anti-depressant Anti-obesity;[53]; potentiating anti-depressant activity of Anti-obesity; anti-depressant 7.49/CB1 [53];antidepressant potentiating drugs activity [123]; of Anti-obesity; anti-depressant [53]; potentiating activity of Others 2290/CB2 3.00 [122] improving albuminuria and 7.49/CB1 antidepressant[53]; potentiating drugs activity [123]; of antidepressant drugs [123]; 7.49/CB1 Others Others2290/CB2[66]7.49/CB1 3.00 [122] improvingrenalantidepressant tubular3.00 albuminuria structure [122 drugs] [123]; [124] and as improving albuminuria and 2290/CB2 [66] Others [66]2290/CB2 3.00 [122] renalwellimproving as tubular recognition albuminuria structure memory [124] and as renal tubular structure [124] as well[125] as recognition memory well as recognition memory (b) [66] renal tubular structure [124] as [125]well as recognition memory [125] (b)AM-251 (b) AM-251 [125] AM-251(b) AM-251 Improving cognitive deficits Improving cognitive deficits [53]; facilitatory effect on Improving cognitive deficits [53]; facilitatory effect on 12.0/CB1 [53];recognitionImproving facilitatory memorycognitive effect [126]; deficitson recognition memory [126]; 12.0/CB1 Others Others12.0/CB14200/CB2 9.91 [122] recognitionameliorating9.91 memory spatial [122] learning[126]; ameliorating spatial learning 4200/CB2 [66] [53]; facilitatory effect on Others 4200/CB2[66]12.0/CB1 9.91 [122] amelioratingandrecognition memory memoryspatialimpairment learning [126]; [127]; and memory impairment [127]; protective effects against Others [66]4200/CB2 9.91 [122] andprotectiveameliorating memory effects impairment spatial against learning [127]; cardiotoxicity [110] [66] protectivecardiotoxicityand memory effects impairment[110] against [127]; (c) (c) AM-281 cardiotoxicityprotective effects [110] against (c)AM-281 cardiotoxicity [110] AM-281 (c) Cl

F AM-281F Cl 0.130, FF F Cl N Anti-obesity [129]; H N C 0.130,0.270/CB1 F N O F F N Others 0.290 [128] Anti-obesitysmoking cessation [129]; [130]; H 170, 310/CB2 O N C 0.270/CB10.130, F N O Others 0.290 [128] smokinganti-nociceptive cessation [131]; [130]; N Anti-obesity [129]; H 170,[104] 310/CB2 C 0.270/CB1 O N anti-nociceptive [131]; N O Others 0.290 [128] smoking cessation [130]; (d) [104]170, 310/CB2 O anti-nociceptive [131]; (d)Taranabant (MK-0364) [104] Taranabant(d) (MK-0364) 0.120 ~ Taranabant (MK-0364) Others 0.1200.700/CB1 ~ 13.1 [122] Anti-obesity [120] Others 0.700/CB1[132,133]0.120 ~ 13.1 [122] Anti-obesity [120] Others [132,133]0.700/CB1 13.1 [122] Anti-obesity [120] [132,133]

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 13 of 34

the development of synthetic CB1-selective antagonists sounded very promising, it remains associated with unideal convoys. Several compounds have been withdrawn from commercial markets and clinical trials [120]. Besides rimonabant, taranabant (MK-0364) (Table 5d) and otenabant (CP-945,598) (Table 5e) were both discontinued in phase III clinical trials for treating obesity due to the risk/reward ratio [17,121] and surinabant (SR147778) (Table 5f) was discontinued from clinical trials for smoking cessation [53]. Moreover, there is no CB1-selective antagonist which is now in the stage of the clinical trial according to ClinicalTrials.gov [70]. Therefore, the current strategy towards tackling these adverse effects may be to restrict binding of CB1 antagonists to CB1 in CNS, limit their crossing of the blood brain barrier, or co-administer CB1 antagonists with drugs blocking side effects.

Table 5. Structures, chemical type and bioactivities of CB1-selective antagonists/inverse agonists.

Chemical IC50 Synthetic Cannabinoids Ki (nM)/CB Bioactivity Type (nM)/CB1

1.80 ~ Anti-obesity, 12.3/CB1 smoking cessation [53]; 5.60 ~ 48.0 Others 702 ~ protective effects of retinal [116,122] 13200/CB2 degeneration [118]; [66] neuroprotective [119]

(a) Rimonabant (SR141716)

Anti-obesity; anti-depressant [53]; potentiating activity of 7.49/CB1 antidepressant drugs [123]; Others 2290/CB2 3.00 [122] improving albuminuria and [66] renal tubular structure [124] as well as recognition memory [125] (b) AM-251

Improving cognitive deficits [53]; facilitatory effect on 12.0/CB1 recognition memory [126]; Int. J. Mol. Sci. 2020, 21, 5064 Others 4200/CB2 9.91 [122] ameliorating spatial learning 13 of 32 [66] and memory impairment [127]; protective effects against Table 5. Cont. cardiotoxicity [110] (c) AM-281 Synthetic Cannabinoids Chemical Type Ki (nM)/CB IC50 (nM)/CB1 Bioactivity

Cl

F F 0.130, Int.F J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 34 N Anti-obesity [129]; H Anti-obesity [129]; C 0.270/CB1 N 0.130, 0.270/CB1 N O OthersOthers 0.290 [128] smoking0.290 cessation [128] [130]; smoking cessation [130]; 170, 310/CB2170, 310 CB2 [104] O 7663/CB2 / anti-nociceptive [131]; Int. J. Mol.Int. J. Sci. Mol. 2020 Sci., 212020, x ,FOR 21, x PEER FOR PEERREVIEW REVIEW 14 of 34 14anti-nociceptive of 34 [131]; [132][104] (d)(d) TaranabantTaranabant (MK-0364) (MK-0364) 7663/CB27663/CB2 [132] [132]0.120 ~ Others 0.700/CB1 13.1 [122] Anti-obesity [120] [132,133] 0.120~0.700/CB1 Others [132,133] 13.1 [122] Anti-obesity [120] 7663/CB2 [132] (e) Otenabant (CP-945,598) (e) (e)(e) OtenabantOtenabant (CP-945,598) (CP-945,598)

O N N H

N N O O 3.50/CB1 Anti-obesity, smoking cessation, N N 3.50/CB1 Cl 9.60 Anti-obesity, smoking cessation, suppressing N N Br Others 9.60 [134] suppressing alcohol preference H H Others 442/CB2 442/CB2 [134] [134] alcohol preference [53] [53] N N [134] N N Cl 3.50/CB13.50/CB1 Cl Cl 9.60 9.60 Anti-obesity,Anti-obesity, smoking smoking cessation, cessation, suppressing suppressing Br OthersBr Others 442/CB2 442/CB2 (f)(f) [134] [134] alcoholalcohol preference preference [53] [53] [134] SurinabantSurinabant (SR147778) [134] Cl Cl (SR147778) K : binding constant; IC : half-maximal inhibitory concentration; NA: not available. (f) (f) i 50 Ki: binding constant; IC50: half-maximal inhibitory concentration; NA: not available. SurinabantSurinabant (SR147778)(SR147778) 4.2.5. CB2-Selective Antagonists/Inverse Agonists 4.2.5. CB2-SelectiveKi: bindingKi: binding constant; constant; Antagonists IC50: half-maximalIC50: half-maximal/Inverse inhibitory inhibitory Agonists concentration; concentration; NA: notNA: available. not available. The most notable CB2-selective antagonists/inverse agonists are the Sanofi-Aventis 4.2.5.diarylpyrazole,4.2.5. CB2-Selective TheCB2-Selective most Antagonists/InverseSR144528 notable Antagonists/Inverse [135] CB2-selective (Table Agonists 6a) Agonists and antagonists 6-iodopravadoline /inverse (AM-630) agonists [115] are (Table the Sanofi-Aventis 6b). Both diarylpyrazole, SR144528compoundsThe Themost most bind [notable135 ]withnotable (Table CB2-selectivemuch 6CB2-selectivea) higher and affinity 6-iodopravadolineantagonists/ antagonists/ to CBinverse2 thaninverse toagonists CB1, (AM-630) agonists exhibit are [markedarethe115 the]Sanofi-Aventis (Tablepotency Sanofi-Aventis6 b).as CB2- Both compounds bind selective antagonists and behave as inverse agonists that can produce inverse cannabimimetic effects diarylpyrazole,withdiarylpyrazole, much SR144528 higher SR144528 [135] affi nity[135] (Table to(Table CB26a) and6a) than 6-iodopravadolineand to 6-iodopravadoline CB1, exhibit (AM-630) marked (AM-630) [115] potency [115] (Table (Table as6b). CB2-selective Both6b). Both antagonists and atcompounds CB2 by themselves bind with [66]. much In fact,higher less affinity attention to CBhas2 beenthan paidto CB1, to CB2-selective exhibit marked antagonists/inverse potency as CB2- compoundsbehave bind asinverse with much agonists higher affinity that can to produceCB2 than to inverse CB1, exhibit cannabimimetic marked potency eff ectsas CB2- at CB2 by themselves [66]. selectiveagonistsselective antagonists comparedantagonists and to behaveand agonists, behave as inverse asas inverseindicated agonists agonists by that a small canthat produce can number produce inverse of inversepatents cannabimimetic cannabimimeticand pharmacological effects effects at CB2Instudiesat CB2by fact, themselves overby less themselves the attention past [66]. few [66].In years. hasfact, In beenfact,less For attentionless example, paid attention to has the CB2-selective beenhasUS patent beenpaid paid tofor CB2-selective antagonistsAM-630to CB2-selective describes antagonists/inverse/inverse antagonists/inverse this compound agonists as compared to agonists, agonistsasaagonists CB2-selective indicated compared compared toantagonist by agonists, ato smallagonists, andas number indicated proposesas indicated ofbythe apatents usebysmall aof small AM-630number and number pharmacologicaloffor patents treatingof patents and or preventingandpharmacological pharmacological studies a disease over the past few years. associated with immune dysfunction such as HIV disease [22]. In addition, AM-630 has been shown studiesForstudies over example, overthe past the pastfew the years.few US years. patent For example, For for example, AM-630 the US the patent US describes patent for AM-630 for this AM-630 describes compound describes this compoundthis as acompound CB2-selective as as antagonist and a CB2-selectivetoa CB2-selectiveeffectively antagonist inhibit antagonist inflammatory and andproposes proposes osteolysis the usethe ofusein AM-630the of differentiationAM-630 for treatingfor treating medium or preventing or preventingsystem a[136] disease a anddisease to proposes the use of AM-630 for treating or preventing a disease associated with immune dysfunction associatedpotentiateassociated with the withimmune activity immune dysfunction of dysfunctionconventional such suchas antidepressant HIV as HIVdisease disease [22]. drugs [22].In addition,in Invivo addition, [26]. AM-630 In AM-630 addition, has been has in been showncontrast shown to to effectivelysuchCB2-selectiveto effectively as inhibit HIV agonists,inhibit disease inflammatory inflammatory no [CB2-selective22]. osteolysis In addition,osteolysis antagonist in the in AM-630 differentiationthe ha sdifferentiation been has in the beenmedium stage medium shown of system clinical system to [136] trial eff [136]ectively soand far andto from inhibitto inflammatory potentiateosteolysisClinicalTrials.govpotentiate the activity the in activity the [70].of diconv offf erentiationconventionalentional antidepressant mediumantidepressant drugs system drugs in vivo[ 136in [26].vivo] and [26].In addition,to In potentiateaddition, in contrast in contrast the to activity to of conventional CB2-selectiveCB2-selective agonists, agonists, no CB2-selective no CB2-selective antagonist antagonist has been has beenin the in stage the stage of clinical of clinical trial trialso far so from far from antidepressantTable 6. Structures, drugs chemicalin vivo type[26 and]. bioactivities In addition, of CB2-selective in contrast antagonists/inverse to CB2-selective agonists. agonists, no CB2-selective ClinicalTrials.govClinicalTrials.gov [70]. [70]. antagonist has been in the stage of clinical trial so far from ClinicalTrials.gov[70]. Synthetic Chemical IC50 Ki (nM)/CB Bioactivity CannabinoidsTableTable 6. Structures, 6. Structures, chemicalType chemical type andtype bioactivities and bioactivities of CB2-selective(nM)/CB2 of CB2-selective antagonists/inverse antagonists/inverse agonists. agonists. Table 6. Structures, chemical type and bioactivities of CB2-selective antagonists/inverse agonists. SyntheticSynthetic ChemicalChemical IC50 IC50 Ki (nM)/CBKi (nM)/CB BioactivityBioactivity CannabinoidsCannabinoids Type Type (nM)/CB2(nM)/CB2 Synthetic Cannabinoids Chemical Type Ki (nM)/CB IC50 (nM)/CB2 Bioactivity 50.3 ~ >10000/CB1 Others 39.0 [135] Anti-nociceptive [53] 0.280 ~ 50.3 50.3 5.60/CB2 [66] ~ >10000/CB1~ >10000/CB1 OthersOthers 39.0 [135]39.050.3~ [135]> Anti-nociceptive 10,000 Anti-nociceptive/CB1 [53] [53] Others0.280 ~ 39.0 [135] Anti-nociceptive [53] (a) 0.280 ~ 0.280~5.60/CB2 [66] 5.60/CB25.60/CB2 [66] [66] SR144528 (a) (a)(a) SR144528 SR144528SR144528

Int. J. Mol. Sci. 2020, 21, 5064 14 of 32

Table 6. Cont. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 34

Synthetic Cannabinoids Chemical Type Ki (nM)/CB IC50 (nM)/CB2 Bioactivity

Inhibiting inflammatory osteolysisInhibiting inflammatory osteolysis [136]; potentiating Amino- 5152/CB1 5152/CB1 [136]; potentiating activity of Amino-alkylindole12.3 [122] 12.3 [122] activity of antidepressant drugs alkylindole 31.2/CB2 [66] 31.2/CB2 [66antidepressant] drugs [26]; improving [26]; improving memory, memory, anti-oxidant [137] anti-oxidant [137] (b)(b) AM-630AM-630

Ki: bindingKi: constant; binding IC constant;50: half-maximal IC50: half-maximal inhibitory concentration; inhibitory NA: concentration; not available. NA: not available.

4.2.6. Allosteric Modulators 4.2.6.Allosteric Allosteric ligands Modulators are ligands binding to a site topographically different from the orthosteric binding site. They can be divided into three groups according to their effects on the orthosteric ligand (bind Allostericto the same site ligands as the endogenous are ligands ligand) binding responses: to a positive site topographically allosteric modulators diff (PAMs),erent from the orthosteric bindingnegative allosteric site. They modulators can be (NAMs), divided and into neutral three allosteric groups ligands according (NALs). toThey their hold e ffgreatects on the orthosteric ligandtherapeutic (bind potential, to the as they same do sitenot possess as the intrin endogenoussic efficacy. Instead, ligand) they responses: enhance or positivediminish the allosteric modulators response of orthosteric ligands at receptors allowing for the tempering of cannabinoid receptor (PAMs),signaling negativewithout the allosteric desensitization, modulators tolerance, (NAMs), and dependence and neutral [103]. allosteric In addition, ligands allosteric (NALs). They hold great therapeuticmodulators of cannabinoid potential, asreceptors they dohave not numerous possess advantages intrinsic over effi cacy.the orthosteric Instead, ligands, they enhancesuch or diminish the responseas a higher ofreceptor orthosteric type selectivity, ligands probe at receptors dependence allowing and biased for thesignaling. tempering Therefore, of cannabinoid they have a receptor signaling withoutgreat potential the desensitization,to separate the therapeutic tolerance, benefits and from dependence side effects [specifically103]. In addition, seen with some allosteric modulators of orthosteric ligands [103]. The notion of an allosteric modulator of CB1 was first reported by Price et cannabinoidal. in 2005 [138]. receptorsThey found have three allosteric numerous modula advantagestors of CB1, over Org29647 the orthosteric (Table 7a), Org27759 ligands, (Table such as a higher receptor type7b) and selectivity, Org27569 (Table probe 7c) which dependence were shown and to enhance biased the signaling. binding of the Therefore, CB1 agonist they CP-55,940, have a great potential to separatewhile reducing the therapeuticthe binding of benefitsthe antagonist/inverse from side eagonistffects specificallySR141716. Among seen them, with Org27569 some orthosteric has ligands [103]. Thethe best notion PAM ofeffect an on allosteric CP-55,940 modulator binding [103]. of Although CB1 was Org27569 first reported acts as PAM by for Price binding, et al. it in 2005 [138]. They reduces CP-55,940-dependent G protein-coupling, producing an insurmountable antagonism of the foundreceptor threefunctionality allosteric and inhibits modulators CP-55,940-depend of CB1,ent Org29647 cAMP inhibition, (Table cons7a),istent Org27759 with a behavior (Table 7b) and Org27569 (Tableas NAM7 c)[103,138]. which Since were then, shown other tosynthetic enhance CB1the allosteric binding modulators of the were CB1 developed, agonist CP-55,940, such as the while reducing the bindingPAMs GAT211 of the (Table antagonist 7d), ZCZ011/inverse (Table agonist 7f), RTI-371 SR141716. (Table 7g), Among and the them,NAM PSNCBAM-1 Org27569 has(Table the best PAM effect on CP-55,9407h). Like Org27569, binding PSCBAM-1 [103]. Although shows a Org27569PAM profile acts in binding as PAM assays for binding,but behaves it reduces as a non- CP-55,940-dependent competitive functional antagonist, decreasing the cellular response induced by orthosteric agonists G[139]. protein-coupling, Among these CB1 producing allosteric modulators, an insurmountable the synthetic antagonism indole Org27569 of the is one receptor of the functionalitymost and inhibits CP-55,940-dependentintensively studied. Recently, cAMP the crystal inhibition, structur consistente of the ternary with complex a behavior of CB1 as with NAM orthosteric [103,138 ]. Since then, other syntheticagonist CP-55,940 CB1 allostericand the allosteric modulators molecule wereOrg2756 developed,9 were reported such for as the the first PAMs time, illustrating GAT211 (Table7d), ZCZ011 (Tablea potential7f), strategy RTI-371 for (Table the drug7g), modulation and the of NAM CB1 and PSNCBAM-1 other class A GPCRs (Table [140].7h). LikeTo date, Org27569, a number PSCBAM-1 shows of Org27569 or PSNCBAM-1 analogs and other types of CB1 allosteric modulators have been adeveloped PAM profile in order in to bindingimprove specificity assays but[12]. behavesHowever, only as a two non-competitive CB1 allosteric modulators, functional ZCZ011 antagonist, decreasing the[141] cellular and the racemic response mix GAT211 induced [142–144], by orthosteric have been demonstrated agonists [139 to ].elicit Among in vivo activity these through CB1 allosteric modulators, thea CB1 synthetic mechanism indole of action. Org27569 Interestingly, is one PAM of theactivity most of intensivelyGAT211 in vivo studied. was recently Recently, shown theto crystal structure of thereside ternary with its complex S-(-)-enantiomer of CB1 (GAT229) with orthosteric (Table 7e), agonist constituting CP-55,940 the first and demonstration the allosteric of molecule Org27569 enantiomer-selective CB1 positive allosteric modulation in animal models [143]. Contrarily, none of werethe other reported CB1 allosteric for the modulators’ first time, CB1-depending illustrating in a vivo potential effects have strategy been demonstrated for the drug to modulationdate of CB1 and other[12,103]. class For example, A GPCRs Org27569 [140]. has To been date, shown a number not to ofalter Org27569 in vivo pharmacological or PSNCBAM-1 effectsanalogs (i.e., and other types ofantinociception, CB1 allosteric catalepsy, modulators and hypothermia) have been of orthosteric developed CB1 agon in orderists (i.e., to AEA, improve CP-55,940, specificity and [12]. However, onlyΔ9-THC) two and CB1 not to allosteric alter the discriminative modulators, stimulus ZCZ011 effects [141 of AEA] and in thefatty racemicacid amide mix hydrolase- GAT211 [142–144], have deficient mice [12]. In conclusion, it remains important to demonstrate the in vivo effects of candidate beencompounds demonstrated producing their to elicitallostericin vivoactionsactivity at CB1. through a CB1 mechanism of action. Interestingly, PAM activityCompared of GAT211 to CB1, onlyin vivo twowas synthetic recently allosteric shown modulators to reside have withbeen developed its S-(-)-enantiomer for CB2 so far. (GAT229) (Table7e), constitutingOne of them is cannabidiol-dimethylheptyl the first demonstration (CBD-D of enantiomer-selectiveMH or HU-219) (Table 7i) CB1which positive has been shown allosteric modulation in animalto act as modelsan allosteric [143 modulator]. Contrarily, for CB2, none reducing of the CP-55,940 other CB1binding allosteric and being modulators’ a PAM of cAMP CB1-depending in vivo e ffects have been demonstrated to date [12,103]. For example, Org27569 has been shown not to alter in vivo pharmacological effects (i.e., antinociception, catalepsy, and hypothermia) of orthosteric CB1 agonists (i.e., AEA, CP-55,940, and ∆9-THC) and not to alter the discriminative stimulus effects of AEA in fatty acid amide hydrolase-deficient mice [12]. In conclusion, it remains important to demonstrate the in vivo effects of candidate compounds producing their allosteric actions at CB1. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 34 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 34 Int.modulation J. Mol. Sci. 2020 but, 21 a, NAMx FOR PEER of β -arrestin1REVIEW recruitment [54]. In addition, CBD-DMH can be considered 16 of 34 as modulationa mixedInt. agonist/PAM J. but Mol. a Sci. NAM 2020 of, of21 CB1 ,β x-arrestin1 FOR [54]. PEER The REVIEWrecruitment other CB2 allosteric[54]. In addition, modulator CBD-DMH is the compound can be considered C2 (Table as7j), 16 of 34 amodulationwhich mixedInt. canagonist/PAM J. Mol. actbut Sci. asa 2020NAM a CB2, of21 ,of CB1xPAM FORβ-arrestin1 [54]. PEER in Thebinding REVIEW recruitmentother assays CB2 allosteric and[54]. displaysIn addition,modulator antinociceptive CBD-DMH is the compound canactivity be consideredC2 in (Table vivo in 7j), asan 16 of 34 whichaexperimental mixed can modulationagonist/PAM act asmouse a CB2 but modelof a PAMCB1 NAM of[54]. in neuropathicof binding Theβ-arrestin1 other assays CB2pain recruitment allostericand [145]. displays Apparently, [54].modulator antinociceptive In addition, all is theCB compoundCBD-DMHallosteric activity modulators C2incan vivo(Table be consideredin 7j), anare as experimentalwhichstill inmodulation can the actstage mouse as ofa but preclinicalCB2 model a NAMPAM of ofstudiesinneuropathic βbinding-arrestin1 instead assays pain recruitment of the[145].and clinical displays Apparently, [54]. trial In antinociceptive addition, according all CB CBD-DMH allostericto ClinicalTrials.gov activity modulators can in bevivo considered in [70].are an as Int. J. Mol. Sci.a mixed 2020, 21 agonist/PAM, x FOR PEER REVIEW of CB1 [54]. The other CB2 allosteric modulator is the compound 16 ofC2 34 (Table 7j), stillexperimental in athe mixedwhich stage mouse agonist/PAMcan of preclinicalact model as a CB2 of studies CB1neuropathic PAM [54]. instead in The binding pain otherof the [145]. assaysCB2 clinical allostericApparently, and trial displays according modulator all antinociceptiveCB to allosteric is ClinicalTrials.gov the compound modulators activity C2 [70].in (Tableare vivo in7j), an Table 7. Structures, chemical type and bioactivities of allosteric modulators. modulationstill inwhich theexperimental stage but can a of NAMact preclinical as mouse of a βCB2-arrestin1 modelstudies PAM of in recruitmentinstead neuropathicbinding of theassays [54]. clinicalpain In and addition,[145]. trial displays Apparently, according CBD-DMH antinociceptive to all ClinicalTrials.gov can CB beallosteric consideredactivity modulators in [70]. vivo as in an are Table 7. Structures, chemical type and bioactivities of allosteric modulators. a mixedSyntheticexperimental agonist/PAMstill Cannabinoids in the stage mouse of CB1 of preclinicalmodel [54]. The of neuropathic otherstudies CB2 instead allosteric Chemicalpain of [145]. the modulator Type clinical Apparently, trial is the according allcompound CB allostericBioactivity to ClinicalTrials.gov C2 (Table modulators 7j), are[70]. Table 7. Structures, chemical type and bioactivities of allosteric modulators. whichSynthetic canstill actCannabinoidsin the as stagea CB2 of PAM preclinical in binding studies assays instead and Chemical of displays the clinicalType antinociceptive trial according activity to Bioactivity ClinicalTrials.gov in vivo in an [70]. Table 7. Structures, chemical type and bioactivities of allosteric modulators. experimentalSynthetic Cannabinoids mouse model of neuropathic pain [145]. Chemical Apparently, Type all CB allosteric Bioactivity modulators are Int. J. Mol. Sci. 2020Table, 21 7., 5064Structures, chemical type and bioactivities of allosteric modulators. 15 of 32 still in the Syntheticstage of preclinical Cannabinoids studies instead of the clinical Chemical trial according Type to ClinicalTrials.gov Bioactivity [70]. Amino-alkylindole NA Synthetic Cannabinoids Chemical Type Bioactivity Table 7. Structures,Table 7. chemicalStructures, type chemical and bioactivitiesAmino-alkylindole type and of bioactivities allosteric modulators. of allostericNA modulators. (a) Amino-alkylindole NA SyntheticOrg29647 Cannabinoids Chemical Type Bioactivity (a) Synthetic Cannabinoids Chemical TypeAmino-alkylindole Bioactivity NA Org29647(a) Amino-alkylindole NA Org29647(a) (a)Org29647 Amino-alkylindoleAmino-alkylindole NA NA Org29647 Amino- (a) NA (a) Amino-alkylindole Org29647 Org29647 NA alkylindoleAmino- (b) NA alkylindoleAmino- (b)Org27759 NA Amino-alkylindole Org27759(b) NA alkylindoleAmino-alkylindole NA Org27759(b) (b) Amino- (b)Org27759 Amino- NA Org27759Org27759 alkylindole Anti-obesity [12] Amino-alkylindole Anti-obesity [12] (b) alkylindoleAmino- (c) Anti-obesity [12] Org27759 alkylindoleAmino- (c)Org27569 Amino-alkylindoleAnti-obesity [12] Anti-obesity [12] Amino-alkylindole Org27569(c) Anti-obesity [12] O alkylindole O Reducing the signs and symptoms of Huntington’s Org27569(c)(c) N O Reducingdisease [146]; the signs and symptoms of Huntington’s Org27569 O Amino- (c)Org27569 N Anti-obesity [12] O Amino- diseaseaugmenting [146]; the pharmacological effects of the CB1 O alkylindole Reducing the signs and symptoms of Huntington’s Org27569 N alkylindole orthosteric agonists [12];Reducing the signs and symptoms of N O Amino- augmenting the pharmacological effects of the CB1 H disease [146]; O Reducing theHuntington’s signs and symptoms disease [of146 Huntington’s]; N alkylindole orthostericanti-nociceptive, agonists [12]; (c) N O Amino- augmenting the pharmacological effects of the CB1 H O Reducingdisease [146];the signsaugmenting and symptoms the pharmacological of Huntington’s eff ects of (d) N Org27569 alkylindole Amino-alkylindoleanti-nociceptive,orthostericanti-inflammatory agonists [142][12]; N Amino- diseaseaugmenting [146]; thethe pharmacological CB1 orthosteric agonists effects of [12 the]; CB1 (d)GAT211 H Amino-alkylindoleanti-inflammatoryanti-nociceptive, augmentingorthosteric [142] the agonistsanti-nociceptive, pharmacological [12]; effects of the CB1 N GAT211 (d) O H Reducing the signs and symptoms of Huntington’s (d) O anti-inflammatory [142] N alkylindole anti-inflammatoryorthostericanti-nociceptive, agonists [142] [12]; GAT211 N H disease [146]; GAT211 (d) anti-nociceptive,anti-inflammatory [142] Amino- augmentingAnti-nociceptive the pharmacological and anti-inflammatory effects of inthe CB1 (d)GAT211 anti-inflammatory [142] alkylindoleAmino- orthostericAnti-nociceptivecombination agonists with and [12];the anti-inflammatoryAnti-nociceptive orthosteric CB1 agonist and in anti-inflammatory [143]; in GAT211N H combination with the orthosteric CB1 Amino-alkylindole anti-nociceptive,combinationAnti-nociceptiveReducing the with signs and the and anti-inflammatoryorthosteric symptoms CB1 of agonist Huntington’s in [143]; Amino-alkylindole agonist [143]; (d) alkylindoleAmino- anti-inflammatoryReducingcombinationdisease [146] the withsigns [142]the and orthosteric symptoms CB1 of Huntington’s agonist [143]; (e) Anti-nociceptivereducing and anti-inflammatory the signs and symptoms in of GAT211 alkylindoleAmino- diseaseReducingAnti-nociceptive [146]combination the signs and with symptomsand the anti-inflammatory orthosteric of Huntington’s CB1 agonistin [143]; (e)GAT229 (e) Huntington’s disease [146] Amino-alkylindoledisease combination [146]Reducing the with signs the andorthosteric symptoms CB1 of agonist Huntington’s [143]; GAT229 GAT229 (e) alkylindole Reducingdisease [146]the signs and symptoms of Huntington’s Anti-nociceptive and anti-inflammatory in GAT229 (e) disease [146] (e)GAT229 Amino- combination with the orthosteric CB1 agonist [143]; Amino- Anti-nociceptive, Anti-nociceptive, Int.GAT229 J. Mol. Sci. 2020, 21, x FOR PEERalkylindole REVIEW Amino-alkylindoleReducing the signs and symptoms of Huntington’s 17 of 34 Amino-alkylindole Anti-nociceptive,anti-inflammatory [139]anti-inflammatory [139] Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW disease [146] 17 of 34 (e) alkylindoleAmino- anti-inflammatoryAnti-nociceptive, [139] (f) (f) GAT229 ZCZ011 alkylindoleAmino- anti-inflammatoryAnti-nociceptive, [139] (f)ZCZ011 Amino-alkylindole Anti-nociceptive,anti-inflammatory [139] ZCZ011(f) alkylindole anti-inflammatory [139] ZCZ011 (f) Blocking cocaine-induced locomotor stimulation Others ZCZ011 Amino- Anti-nociceptive, (f) Blocking[147] cocaine-induced locomotor stimulation alkylindoleOthers anti-inflammatory [139]Blocking cocaine-induced locomotor ZCZ011 Others[147] stimulation [147] (f) (g) ZCZ011 (g) RTI-371 (g) RTI-371 RTI-371

Others OthersAnti-obesity [139] Anti-obesity [139] Others Anti-obesity [139] (h)(h) PSNCBAM-1 (h) PSNCBAM-1 PSNCBAM-1

Anti-inflammatory [148], Nonclassical Anti-inflammatoryanxiolytic [149] [148], Nonclassical anxiolytic [149] (i) (i) CBD-DMH (HU-219) CBD-DMH (HU-219)

Others Anti-nociceptive [145] Others Anti-nociceptive [145]

(j) (j) Compound C2 Compound C2 NA: not available. NA: not available. 5. Novel Cannabinoids from Animal Sources 5. Novel Cannabinoids from Animal Sources GPCRs mediate a series of signal processes in the human body [150]. One third of clinically used GPCRsdrugs target mediate these a series receptors of signal [150,151]. processes Considering in the human that bodymany [150]. of the One natural third endogenous of clinically usedligands of drugsGPCRs target theseare peptides receptors (comprising [150,151]. Considering 50 or fewer that amino many acids), of the naturaldrug development endogenous basedligands on of these GPCRspeptides are peptides is suggested (comprising as an attracting50 or fewer and amino prom isingacids), way drug to producedevelopment drugs basedfor the on treatment these of peptidesdiseases is suggested in which as the an GPCR attracting signaling and pathwaypromising plays way a topivotal produce role drugs[150]. Inspiredfor the treatment by the successful of diseasesmedicinal in which use the of GPCR the endogenous signaling pathway peptide playsinsulin, a pivotal researchers role [150]. began Inspired to focus by on the research successful and the medicinaldevelopment use of the of endogenous peptide-type peptide drugs insulin,[151]. A researchers milestone beganof the toresearch focus on on research natural andendogenous the developmentpeptides ofis thepeptide-type discovery ofdrugs hemopressin [151]. A (Hp) mile andstone its of derivates the research RVD-Hp onα natural and VD-Hp endogenousα which have peptidesbeen is theproven discovery to be of ligands hemopressin of cannabinoid (Hp) and its receptors derivates RVD-Hpinvolvingα andnumerous VD-Hp αphysiological which have and been pathologicalproven to beprocesses ligands [152,153]. of cannabinoid These peptides receptors can beinvolving regarded numerousas novel cannabinoids physiological because and they pathologicalexhibit processesactivity at [152,153]. cannabinoid These receptors peptides and can have be regarded distinct asstructures novel cannabinoids from phytocannabinoids because they and exhibitsynthetic activity cannabinoids.at cannabinoid Their receptors actions and at havecannabinoid distinct receptorsstructures and from the phytocannabinoids associated pharmacological and syntheticeffects cannabinoids. have attracted Their considerable actions at attentioncannabinoid since receptors their discovery, and the leadingassociated to manypharmacological studies showing effectssatisfying have attracted results considerable in vitro and inattention vivo. A fewsince years their ago, discovery, a novel leading peptide to Pep19 many derived studies from showing peptidyl- satisfyingprolyl results cis-trans in vitro isomerase and in vivoA in. Ahumans few years was ago, reported a novel to peptideact as a CB1Pep19 ligand derived and from showed peptidyl- surprising prolylin cis-trans vivo results isomerase that are A relatedin humans to CB1 was [35]. reported In this to part,act as we a CB1 briefly ligand introduce and showed the reported surprising peptides in vivotargeting results thatCB1/CB2. are related Additionally, to CB1 [35].from In a perspectivethis part, we point briefly of view,introduce the potential the reported of venom peptides peptides targetingas a CB1/CB2.source for Additionally, novel cannabinoids from a perspective is demonstrat pointed, of since view, they the potentialcurrently ofaccount venom forpeptides the largest as a sourceportion for of discoverednovel cannabinoids nature-derived is demonstrat peptidesed, targeting since they GPCRs currently [37]. account for the largest portion of discovered nature-derived peptides targeting GPCRs [37].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 17 of 34 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 17 of 34

Blocking cocaine-induced locomotor stimulation Others [147]Blocking cocaine-induced locomotor stimulation Others [147]

(g) RTI-371(g) RTI-371

Int. J. Mol. Sci. 2020, 21, 5064 16 of 32 Others Anti-obesity [139] Others Anti-obesity [139] (h) Table 7. Cont. PSNCBAM-1(h) PSNCBAM-1Synthetic Cannabinoids Chemical Type Bioactivity

Anti-inflammatory [148], Nonclassical anxiolytic [149] Anti-inflammatoryAnti-inflammatory [148], [148], NonclassicalNonclassical anxiolytic [149] anxiolytic [149] (i) CBD-DMH(i) (i) (HU-219) CBD-DMHCBD-DMH (HU-219) (HU-219)

Others OthersAnti-nociceptive [145] Anti-nociceptive [145] Others Anti-nociceptive [145]

(j) (j) Compound C2 Compound(j) C2 NA: not available. Compound C2 NA: not available. NA: not available. 5. Novel Cannabinoids from Animal Sources 5. NovelCompared Cannabinoids to CB1, from only Animal two synthetic Sources allosteric modulators have been developed for CB2 so far. GPCRs mediate a series of signal processes in the human body [150]. One third of clinically used One of them is cannabidiol-dimethylheptyl (CBD-DMH or HU-219) (Table7i) which has been shown drugs target these receptors [150,151]. Considering that many of the natural endogenous ligands of toGPCRs act as an mediate allosteric a series modulator of signal forprocesses CB2, reducingin the human CP-55,940 body [150]. binding One third and beingof clinically a PAM used of cAMP GPCRsdrugs are target peptides these (comprisingreceptors [150,151]. 50 or fewer Considering amino thatacids), many drug of thedevelopment natural endogenous based on ligandsthese of modulation but a NAM of β-arrestin1 recruitment [54]. In addition, CBD-DMH can be considered as a peptidesGPCRs is suggestedare peptides as an(comprising attracting 50and or prom fewerising amino way acids),to produce drug drugsdevelopment for the treatmentbased on ofthese mixed agonist/PAM of CB1 [54]. The other CB2 allosteric modulator is the compound C2 (Table7j), diseasespeptides in which is suggested the GPCR as signaling an attracting pathway and playsprom isinga pivotal way role to produce[150]. Inspired drugs byfor the the successful treatment of which can act as a CB2 PAM in binding assays and displays antinociceptive activity in vivo in an medicinaldiseases use in ofwhich the endogenousthe GPCR signaling peptide pathwayinsulin, researchersplays a pivotal began role to [150]. focus Inspired on research by the and successful the experimental mouse model of neuropathic pain [145]. Apparently, all CB allosteric modulators are still developmentmedicinal ofuse peptide-type of the endogenous drugs [151].peptide A insulin,milestone researchers of the research began toon focus natural on researchendogenous and the peptidesdevelopmentin theis the stage discovery of preclinicalpeptide-type of hemopressin studies drugs instead (Hp)[151]. and ofA its themile derivates clinicalstone of trialRVD-Hp the according researchα and VD-Hp toon ClinicalTrials.gov naturalα which endogenous have [70]. beenpeptides proven is to the be discovery ligands ofof hemopressin cannabinoid (Hp) receptors and its derivatesinvolving RVD-Hp numerousα and physiological VD-Hpα which and have 5. Novel Cannabinoids from Animal Sources pathologicalbeen proven processes to be [152,153]. ligands Th ofese cannabinoid peptides can receptorsbe regarded involving as novel cannabinoidsnumerous physiological because they and exhibitpathological activityGPCRs at processes cannabinoid mediate [152,153]. a series receptors ofTh signalese and peptides processeshave distinct can inbe the regardedstructures human as frbody novelom phytocannabinoids [ 150cannabinoids]. One third because of and clinically they used syntheticexhibitdrugs cannabinoids. activity target theseat cannabinoid Their receptors actions receptors [150 at ,cannabinoid151]. and Considering have receptors distinct that structuresand many the ofassociated fr theom natural phytocannabinoids pharmacological endogenous and ligands of effectssyntheticGPCRs have attracted cannabinoids. are peptides considerable (comprising Their actionsattention 50 at or sincecannabinoid fewer their amino discovery, receptors acids), leading drug and developmentthe to associated many studies basedpharmacological showing on these peptides satisfyingeffectsis suggested results have attracted in asvitro an and considerable attracting in vivo. A and attentionfew promising years since ago, way atheir novel to discovery, producepeptide Pep19leading drugs derived forto many the from treatment studies peptidyl- showing of diseases in prolylsatisfyingwhich cis-trans the results isomerase GPCR in signalingvitro A inand humans in pathway vivo .was A playsfew reported years a pivotal ago, to act a role novelas a [150 CB1 peptide]. ligand Inspired Pep19 and by derivedshowed the successful fromsurprising peptidyl- medicinal use in vivoprolylof results the cis-trans endogenous that areisomerase related peptide Ato in CB1 insulin,humans [35]. In researcherswas this reported part, we began to brieflyact toas focus aintroduce CB1 on ligand research the and reported showed and the peptides surprising development of targetinginpeptide-type vivo CB1/CB2. results that drugsAdditionally, are [ 151related]. A from to milestone CB1 a perspective [35]. of In the this research point part, of we onview, briefly natural the introducepotential endogenous of the venom peptidesreported peptides ispeptides the discovery as a source for novel cannabinoids is demonstrated, since they currently account for the largest targetingof hemopressin CB1/CB2. (Hp) Additionally, and its derivates from a RVD-Hpperspectiveα and point VD-Hp of view,α which the potential have been of provenvenom peptides to be ligands of portionas a of source discovered for novel nature-derived cannabinoids peptides is demonstrat targetinged, GPCRs since [37]. they currently account for the largest cannabinoid receptors involving numerous physiological and pathological processes [152,153]. These portion of discovered nature-derived peptides targeting GPCRs [37]. peptides can be regarded as novel cannabinoids because they exhibit activity at cannabinoid receptors

and have distinct structures from phytocannabinoids and synthetic cannabinoids. Their actions at

cannabinoid receptors and the associated pharmacological effects have attracted considerable attention since their discovery, leading to many studies showing satisfying results in vitro and in vivo. A few years ago, a novel peptide Pep19 derived from peptidyl-prolyl cis-trans isomerase A in humans was reported to act as a CB1 ligand and showed surprising in vivo results that are related to CB1 [35]. In this part, we briefly introduce the reported peptides targeting CB1/CB2. Additionally, from a perspective point of view, the potential of venom peptides as a source for novel cannabinoids is demonstrated, since they currently account for the largest portion of discovered nature-derived peptides targeting GPCRs [37]. Int. J. Mol. Sci. 2020, 21, 5064 17 of 32

5.1. Peptides Targeting CB1/CB2

5.1.1. Hemopressin and its Derivates VD-Hpα and RVD-Hpα Hemopressin (Hp or rat Hp, sequence: PVNFKFLSH) (Table8a) and related peptides VD-Hpα (pepcan-11, sequence: VDPVNFKLLSH) (Table8b) and RVD-Hp α (pepcan-12, sequence: RVDPVNFKLLSH) (Table8c) are endogenous peptides that have been found to bind to cannabinoid receptors. Among them, Hp was first discovered and identified in rat brain and spleen [153]. Thereafter, VD-Hpα and RVD-Hpα were identified in mouse brain as well as mouse and human plasma [152]. It was found that they are fragments of α-hemoglobin in these animals. Because CB1 is primarily expressed in the CNS, these peptides were investigated as endogenous ligands modulating CB1 activity.

Table 8. Sequences, bioactivities and sources of novel peptide-type cannabinoids.

Peptides Sequence Bioactivity Source Neuromodulatory, anorexigenic, (a) PVNFKFLSH alleviating liver fibrosis, α-Hemoglobin in rat Hemopressin (Hp) antinociceptive [36] Neuromodulatory, antinociceptive, (b) inhibiting gastrointestinal α-Hemoglobin in human VDPVNFKLLSH VD-Hpα (pepcan-11) mobility, inducing tolerance to and mouse thermal antinociception, orexigenic [36] (c) Neuromodulatory, anxiolytic, α-Hemoglobin in human RVDPVNFKLLSH RVD-Hpα (pepcan-12) anti-depressant [36] and mouse (d) Reducing body weight, improving Peptidyl-prolyl cis-trans DIIADDEPLT Pep19 metabolic parameters [35] isomerase A in human

Hp was originally identified as an inverse agonist/antagonist of CB1 [154]. A few years ago, a study revealed that Hp slightly activated G proteins and functioned as a very weak agonist, proposing a hypothesis that Hp indirectly interacts with CB1 [155]. In addition, it is confirmed that Hp does not function as a NAM of CB1 in a study using electrophysiology [156]. In contrast to Hp, VD-Hpα and RVD-Hpα were initially suggested to be agonistic ligands of CB1 [152]. Later on, RVD-Hpα was reported as a potential NAM of CB1 in the brain [157]. Soon after, it was identified as a PAM of CB2 in peripheral tissues [158]. Moreover, RVD-Hpα was demonstrated to exhibit the effect of CB2-mediated G-protein recruitment and cAMP inhibition without affecting β-arrestin-2 recruitment and receptor internalization [158]. Some pharmacological effects of Hp, VD-Hpα and RVD-Hpα related to cannabinoid receptors have also been characterized. Hp and the extended peptides VD-Hpα and RVD-Hpα can cause CB1-mediated neuromodulation. This effect was shown in a study which demonstrated that Hp blocked CB1 agonist-mediated neurite outgrowth in Neuro-2A cells (a mouse neuroblastoma cell line with neuronal and amoeboid stem cell morphology) expressing CB1 in a manner similar to that of SR141716 [154], while VD-Hpα and RVD-Hpα promoted neurite outgrowth in a manner similar to that of the CB1 agonist HU-210 in Neuro-2A cells. This effect could be inhibited by pretreatment with the antagonist SR141716 [152]. In addition, Hp acts as an endogenous functional antagonist of CB1 and modulates the activity of appetite pathways in the brain. It was shown that Hp dose-dependently decreased night-time food intake in normal male rats and mice, as well as in obese male mice, without causing any obvious side effects [159]. The anorectic effect was absent in CB1 null mutant male mice, and Hp could block CB1 agonist CP-55,940-induced hyperphagia in male rats, providing strong evidence for antagonism of CB1 in vivo [159]. Moreover, Hp was shown to reduce hepatic collagen deposition, downregulate CB1 and CB2 expression, and increase matrix metalloproteinase-1 expression by the per os administration of Hp for 2 weeks [160]. Consequently, Hp alleviated liver fibrosis in the bile duct-ligated rats in comparison with the CB2 agonist β-caryophyllene. This could be partly Int. J. Mol. Sci. 2020, 21, 5064 18 of 32 attributed to its antagonism/inverse agonism at CB1 [160]. Regarding VD-Hpα, it was proven to possess antinociceptive effects mediated by CB1. It is a dose-dependent antinociceptive effect achieved at the levels of the spinal cord [161]. This antinociceptive effect can be blocked by the CB1 antagonist AM-251 but not the CB2 antagonist AM-630 or by naloxone, suggesting that the mechanism is related to CB1 rather than CB2 or opioid receptors [161]. Furthermore, VD-Hpα can inhibit gastrointestinal transit via the activation of CB1 located in the brain, because VD-Hpα via intracerebroventricular administration inhibits upper gastrointestinal transit and colonic expulsion [162]. These effects are prevented by AM-251 instead of AM-630 [162]. Notably, the side effects of Hp, VD-Hpα and RVD-Hpα cannot be ignored. Intracerebroventricular administration of VD-Hpα or RVD-Hpα impairs the memory of normal mice [163]. In the case of Hp, the side effects so far found are of psychiatric nature. Hp administration induced anxiogenic and depressive behavior, decreased monoamine steady state levels in prefrontal cortex, and increased the gene expression of the enzymes involved in the catabolism in rats [164]. The mechanism of these side effects has not been completely clarified.

5.1.2. Pep19 In 2017, Reckziegel et al. reported a novel peptide Pep19 (Sequence: DIIADDEPLT) (Table8d) exhibiting inverse agonistic activity at CB1 [35]. Pep19 is a peptide resulting from the rational modification of the original peptide (sequence: DITADDEPLT) which is derived from peptidyl-prolyl cis-trans isomerase A in humans. The inverse agonistic activity of Pep19 is greatly enhanced compared with the original peptide which shows very weak inverse agonistic activity at CB1 [35]. Oral administration of Pep19 to rats improved several metabolic parameters, including a reduction in the serum glucose, triacylglycerol and blood pressure, without changing heart rate, as well as reducing the whole adiposity index and the mass of gonadal and mesenteric adipose tissues [35]. Moreover, oral administration of Pep19 significantly increased the expression of uncoupling protein 1 in specific cells of the inguinal adipose tissue [35]. This effect was proven again in both white adipose tissue and 3T3-L1 differentiated adipocytes, and was blocked by AM-251, a CB1 antagonist [35]. Surprisingly, Pep19 did not exhibit cellular toxicity or typical undesired CNS effects related to cannabinoid receptors in rodents, even after 10 days of chronic treatment with Pep19 [35]. Although Pep19 was shown to work similarly to Hp in this study, reducing body weight and improving metabolic parameters, it has the advantage of lacking the undesired side effects associated with CB1 inverse agonists such as rimonabant and Hp [35].

5.2. Potential of Animal Venoms as a Source for Novel Cannabinoids Amid the vast animal sources on planet Earth, animal venoms are regarded as a tremendous treasure-house of venom toxins (i.e., venom peptides) that specifically, potently, stably and speedily manipulate physiological targets including ion channels and receptors. Animal venoms, like parts of the plant Cannabis sativa, have been used to treat some ailments throughout human history. Since the 7th century BCE, snake venom has been used in Ayurvedic medicine to prolong life and treat arthritis and gastrointestinal ailments, while tarantulas are used in the traditional medicine of indigenous populations of Mexico and Central and South America [38]. In the 1970s, an active peptide was found in the venom of the Brazilian snake Bothrops jaracaca. On this basis, the blockbuster drug captopril was developed. It is an inhibitor of the angiotensin converting enzyme and is now used clinically as an antihypertensive drug. Since then, drug discovery based on animal toxins has increased significantly. Moreover, the advancement of technologies has enabled the study of venoms from animals that are small, rare, or hard to maintain in the lab, which greatly facilitates the high-throughput screening of animal venoms and the characterization of venom peptides’ structure and function [38]. The research on venom-derived drugs has so far yielded at least 10 registered and deposited drugs on the market [165], examples of which include captopril (Capoten®), tirofiban (Aggrastat®) and eptifibatide (Integrilin®). Although humans have been using venoms for thousands of years, only for the past Int. J. Mol. Sci. 2020, 21, 5064 19 of 32

five or six decades scientists have been studying the venoms at the molecular level using modern biochemistry, physiology and biophysics and other technologies [38]. So, enormous strides in the research of animal toxins have been made over the past half a century [38]. Venom peptides, e.g., from cone-snails, snakes, spiders and scorpions, are currently of particular interest as a source of lead compounds for the development of GPCR ligands among naturally occurring compounds, since they cover a chemical space, which differs from that of synthetic small molecules [37]. They are recognized as reliable alternatives for small molecules, owing to their higher selectivity [166]. In addition, peptides may be metabolized and cleared without accumulation in body tissues, thereby minimizing the occurrence of side effects [167]. Many GPCR-targeting venom-derived peptides were discovered to be beneficial in diverse pathological animal models [37]. For example, mambaqauretin-1 was recently isolated from the venom of a green mamba (Dendroaspis angusticeps), exhibiting high affinity and inhibitory action on the vasopressin type 2 receptor [168]. Further in vivo experiments highlighted the potential usefulness of mambaquaretin-1 for the treatment of polycystic kidney disease [168]. Exenatide derived from exendin-4 which was isolated from the venom of the Gila monster (Heloderma suspectum) is an example of a GPCR-targeting nature-derived peptide drug (Byetta®) as described in the “Introduction”. It was introduced in 2005 to treat diabetes mellitus type 2 and acts as an agonist of the glucagon-like peptide-1 receptor [38]. Another example is the National Medical Products Administration of China (NMPA)-approved drug cobratide, derived from venoms of Chinese cobra (Naja atra). This is an analgesic peptide targeting the acetylcholine receptor. Interestingly, δ-CNTX-Pn1a, a peptide from Phoneutria nigriventer spider venom (also called PnTx4(6-1) or δ-Ctenitoxin-Pn1a), has been reported to induce antinociception in rat models of pain, involving both opioid and cannabinoid systems [169]. It was also reported that peripheral interactions between opioid and cannabinoid systems contribute to antinociception of a peptide, crotalphine, of which the sequence is based on the structure of the natural analgesic factor isolated from the venom of the South American rattle-snake Crotalus durissus terrificus [170]. Moreover, PnPP-19 derived from venom of the spider Phoneutria nigriventer was shown to induce central and peripheral antinociception through both opioid and cannabinoid systems in in vivo pain models [171,172]. This peptide displayed long-lasting analgesic activity after oral administration in rats [173]. However, the detailed action mechanisms of these peptides on opioid or cannabinoid receptors remain unknown. In the search for novel compounds that produce analgesia via GPCR modulation, animal venoms offer an enormous and mostly untapped source of potent and selective peptide molecules [174]. Another example related to this is that the GABAB receptor (GABABR) appears to play a critical role in analgesia of α-conotoxin Vc1.1 in rodent models of pain, while the precise nature of α-conotoxin Vc1.1 binding to GABABR is currently unknown [174]. Additionally, it is evident that cannabinoid receptors are one of the primary GPCR targets of natural products based [37]. Thus, targeting the GPCRs, i.e., cannabinoid receptors, shows a great prospective potential for medical uses of animal venoms which indeed represent a promising and uncharted pharmacopeia.

6. Expression Systems of Cannabinoid Receptors Used for Screening Natural Products In Vitro The classical expression system of cannabinoid receptors used for rapidly analyzing the binding and function of phytocannabinoids, synthetic cannabinoids and novel peptide-type cannabinoids in vitro uses mammalian cells transfected with human CB1 or CB2, e.g., Chinese hamster ovary (CHO) cells and embryonic mouse fibroblast (3T3-L1 cells) [14,35,157,158]. These mammalian transient expression systems enable the flexible and rapid production of membrane-bound cannabinoid receptor proteins. They are ideal for the expression of these human proteins because these systems generate recombinant proteins with more native folding and post-translational modifications—such as glycosylation—than expression systems based on hosts such as E. coli, yeast, or insect cells. In addition, certain cultured cell lines that express cannabinoid receptors naturally, and cannabinoid receptor-containing membrane preparations obtained from tissues (such as brain and spleen), are also commonly used [61]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 21 of 34 products.Int. J. Mol. Sci.The2020 direct, 21, 5064 electrophysiological measurement of transmembrane K+ currents is an effective20 of 32 method to study the effects of compounds on membrane-bound cannabinoid receptors. As shown in Figure 3, a typical trace of K+ current evoked by the cannabinoid receptor agonist WIN-55,212-2 The Xenopus laevis oocyte transfected with human CB1 or CB2 and the accessory G protein-activated (WIN) (0.5 μM) is shown. An advantage of the Xenopus oocytes is that they are able to properly inwardly rectifying potassium (GIRK) channels 1/2 or GIRK1/4 as well as regulator of G-protein signaling assemble and incorporate functionally active proteins, e.g., cannabinoid receptors, into their plasma 4 (RGS 4) is also a robust and reliable system for screening unlabeled natural products. The direct membranes, which is the reason that the Xenopus oocytes are widely used as a heterologous electrophysiological measurement of transmembrane K+ currents is an effective method to study the expression system [175]. Using this system, it is also possible to functionally investigate the effects of compounds on membrane-bound cannabinoid receptors. As shown in Figure3, a typical trace membrane protein CB1 or CB2 in combination with its accessory proteins, in order to screen potential of K+ current evoked by the cannabinoid receptor agonist WIN-55,212-2 (WIN) (0.5 µM) is shown. An drugs [175]. Another advantage of using the heterologous Xenopus oocyte expression system is the advantage of the Xenopus oocytes is that they are able to properly assemble and incorporate functionally freedom to co-express different combinations of membrane-bound proteins: one can, for instance, co- active proteins, e.g., cannabinoid receptors, into their plasma membranes, which is the reason that the inject mRNA encoding CB1 and TRPV1 (the vanilloid receptor, also the target of some cannabinoid Xenopus oocytes are widely used as a heterologous expression system [175]. Using this system, it is also ligands), or CB2 and mu opioid receptors, in order to check the possible promiscuity of ligands, or possible to functionally investigate the membrane protein CB1 or CB2 in combination with its accessory CB1 with different types of voltage-gated ion channels (Na, K, Ca, Cl). Furthermore, it also allows proteins, in order to screen potential drugs [175]. Another advantage of using the heterologous Xenopus researchers to carry out structure–function research, based on the expression of mutant CB1/CB2. As oocyte expression system is the freedom to co-express different combinations of membrane-bound such, this functional bioassay can unravel hitherto unknown coupling(s) of signal-transmission proteins: one can, for instance, co-inject mRNA encoding CB1 and TRPV1 (the vanilloid receptor, pathways, e.g., the cannabinoid and opioid pathways, allowing us to better understand the analgesic also the target of some cannabinoid ligands), or CB2 and mu opioid receptors, in order to check the properties of some cannabinoids. It is notable that this system also nicely mimics the physiological realitypossible of promiscuityCB1 or CB2. ofThis ligands, is because or CB1 these with receptors different have types been of shown voltage-gated to couple ion to channels GIRK channels (Na, K, [176].Ca, Cl). The Furthermore, activation of itthe also cannabinoid allows researchers receptors toaffects carry GIRK out structure–function channels [177], so that research, they can based exert on thethe inhibitory expression regulation of mutant of CB1 neuronal/CB2. Asexcitability such, this in functional most brain bioassay regions canand unravelheart rate hitherto [176]. Neuronal unknown GIRKcoupling(s) channels of signal-transmissionare predominantly heteromultimers pathways, e.g., composed the cannabinoid of GIRK1 and and opioid GIRK2 pathways, subunits allowing in most brainus to betterregions, understand whereas atrial the analgesic GIRK channels properties are ofheteromultimers some cannabinoids. composed It is notable of GIRK1 that and this GIRK4 system subunitsalso nicely [176]. mimics the physiological reality of CB1 or CB2. This is because these receptors have been shownIn addition, to couple when to GIRK using channels these [expression176]. The activationsystems, it of is the also cannabinoid important receptorsto pay attention affectsGIRK to a phenomenonchannels [177 ],called so that shear they stress, can exert acting the on inhibitory the cell regulationand which of can neuronal rupture excitability the fragile in membrane, most brain therebyregions releasing and heart the rate intracellular [176]. Neuronal material. GIRK Since channels there areis no predominantly cell wall in animal heteromultimers and mammalian composed cells, theyof GIRK1 rely on and a GIRK2plasmasubunits membrane in most to keep brain the regions, intracellular whereas contents atrial GIRK stay intact. channels are heteromultimers composed of GIRK1 and GIRK4 subunits [176].

ND96 HK ND96

WIN 0.5 μM

2μA

100 sec

Figure 3. Cannabinoid agonist WIN-55,212-2 (WIN) activated inwardly rectifying potassium currents in Figureoocytes 3. co-expressing Cannabinoid GIRK1agonist/2 WIN-55,212-2+ RGS4 with CB1. (WIN) Currents activate wered inwardly induced rectifying by exchanging potassium ND96 currents with HK inwhile oocytes the oocytesco-expressing were voltage-clamped GIRK1/2 + RGS4 atwith90 CB1. mV. Currents Current were enhancement induced observedby exchanging on application ND96 with of − HK0.5 µwhileM WIN the in theoocytes presence were of HK.voltage-clamped In the final, oocytes at −90 were mV. perfused Current with enhancement ND96. The experiment observed wason applicationrepeated in of at 0.5 least μM 4 oocytes.WIN in the In thepresence case of of CB2, HK. aIn similar the final, approach oocytes is were followed. perfused ND96: with physiological ND96. The

experimentsalt buffer solutionwas repeated (96 mM in NaCl, at least 2 mM 4 oocytes. MgCl2 ,In 2 mMthe case KCl, of 5 mM CB2, HEPES, a similar and approach 1.8 mM CaClis followed.2, with a ND96:final pH physiological of 7.5), HK: salt high buffer potassium solution solution (96 mM for NaCl, the measurement 2 mM MgCl2 of, 2 KmM+ currents KCl, 5 mM through HEPES, GIRK1 and/2 + 1.8channels mM CaCl (962 mM, with KCl, a final 2 mM pH NaCl,of 7.5), 1 HK: mM high MgCl pota2, 1.8ssium mM solution CaCl2, 5 for mM the HEPES measurement with a final of K pH currents of 7.5). through GIRK1/2 channels (96 mM KCl, 2 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM HEPES with aIn final addition, pH of 7.5). when using these expression systems, it is also important to pay attention to a phenomenon called shear stress, acting on the cell and which can rupture the fragile membrane, thereby

Int. J. Mol. Sci. 2020, 21, 5064 21 of 32 releasing the intracellular material. Since there is no cell wall in animal and mammalian cells, they rely on a plasma membrane to keep the intracellular contents stay intact.

7. Conclusions and Perspectives Starting from the research on the medicinal cannabis plant, hundreds of different phyto- and synthetic cannabinoids targeting CB1 and/or CB2 have emerged with diverse pharmacological effects, for either medicinal or recreational use. CB1 and CB2, as the earliest-identified targets of the phytocannabinoids in the human body, have received considerable attention in the field of therapeutics. In general, agonists of the cannabinoid receptors are theoretically important for ameliorating neurological or brain diseases, treating pain and inflammation as well as many cancers. On the other hand, antagonists/inverse agonists have been shown to primarily play a role in weight loss, diabetes and treating feeding disorders. However, to date, both natural and synthetic ligands of the cannabinoid receptors show only a few clinical applications. This indeed requires more mechanistic investigations in a systematic way in order to delineate the mode of action of the phytocannabinoids and synthetic cannabinoids. In this case, the ligand-bound cryo-EM structures of the active cannabinoid receptors in complex with G-protein are necessary and could assist in identifying more drug leads, especially in light of the withdrawals often occurring for many drugs targeting cannabinoid receptors. The activation mechanisms of CB1 and CB2 have been recently revealed thanks to the powerfulness of cryo-EM, which may enable rational, structure-guided cannabinoids design. According to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), over 620 new psychoactive substances (NPS) are currently being monitored through the EU Early Warning System. One hundred and sixty-nine of these are synthetic cannabinoid receptor agonists, with 14 recognizable chemical families. More and more ligands of CB1 or CB2 with new cannabinoid scaffolds showing potential therapeutic activity in vivo have emerged over the past decade [78,178–181]. The structures of many synthetic cannabinoids can be categorized into four components: tail, core, linker and linked group. Assigning each component, a code name allows the chemical structure of the cannabinoid to be identified without the long chemical name. From all the possible combinations, it is clear that structure activity − relationships (SAR) of these compounds is almost endless, hard to oversee and that many more NPS compounds will continue to emerge [182]. The novel cannabinoids—peptides—from animal sources draws a brighter future for therapeutically targeting of CB1/CB2. The four peptide-type ligands of cannabinoid receptors found so far have shown potential therapeutic effects depending on CB1 in vivo, and one of them has shown no CNS side effects. For future perspectives, it is promising to discover more novel cannabinoids with less or without CNS side effects from animals living on planet Earth. In particular, animal venoms contain a true pharmacopeia of venom peptides targeting ion channels or receptors with incredible selectivity, potency and speed. This makes them promising and ideal drug leads. Venomous animal species account for ~15% of all described animal biodiversity on Earth, yet we know very little about their venoms [38]. For rapidly determining the effects of the natural products for CB1 and/or CB2 in vitro, the robust cannabinoid receptor expression systems mimicking their physiological reality are commonly relied on. Hence, we are convinced that there will be more exciting discoveries in the future. The field of the development of venom drugs targeting CB1/CB2 is starting a new chapter.

Author Contributions: Conceptualization, D.A. and J.T.; writing-original draft preparation, D.A.; writing—review and editing, D.A., J.T., S.P. and L.A.H.; visualization, D.A.; supervision, J.T.; project administration, D.A., J.T. and S.P.; funding acquisition, D.A., J.T. and S.P. All authors have read and agreed to the published version of the manuscript. Funding: This project was funded by Chinese Scholarship Council (grant number 201907060021, awarded to D.A.), the F.W.O. Vlaanderen (grant numbers G0E7120N, GOC2319N and GOA4919N, awarded to J.T.) and KU Leuven (grant number PDM/19/164, awarded to S.P.). Acknowledgments: The authors would like to acknowledge figures of CB1/CB2-Gi structures from Tian Hua et al. and Changrui Xing et al. Int. J. Mol. Sci. 2020, 21, 5064 22 of 32

Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

∆9-THC ∆9-tetrahydrocannabinol ∆9-THCP ∆9-tetrahydrocannabiphorol ACEA Arachidonyl-20-chloroethylamide ACPA Arachidonylcyclopropylamide AEA Anandamide or arachidonoylethanolamide AM-251 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-piperidin-1-ylpyrazole-3-carboxamide AM-281 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-morpholin-4-ylpyrazole-3-carboxamide AM-630 [6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone or 6-iodopravadoline CBD Cannabidiol CBDP Cannabidiphorol Compound C2 N-[5-bromo-1,2-dihydro-1-(40-fluorobenzyl)-4-methyl-2-ox-opyridin-3yl] cycloheptanecarboxamide CP-55,940 2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol GAT211 3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole GAT229 S-(-)-3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole GW-405833 (2,3-dichlorophenyl)-[5-methoxy-2-methyl-3-(2-morpholin-4-ylethyl)indol-1-yl]methanone HK High potassium solution (96mM KCl, 2 mM NaCl, 1 mMMgCl2, 1.8mM CaCl2, 5mM HEPES with a final pH of 7.5) HU-210 (6aR,10aR)-9-(hydroxymethyl)-6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a tetrahydrobenzo[c]chromen-1-ol or 11-hydroxy-∆8-THC-dimethylheptyl HU-308 [(1S,4S,5S)-4-[2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl]-6,6-dimethyl-2-bicyclo [3.1.1]hept-2-enyl]methanol JWH-015 (2-methyl-1-propylindol-3-yl)-naphthalen-1-ylmethanone JWH-133 (6aR,10aR)-6,6,9-trimethyl-3-(2-methylpentan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromene L-759633 (6aR,10aR)-1-methoxy-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c] chromene L-759656 (6aR,10aR)-1-methoxy-6,6-dimethyl-9-methylidene-3-(2-methyloctan-2-yl)-7,8,10,10a- tetrahydro-6aH-benzo[c]chromene ND96 Physiological salt buffer solution (96 mM NaCl, 2 mM MgCl2, 2 mM KCl, 5 mM HEPES, and 1.8 mM CaCl2, with a final pH of 7.5) Org27569 5-chloro-3-ethyl-N-[2-(4-piperidin-1-ylphenyl)ethyl]-1H-indole-2-carboxamide Org27759 N-[2-[4-(dimethylamino)phenyl]ethyl]-3-ethyl-5-fluoro-1H-indole-2-carboxamide Org29647 N-[(3R)-1-benzylpyrrolidin-3-yl]-5-chloro-3-ethyl-1H-indole-2-carboxamide;(E)-but-2-enedioic acid PM-226 9-methoxy-4,4-dimethyl-7-(2-methyloctan-2-yl)chromeno[3,4-d][1,2]oxazole PSNCBAM-1 1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea Rimonabant or 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-piperidin-1-ylpyrazole-3-carboxamide SR141716 RTI-371 3-(4-chlorophenyl)-5-[(1R,2S,3S,5S)-8-methyl-3-(4-methylphenyl)-8-azabicyclo[3.2.1]octan-2 -yl]-1,2-oxazole SR144528 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethyl-2- bicyclo[2.2.1]heptanyl]pyrazole-3-carboxamide Surinabant or 5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-piperidin-1-ylpyrazole-3-carboxamide SR147778 Taranabant or N-[(2S,3S)-4-(4-chlorophenyl)-3-(3-cyanophenyl)butan-2-yl]-2-methyl-2-[5- MK-0364 (trifluoromethyl)pyridin-2-yl]oxypropanamide THC Tetrahydrocannabinol WIN-55,212-2 [(11R)-2-methyl-11-(morpholin-4-ylmethyl)-9-oxa-1-azatricyclo[6.3.1.04,12]dodeca- 2,4(12),5,7-tetraen-3-yl]-naphthalen-1-ylmethanone ZCZ011 6-methyl-3-(2-nitro-1-thiophen-2-ylethyl)-2-phenyl-1H-indole Int. J. Mol. Sci. 2020, 21, 5064 23 of 32

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