NMR characterization of recombinant transmembrane protein CB2 fragment CB2(180-233)

The expression of membrane proteins has been the bottleneck for their structural studies. Recently, we developed a method to obtain milligram quantities of isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor fragment in E. coli. In order to verify this method and confirm the recombinant isotope-labeled CB2 fragment, 3D hetero-nuclear NMR techniques were used to analyze the structure of the fragment CB2(180-233) in DMSO-d6 solvent. The sequential assignments of TM5 and intra-cellular loop 3 were accomplished, which confirmed the experimental protocols of isotope-labeled recombinant protein expression, fusion protein cleavage, and membrane protein purification. The obtained structure also showed alpha-helix in the TM5 region, but it was interrupted by a disordered region (Gly204_ILe206). These results further revealed that our established approach is a promising method to express recombinant membrane proteins for their structural studies.     

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure–activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 μM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.     

Structure-activity relationships for 1-alkyl-3-(1-naphthoyl)indoles at the cannabinoid CB(1) and CB(2) receptors: steric and electronic effects of naphthoyl substituents. New highly selective CB(2) receptor agonists

In an effort to improve indole-based CB(2) cannabinoid receptor ligands and also to develop SAR for both the CB(1) and CB(2) receptors, 47 indole derivatives were prepared and their CB(1) and CB(2) receptor affinities were determined. The indole derivatives include 1-propyl- and 1-pentyl-3-(1-naphthoyl)indoles both with and without a 2-methyl substituent. Naphthoyl substituents include 4- and 7-alkyl groups as well as 2-, 4-, 6-, 7-methoxy and 4-ethoxy groups. The effects of these substituents on receptor affinities are discussed and structure-activity relationships are presented. In the course of this work three new highly selective CB(2) receptor agonists were identified, 1-propyl-3-(4-methyl-1-naphthoylindole (JWH-120), 1-propyl-2-methyl-3-(6-methoxy-1-naphthoylindole (JWH-151), and 1-pentyl-3-(2-methoxy-1-naphthoylindole (JWH-267). GTPgammaS assays indicated that JWH-151 is a full agonist at CB(2), while JWH-120 and JWH-267 are partial agonists. Molecular modeling and receptor docking studies were carried out on a set of 3-(4-propyl-1-naphthoyl)indoles, a set of 3-(6-methoxy-1-naphthoyl)indoles and the pair of N-pentyl-3-(2-methoxy-1-naphthoyl)indoles. Docking studies indicated that the CB(1) receptor affinities of these compounds were consistent with their aromatic stacking interactions in the aromatic microdomain of the CB(1) receptor.     

New Natural Noncannabinoid Ligands for Cannabinoid Type-2 (CB2) Receptors

Since the discovery that Δ ⁹-tetrahydrocannabinol and related cannabinoids from Cannabis sativa L. act on specific physiological receptors in the human body and the subsequent elucidation of the mammalian endogenous cannabinoid system, no other natural product class has been reported to mimic the effects of cannabinoids. We recently found that N-alkyl amides from purple coneflower (Echinacea spp.) constitute a new class of cannabinomimetics, which specifically engage and activate the cannabinoid type-2 (CB₂) receptors. Cannabinoid type-1 (CB₁) and CB₂ receptors belong to the family of G protein-coupled receptors and are the primary targets of the endogenous cannabinoids N-arachidonoyl ethanolamine and 2-arachidonoyl glyerol. CB₂ receptors are believed to play an important role in distinct pathophysiological processes, including metabolic dysregulation, inflammation, pain, and bone loss. CB₂ receptors have, therefore, become of interest as new targets in drug discovery. This review focuses on N-alkyl amide secondary metabolites from plants and underscores that this group of compounds may provide novel lead structures for the development of CB₂-directed drugs.     

Expression, purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233)

To develop an approach to obtain milligram quantities of purified isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor for NMR structural analysis, we chose a truncated CB receptor fragment, CB2(180-233), spanning from the fifth transmembrane domain (TM5) to the associated loop regions of cannabinoid CB2 receptor. This highly hydrophobic membrane protein fragment was pursued for developmental studies of membrane proteins through expression and purification in Escherichia coli. The target peptide was cloned and over-expressed in a preparative scale as a fusion protein with a modified TrpDeltaLE1413 (TrpLE) leader sequence and a nine-histidine tag at its N-terminal. An experimental protocol for enzyme cleavage was developed by using Factor Xa to remove the TrpLE tag from the fusion protein. A purification process was also established using a nickel affinity column and reverse-phase HPLC, and then monitored by SDS-PAGE and MS. This expression level is one of the highest reported for a G-protein coupled receptor and fragments in E. Coli, and provided a sufficient amount of purified protein for further biophysical studies.     

New natural noncannabinoid ligands for cannabinoid type-2 (CB2) receptors

Since the discovery that Delta 9-tetrahydrocannabinol and related cannabinoids from Cannabis sativa L. act on specific physiological receptors in the human body and the subsequent elucidation of the mammalian endogenous cannabinoid system, no other natural product class has been reported to mimic the effects of cannabinoids. We recently found that N-alkyl amides from purple coneflower (Echinacea spp.) constitute a new class of cannabinomimetics, which specifically engage and activate the cannabinoid type-2 (CB2) receptors. Cannabinoid type-1 (CB1) and CB2 receptors belong to the family of G protein-coupled receptors and are the primary targets of the endogenous cannabinoids N-arachidonoyl ethanolamine and 2-arachidonoyl glyerol. CB2 receptors are believed to play an important role in distinct pathophysiological processes, including metabolic dysregulation, inflammation, pain, and bone loss. CB2 receptors have, therefore, become of interest as new targets in drug discovery. This review focuses on N-alkyl amide secondary metabolites from plants and underscores that this group of compounds may provide novel lead structures for the development of CB2-directed drugs.     

CB(2) cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of CB(2) receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.     

Inhibition of pain responses by activation of CB(2) cannabinoid receptors

Cannabinoid receptor agonists diminish responses to painful stimuli. Extensive evidence demonstrates that CB(1) cannabinoid receptor activation inhibits pain responses. Recently, the synthesis of CB(2) cannabinoid receptor-selective agonists has allowed testing whether CB(2) receptor activation inhibits pain. CB(2) receptor activation is sufficient to inhibit acute nociception, inflammatory hyperalgesia, and the allodynia and hyperalgesia produced in a neuropathic pain model. Studies using site-specific administration of agonist and antagonist have suggested that CB(2) receptor agonists inhibit pain responses by acting at peripheral sites. CB(2) receptor activation also inhibits edema and plasma extravasation produced by inflammation. CB(2) receptor-selective agonists do not produce central nervous system (CNS) effects typical of cannabinoids retaining agonist activity at the CB(1) receptor. Peripheral antinociception without CNS effects is consistent with the peripheral distribution of CB(2) receptors. CB(2) receptor agonists may have promise for the treatment of pain and inflammation without CNS side effects.     

Development and characterization of immobilized cannabinoid receptor (CB1/CB2) open tubular column for on-line screening

Cannabinoid receptors, CB1 and CB2, are therapeutic targets in the treatment of anxiety, obesity, movement disorders, glaucoma, and pain. We have developed an on-line screening method for CB1 and CB2 ligands, where cellular membrane fragments of a chronic myelogenous leukemia cell line, KU-812, were immobilized onto the surface of an open tubular (OT) capillary to create a CB1/CB2–OT column. The binding activities of the immobilized CB1/CB2 receptors were established using frontal affinity chromatographic techniques. This is the first report that confirms the presence of functional CB1 and CB2 receptors on KU-812 cells. The data from this study confirm that the CB1/CB2–OT column can be used to determine the binding affinities (K_i values) for a single compound and to screen individual compounds or a mixture of multiple compounds. The CB1/CB2–OT column was also used to screen a botanical matrix, Zanthoxylum clava-herculis, where preliminary results suggest the presence of a high-affinity phytocannabinoid.     

Optimisation of a novel series of selective CNS penetrant CB(2) agonists

A series of benzimidazole CB₂ receptor agonists were prepared and their properties investigated. Optimisation of the three benzimidazole substituents led to the identification of compound 23, a potent CB₂ full agonist (EC₅₀ 2.7 nM) with excellent selectivity over the CB₁ receptor (>3000-fold). Compound 23 demonstrated good CNS penetration in rat. Further optimisation led to the identification of compound 34 with improved selectivity over hERG and excellent CNS penetration in rat.     

Synthesis and CB1 receptor activities of dimethylheptyl derivatives of 2-arachidonoyl glycerol (2-AG) and 2-arachidonyl glyceryl ether (2-AGE)

Results from a factor analysis and activity studies of commercially available endocannabinoid-type compounds set the starting point for the current study where dimethylheptyl (DMH) analogues of two endocannabinoids, 2-arachidonoyl glycerol (2-AG) and 2-arachidonyl glyceryl ether (2-AGE), were synthesized and their ability to activate the CB1 receptors was determined by the [35S]GTPgammaS binding assay using rat cerebellar membranes. The main goal of the study was to examine how the DMH end tail affects the activity properties of 2-AG (1) and its stable ether (2) and urea analogues (5). The importance of the chain length was also explored by synthesizing 2-AG and 2-AGE derivatives (3 and 4) possessing the chain length C21 instead of C22. Replacement of the pentyl end chain with the DMH resulted in distinct potency decrease as compared to the reference compounds. The modification did not have such a strong impact on the efficacy values. In fact, the efficacy of the derivatives of 2-AGE (2 and 4) was comparable or even improved. Introducing a more stable and hydrophilic urea bond led to a dramatic decrease in biological activity.     

Discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB2 agonists

Recently sulfamoyl benzamides were identified as a novel series of cannabinoid receptor ligands. Replacing the sulfonamide functionality and reversing the original carboxamide bond led to the discovery of N-(3-(morpholinomethyl)-phenyl)-amides as potent and selective CB₂ agonists. Selective CB₂ agonist 31 (K_i = 2.7; CB₁/CB₂ = 190) displayed robust activity in a rodent model of postoperative pain.     

1-Alkyl-2-aryl-4-(1-naphthoyl)pyrroles: new high affinity ligands for the cannabinoid CB1 and CB2 receptors

Two series of 1-alkyl-2-aryl-4-(1-naphthoyl)pyrroles were synthesized and their affinities for the cannabinoid CB(1) and CB(2) receptors were determined. In the 2-phenyl series (5) the N-alkyl group was varied from n-propyl to n-heptyl. A second series of 23 1-pentyl-2-aryl-4-(1-naphthoyl)-pyrroles (6) was also prepared. Several compounds in both series have CB(1) receptor affinities in the 6-30nM range. The high affinities of these pyrrole derivatives relative to JWH-030 (1, R=C(5)H(11)) support the hypothesis that these pyrroles interact with the CB(1) receptor primarily by aromatic stacking.     

Arylsulfonamide CB2 receptor agonists: SAR and optimization of CB2 selectivity

A high-throughput screening campaign resulted in the discovery of a highly potent dual cannabinoid receptor 1 (CB1) and 2 (CB2) agonist. Following a thorough SAR exploration, a series of selective CB2 full agonists were identified.     

The discovery of novel indole-2-carboxamides as cannabinoid CB(1) receptor antagonists

The discovery and structure-activity relationship of a novel series of indole-2-carboxamide antagonists of the cannabinoid CB(1) receptor is disclosed. Compound 26i was found to be a high potency, selective cannabinoid CB(1) antagonist.     

Cannabinoid CB2 receptor (CB2R) stimulation delays rubrospinal mitochondrial-dependent degeneration and improves functional recovery after spinal cord hemisection by ERK1/2 inactivation

Spinal cord injury (SCI) is a devastating condition of CNS that often results in severe functional impairments for which there are no restorative therapies. As in other CNS injuries, in addition to the effects that are related to the primary site of damage, these impairments are caused by degeneration of distal regions that are connected functionally to the primary lesion site. Modulation of the endocannabinoid system (ECS) counteracts this neurodegeneration, and pharmacological modulation of type-2 cannabinoid receptor (CB2R) is a promising therapeutic target for several CNS pathologies, including SCI. This study examined the effects of CB2R modulation on the fate of axotomized rubrospinal neurons (RSNs) and functional recovery in a model of spinal cord dorsal hemisection (SCH) at the cervical level in rats. SCH induced CB2R expression, severe atrophy, and cell death in contralateral RSNs. Furthermore, SCH affected molecular changes in the apoptotic cascade in RSNs – increased cytochrome c release, apoptosome formation, and caspase-3 activity. CB2R stimulation by its selective agonist JWH-015 significantly increased the bcl-2/bax ratio, reduced cytochrome c release, delayed atrophy and degeneration, and improved spontaneous functional recovery through ERK1/2 inactivation. These findings implicate the ECS, particularly CB2R, as part of the endogenous neuroprotective response that is triggered after SCI. Thus, CB2R modulation might represent a promising therapeutic target that lacks psychotropic effects and can be used to exploit ECS-based approaches to counteract neuronal degeneration.Spinal cord injury (SCI) is a devastating neurological disease that results in severe functional impairments for which there are no restorative therapies. In addition to the primary injury, functional impairments following SCI are attributed to degenerative events in regions that are remote but functionally connected to the primary lesion site – that is, supraspinal structures. These events include cell death and structural changes and are important predictors of outcome.^{1, 2} However, few studies have examined the molecular and biochemical changes in remote neurons after SCIs as targets for therapeutic interventions.The spinal cord hemisection (SCH) model is a sensitive and reliable paradigm that has been used to evaluate forelimb motor functions and changes in remote supraspinal areas that are functionally related to the primary site of injury.³ When performed at the cervical level, an SCH in rodents axotomizes nearly the entire contralateral neuronal population of the magnocellular component of the red nucleus and mimics Brown–Séquard syndrome in humans.^{3, 4}The endocannabinoid system (ECS) is a ubiquitous lipid signaling system that has homeostatic functions and comprises two types of G protein-coupled receptors, CB1R and CB2R, their endogenous ligands (arachidonoyl ethanolamide or anandamide and 2-arachidonoylglycerol), and the enzymatic machinery responsible for their synthesis and degradation.⁵ In the brain, the ECS acts like a neurotransmitter system that governs neuronal excitability at various synapses, regulating such processes as pain, mood, appetite, memory, and motor activity.^{6, 7} Unlike classical neurotransmitters, endocannabinoids are not stored in vesicles but are produced on demand in response to various stimuli.^{6, 7}The ECS is modulated by many neurological insults, such as cerebral ischemia,⁸ traumatic,⁹ and focal brain injury,¹⁰ and it is increasingly considered a promising therapeutic target in several CNS pathologies^{9, 10, 11, 12, 13} including SCI.^{14, 15, 16, 17}After an incomplete SCI by concussion, the rapid postlesional activation of cannabinoid receptors that occurs is considered an endogenous protective mechanism.¹⁴ Simultaneous stimulation of CB1R and CB2R early before injury impedes expansion of the lesion and white matter at the epicenter of damage – effects that are maintained for up to 28 days after injury.¹⁵ This limited damage is also accompanied by greater recovery of locomotor function.¹⁶ These neuroprotective effects are prevented by simultaneous blockade of CB1R and CB2R but not of either receptor alone. In the same SCI model, Adhikary et al.¹⁷ demonstrated that a selective CB2R agonist significantly modulated immune responses at the lesion site and improved motor and autonomic functions. The potential of endocannabinoids to limit damage at the primary site has also been highlighted in many studies.^{8, 9, 14, 15, 16, 17} Recent reports have implicated the ECS in remote damage,¹⁰ but the function of the ECS in supraspinal structures after SCI has not been examined.In this study, we determined the effects of CB2R modulation on the fate of the axotomized rubrospinal neurons (RSNs) and on spontaneous functional recovery after SCH at the cervical level in rats. Beginning 7 days after damage, SCH induced severe atrophy and cell loss and upregulated CB2R in axotomized RSNs. Notably, neuronal degeneration proceeded concomitantly with molecular changes in the apoptotic cascade – that is, greater cytochrome c (cyt-c) release from damaged mitochondria, cyt-c/Apaf-1 binding, and caspase-3 activity. CB2R stimulation by its selective agonist JWH-015 (JWH) significantly increased the bcl-2/bax ratio, reduced cyt-c release, delayed atrophy and degeneration, and improved spontaneous functional recovery through ERK1/2 inactivation. Thus, CB2R modulation is a therapeutic target that might counteract the remote degeneration of supraspinal regions after SCI.