The Cannabinoid Receptor Type 1 Positive Allosteric Modulator ZCZ011 Attenuates Naloxone-Precipitated Diarrhea and Weight Loss in Oxycodone-Dependent Mice.

Opioid use disorder reflects a major public health crisis of morbidity and mortality in which opioid withdrawal often contributes to continued use. However, current medications that treat opioid withdrawal symptoms are limited by their abuse liability or lack of efficacy. Although cannabinoid 1 (CB1) receptor agonists, including Δ9-tetrahydrocannabinol, ameliorate opioid withdrawal in both clinical and preclinical studies of opioid dependence, this strategy elicits cannabimimetic side effects as well as tolerance and dependence after repeated administration. Alternatively, CB1 receptor positive allosteric modulators (PAMs) enhance CB1 receptor signaling and show efficacy in rodent models of pain and cannabinoid dependence but lack cannabimimetic side effects. We hypothesize that the CB1 receptor PAM ZCZ011 attenuates naloxone-precipitated withdrawal signs in opioid-dependent mice. Accordingly, male and female mice given an escalating dosing regimen of oxycodone, a widely prescribed opioid, and challenged with naloxone displayed withdrawal signs that included diarrhea, weight loss, jumping, paw flutters, and head shakes. ZCZ011 fully attenuated naloxone-precipitated withdrawal-induced diarrhea and weight loss and reduced paw flutters by approximately half, but its effects on head shakes were unreliable, and it did not affect jumping behavior. The antidiarrheal and anti-weight loss effects of ZCZ0111 were reversed by a CB1 not a cannabinoid receptor type 2 receptor antagonist and were absent in CB1 (-/-) mice, suggesting a necessary role of CB1 receptors. Collectively, these results indicate that ZCZ011 completely blocked naloxone-precipitated diarrhea and weight loss in oxycodone-dependent mice and suggest that CB1 receptor PAMs may offer a novel strategy to treat opioid dependence. SIGNIFICANCE STATEMENT: Opioid use disorder represents a serious public health crisis in which current medications used to treat withdrawal symptoms are limited by abuse liability and side effects. The CB1 receptor positive allosteric modulator (PAM) ZCZ011, which lacks overt cannabimimetic behavioral effects, ameliorated naloxone-precipitated withdrawal signs through a CB1 receptor mechanism of action in a mouse model of oxycodone dependence. These results suggest that CB1 receptor PAMs may represent a viable strategy to treat opioid withdrawal.

Structure-Activity Relationship in the Leucettine Family of Kinase Inhibitors.

The protein kinase DYRK1A is involved in Alzheimer's disease, Down syndrome, diabetes, viral infections, and leukemia. Leucettines, a family of 2-aminoimidazolin-4-ones derived from the marine sponge alkaloid Leucettamine B, have been developed as pharmacological inhibitors of DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). We report here on the synthesis and structure-activity relationship (SAR) of 68 Leucettines. Leucettines were tested on 11 purified kinases and in 5 cellular assays: (1) CLK1 pre-mRNA splicing, (2) Threonine-212-Tau phosphorylation, (3) glutamate-induced cell death, (4) autophagy and (5) antagonism of ligand-activated cannabinoid receptor CB1. The Leucettine SAR observed for DYRK1A is essentially identical for CLK1, CLK4, DYRK1B, and DYRK2. DYRK3 and CLK3 are less sensitive to Leucettines. In contrast, the cellular SAR highlights correlations between inhibition of specific kinase targets and some but not all cellular effects. Leucettines deserve further development as potential therapeutics against various diseases on the basis of their molecular targets and cellular effects.

Effects of a CB2 Subtype Selective Agonist ABK5-1 on Cytokine Production in Microglia.

BACKGROUND AND OBJECTIVES: Neuroinflammation is closely associated with various diseases including neuropathic pain. Microglia are immune cells in the central nervous system which are the main players of immunity and inflammation. Since microglia are activated by nerve injury, and they produce proinflammatory mediators to cause neuropathic pain, targeting activated microglia is considered to be a strategy for treating neuropathic pain. Activation of the cannabinoid CB2 receptor is known to have anti-inflammatory effects in microglia. ABK5-1 is a CB2 subtype selective agonist which inhibits IL-1ß and IL-6 production in the microglia cell line BV-2. The purpose of the current study is to further analyze anti-inflammatory effects of ABK5 in terms of different cytokines and the possible pathway involved in the effect in the BV-2 cell line. METHODS: A cytokine array was performed to screen the effect of ABK5-1 on forty inflammatory mediators in BV-2 cells. Changes of the inflammatory mediators was further supported by mRNA analysis, and a possible signaling molecule that involved the observation was evaluated by western blot. RESULTS: Stimulating BV-2 cells by lipopolysaccharide increased expression of eleven inflammatory mediators, and ABK5-1 treatment resulted in more than a 50% decrease of sICAM1, IL-6, and RANTES. Real-time PCR results showed a decrease of G-CSF, ICAM1, MCP-1, MIP-1α, and MIP-1ß mRNA levels. Western blot analysis showed that ABK5-1 inhibited LPS-induced ERK phosphorylation, which can be a mechanism of ABK5-1-mediated anti-inflammatory effect. CONCLUSIONS: Our current results support the possibility that ABK5-1 is an anti-inflammatory drug for microglia.

Characterization of Subtype Selective Cannabinoid CB2 Receptor Agonists as Potential Anti-Inflammatory Agents.

Activation of the CB2 receptor has been shown to have anti-inflammatory and antinociceptive effects without causing psychoactive effects. Previously, we reported that the compound ethyl 2(2-(N-(2,3-dimethylphenyl) phenylsulfonamido)acetamido)benzoate (ABK5) is a CB2 subtype selective agonist with anti-inflammatory and antinociceptive effects. In the present study, we tested four ABK5 derivatives, ABK5-1, ABK5-2, ABK5-5, and ABK5-6, to analyze the structure of ABK5 to obtain CB2-selective agonists with higher affinity and efficacy. Affinity, subtype selectivity, and G-protein coupling were determined by radioligand binding assays. Selected compounds were then subjected to evaluation of anti-inflammatory effects using two different cell lines, Jurkat (ABK5-1 and 5-2) and BV-2 cells (ABK5-1), which are models of T cells and microglia, respectively. ABK5-1, ABK5-2, and ABK5-6 had comparable CB2 binding affinity with ABK5 (and stimulated G-protein coupling), while only ABK5-1 and ABK5-2 maintained CB2-subtype selectivity. ABK5-5 did not bind CB2 in the detectable range. RT-PCR and ELISA analysis showed that the two compounds also inhibit IL-2 and TNF-α production, and they were more efficacious than ABK5 in inhibiting TNF-α production. CXCL-12 mediated chemotaxis was also evaluated by the transwell migration assay, and both ABK5-1 and ABK5-2 inhibited chemotaxis with a stronger effect observed in ABK5-1. In the microglia cell line BV-2, ABK5-1 inhibited IL-1ß and IL-6 production, which suggests this compound has anti-inflammatory effects through targeting multiple immune cells, and may be a candidate for treatment of inflammation.

Anti-inflammatory and antinociceptive effects of the selective cannabinoid CB2 receptor agonist ABK5.

Cannabinoid receptors are a potential target for anti-inflammatory and pain therapeutics. There are two subtypes, CB1 and CB2, and Δ9-tetrahydrocannabinol activates both of them, providing an analgesic effect but also psychoactive side effects. The psychoactive side effects are considered to be caused by activation of CB1, but not CB2. ABK5 is a CB2 subtype selective agonist that has a very different structure from known cannabinoid receptor agonists. Here, we report anti-inflammatory effects of ABK5 using the T-cell line Jurkat cells, and antinociceptive effect in an inflammatory pain model in rats. Production of the cytokines IL-2 and TNF-α was measured in stimulated Jurkat cells and MOLT-4 cells, and CXCL12-mediated chemotaxis of Jurkat cells was evaluated by a transwell migration assay. Anti-inflammatory and antinociceptive effects of ABK5 were also evaluated in a hindpaw CFA model in rats. ABK5 significantly decreased production of IL-2 and TNF-α measured as both mRNA and protein levels, and reduced chemotaxis towards CXCL12. It also attenuated edema and increased mechanical threshold in the hindpaw of CFA-treated rats. These results suggest that ABK5 is a good lead compound for the development of potential anti-inflammatory and analgesic agents.

Molecular Mechanism and Cannabinoid Pharmacology.

Since antiquity, Cannabis has provoked enormous intrigue for its potential medicinal properties as well as for its unique pharmacological effects. The elucidation of its major cannabinoid constituents, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), led to the synthesis of new cannabinoids (termed synthetic cannabinoids) to understand the mechanisms underlying the pharmacology of Cannabis. These pharmacological tools were instrumental in the ultimate discovery of the endogenous cannabinoid system, which consists of CB1 and CB2 cannabinoid receptors and endogenously produced ligands (endocannabinoids), which bind and activate both cannabinoid receptors. CB1 receptors mediate the cannabimimetic effects of THC and are highly expressed on presynaptic neurons in the nervous system, where they modulate neurotransmitter release. In contrast, CB2 receptors are primarily expressed on immune cells. The endocannabinoids are tightly regulated by biosynthetic and hydrolytic enzymes. Accordingly, the endocannabinoid system plays a modulatory role in many physiological processes, thereby generating many promising therapeutic targets. An unintended consequence of this research was the emergence of synthetic cannabinoids sold for human consumption to circumvent federal laws banning Cannabis use. Here, we describe research that led to the discovery of the endogenous cannabinoid system and show how knowledge of this system benefitted as well as unintentionally harmed human health.

Structural Optimization of the Diarylurea PSNCBAM-1, an Allosteric Modulator of Cannabinoid Receptor 1.

BACKGROUND: Structure-activity relationship studies improve the pharmacological and pharmacokinetic properties of a lead compound such as PSNCBAM-1, an allosteric modulator of the cannabinoid receptor 1. OBJECTIVES: Here, several derivatives of PSNCBAM-1 were synthesized with the aim of reducing the number of rings within its structure and enhancing the solubility of the compounds. The derivatives studied contain substituents previously shown to enhance binding of agonists (ie, a cyano group and a pyrimidine ring), with a reduced number of rings compared with the parent compound, PSNCBAM-1. METHODS: The synthesized compounds were tested for the enhancement of the binding of orthosteric cannabinoid receptor 1 agonist CP55,940 in the presence of varying concentrations of each test compound. Select compounds were also tested for their effects on cannabinoid receptor 1 inverse agonist SR141716A binding. The compounds were also subjected to computational analysis of drug-like properties and solubility. RESULTS: Consistent with a positive allosteric modulator for orthosteric ligand binding, compounds LDK1317 (12a), LDK1320 (12b), LDK1321 (6a), LDK1323 (8a), and LDK1324 (6b) all enhanced the binding of agonist CP55,940 to some degree. Reduction in the number of rings did not abolish the activity. The new lead compounds LDK1317 (12a) and LDK1321 (6a) showed improved drug-like properties and enhanced solubility in silico. CONCLUSIONS: In contrast to PSNCBAM-1, the synthesized compounds are analogs with fewer rings. The compounds LDK1317 (12a) and LDK1321 (6a) contained only 2 or 3 rings, respectively, and showed the binding parameters (KB = 110 nM, α = 2.3, and KB = 85 nM, α = 5.9). Further, the computationally predicted drug-like properties and solubility suggest these compounds are acceptable new lead compounds for further development of cannabinoid receptor 1 allosteric modulators. (Curr Ther Res Clin Exp. 2020; 81:XXX-XXX).

In vivo evaluation of the CB1 allosteric modulator LDK1258 reveals CB1-receptor independent behavioral effects.

In the present study, we examined whether LDK1258, which produces strong CB1 receptor allosteric effects in in vitro assays, would elicit in vivo effects consistent with allosteric activity. In initial studies, LDK1258 reduced food consumption and elicited delayed antinociceptive effects in the chronic constrictive injury of the sciatic nerve (CCI) model of neuropathic pain, which unexpectedly emerged 4 h post-injection. UPLC-MS/MS analysis quantified significant levels of LDK1258 in both blood and brain tissue at 30 min post-administration that remained stable up to 4 h. The observation that LDK1258 also produced respective antinociceptive and anorectic effects in rimonabant-treated wild type mice and CB1 (-/-) mice suggests an off-target mechanism of action. Likewise, LDK1258 produced a partial array of common cannabimimetic effects in the tetrad assay, which were not CB1 receptor mediated. Additionally, LDK1258 did not substitute for the CB1 receptor orthosteric agonists CP55,940 or anandamide in the drug discrimination paradigm. In other in vivo assays sensitive to CB1 receptor allosteric modulators, LDK1258 failed to shift the dose-response curves of either CP55,940 or anandamide in producing thermal antinociception, catalepsy, or hypothermia, and did not alter the generalization curve of either drug in the drug discrimination assay. Thus, this battery of tests yielded results demonstrating that LDK1258 produces antinociceptive effects in the CCI model of neuropathic pain, anorectic effects, and other in vivo pharmacological effects in a manner inconsistent with CB1 receptor allosterism. More generally, this study offers a straightforward screening assay to determine whether newly synthesized CB1 receptor allosteric modulators translate to the whole animal.

Allosteric modulators restore orthosteric agonist binding to mutated CB1 receptors.

OBJECTIVES: To determine if diminished orthosteric agonist binding due to mutations in extracellular loops 1 or 2 of the cannabinoid receptor 1 (CB1 ) can be overcome by an allosteric modulator and restore agonist binding. METHODS: Binding assays were performed using a range of concentrations of orthosteric compound, in the presence or absence of a set concentration of the allosteric modulator PSNCBAM-1 to determine the EC50 in its absence or presence. KEY FINDINGS: Single mutations in extracellular loop 1 or 2 of CB1 showed weak or no binding of agonist CP55940 to the receptor. Interestingly, upon addition of the allosteric modulator PSNCBAM-1, this binding was restored typically to wild-type CB1 levels. In a few cases, the allosteric modulator ORG27569 was compared with PSNCBAM-1 for CP55940 binding and it also restored binding. Further, wild-type levels of inverse agonist bound the CB1 mutants in the absence of modulator, suggesting the mutants were originally folded like the wild type. CONCLUSIONS: Based on our findings, we provide evidence of a therapeutic application for allosteric modulators in situations where a mutation in the receptor may hinder its function. By utilizing allosteric modulators, restoration of orthosteric binding may be possible.

Identification and biochemical analyses of selective CB2 agonists.

Cannabinoid CB1 and CB2 receptors are activated by Δ9-tetrahydrocannabinol, a psychoactive component of marijuana. The cannabinoid CB1 receptor is primarily located in the brain and is responsible for the psychoactive side effects, whereas the cannabinoid CB2 receptor is located in immune cells and is an attractive target for immune-related maladies. We identify small molecules that selectively bind to the cannabinoid CB2 receptor and can be further developed into therapeutics. The affinity of three molecules, ABK5, ABK6, and ABK7, to the cannabinoid CB2 receptor was determined with radioligand competition binding. The potency of G-protein coupling was determined with GTPγS binding. The three compounds bound selectively to the cannabinoid CB2 receptor, and no binding to the cannabinoid CB1 receptor was detected up to 10 µM. Immunoblotting studies show that the amount of ERK1/2 and MEK phosphorylation increased in a Gi/o-dependent manner. Furthermore, an immune cell line (Jurkat cells) was treated with ABK5, and as a result, inhibited cell proliferation. These three compounds are novel cannabinoid CB2 receptor agonists and hold promise to be further developed to treat inflammation and the often-associated pain.

Translational potential of allosteric modulators targeting the cannabinoid CB1 receptor.

The cannabinoid type-1 (CB1) receptor, a G-protein-coupled receptor, is an attractive target for drug discovery due to its involvement in many physiological processes. Historically, drug discovery efforts targeting the CB1 receptor have focused on the development of orthosteric ligands that interact with the active site to which endogenous cannabinoids bind. Research performed over the last several decades has revealed substantial difficulties in translating CB1 orthosteric ligands into druggable candidates. The difficulty is mainly due to the adverse effects associated with orthosteric CB1 ligands. Recent discoveries of allosteric CB1 modulators provide tremendous opportunities to develop CB1 ligands with novel mechanisms of action; these ligands may potentially improve the pharmacological effects and enhance drug safety in treating the disorders by regulating the functions of the CB1 receptor. In this paper, we review and summarize the complex pharmacological profiles of each class of CB1 allosteric modulators, the development of new classes of CB1 allosteric modulators and the results from in vivo assessments of their therapeutic value.

Pyrimidinyl Biphenylureas Act as Allosteric Modulators to Activate Cannabinoid Receptor 1 and Initiate ß-Arrestin-Dependent Responses.

Cannabinoid receptor 1 (CB1) is a G-protein-coupled receptor that is abundant in the central nervous system. It binds several compounds in its orthosteric site, including the endocannabinoids, arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, and the plant-derived Δ9-tetrahydrocannabinol, one of the main psychoactive components of marijuana. It primarily couples to Gi/o proteins to inhibit adenylate cyclase activity and typically induces downstream signaling that is Gi-dependent. Since this receptor is implicated in several maladies, such as obesity, pain, and neurodegenerative disorders, there is interest in developing therapeutics that selectively target this receptor. Allosteric modulators of CB1 offer one new approach that has tremendous therapeutic potential. Here, we reveal receptor- and cellular-level properties consistent with receptor activation by a series of pyrimidinyl biphenylureas (LDK1285, LDK1288, LDK1305, and PSNCBAM1), including promoting binding of the agonist CP55940 with positive cooperativity and inhibiting binding of the inverse agonist SR141716A with negative cooperativity, demonstrated via radioligand binding studies. Consistent with these findings, the allosteric modulators induced cellular internalization of the receptor and recruitment of ß-arrestin 2 in human embryonic kidney cell line 293 cells monitored with confocal and total internal reflective fluorescence microscopy, respectively. These allosteric modulators, however, caused G-protein-independent but ß-arrestin 1-dependent phosphorylation of the downstream kinases extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Src, shown by immunoblotting studies. These results are consistent with the involvement of ß-arrestin and suggest that these allosteric modulators induce biased signaling.

Exploring 6-Azaindole and 7-Azaindole Rings for Developing Cannabinoid Receptor 1 Allosteric Modulators.

Introduction and Objective: Org27569 is a prototypical allosteric modulator of the cannabinoid receptor 1 (CB1). It belongs to the indole-2-carboxamide scaffold and has been intensively investigated in pharmacology and in structure-activity relationship (SAR) studies. Although azaindoles are rare in natural products and differ only by the presence of an extra ring nitrogen, they were demonstrated as valuable bioisosteres in many pharmacologically important molecules. To extend the SAR investigation of the indole-2-carboxamide class of CB1 allosteric modulators, azaindole (pyrrolopyridine) rings were used to replace the indole ring of Org27569 analogs to explore the potential of azaindole-2-carboxamides as CB1 allosteric modulators. Using 6- and 7-azaindole in lieu of the indole moiety within this class of CB1 allosteric modulators indeed improved the aqueous solubility. Materials and Methods: We synthesized 6- and 7-azaindole-2-carboxamides and their indole-2-carboxamide counterparts. The molecules were evaluated by [3H]CP55,940 binding and [35S]GTPγS binding assays for their allosteric modulation of the CB1 receptor. Results: The 7-azaindole-2-carboxamides lost the ability to bind to the CB1 receptor. The 6-azaindole-2-carboxamides (e.g., 3c and 3d) showed markedly reduced binding affinities to the CB1 receptor in comparison with their indole-2-carboxamide counterparts. However, they behaved similarly as indole-2-carboxamides in potentiating the orthosteric agonist binding and inhibiting the orthosteric agonist-induced G-protein coupling. The results indicated that some azaindole scaffolds (e.g., 6-azaindole) are worth further exploration, whereas the 7-azaindole ring is not a viable bioisostere of the indole ring in the Org27569 class of CB1 allosteric modulators.

Allosteric modulators of cannabinoid receptor 1: developing compounds for improved specificity.

The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor (GPCR) that is located primarily in the central nervous system. CB1 is a therapeutic target which may impact pathways to mediate pain, neurodegenerative disorders, hunger, and drug-seeking behavior. Despite these benefits, development of orthosteric therapeutic compounds, which target the endogenous ligand-binding site of CB1, has been challenging due to detrimental side effects including psychoactivity, depression, and suicidal thoughts. However, CB1 also has an allosteric binding site(s), which is topographically distinct from the orthosteric site. Allosteric modulation of CB1 has a number of potential advantages including providing a mechanism for more precise control of downstream pathways and circumventing these side effects. In this review, we summarize the concept of allosteric modulation and focus on the structure-activity relationship studies of the well-characterized allosteric modulators, ORG27569 and PSNCBAM-1 and their derivatives, and a few other recent modulators. We review studies on the properties of these modulators on CB1 signaling in cells and their effects in vivo. While many current allosteric modulators also produce complex outcomes, they provide new advances for the design of CB1 centered therapeutics.

Discovery of Selective Cannabinoid CB2 Receptor Agonists by High-Throughput Screening.

The endocannabinoid system (ECS) plays a diverse role in human physiology ranging from the regulation of mood and appetite to immune modulation and the response to pain. Drug development that targets the cannabinoid receptors (CB1 and CB2) has been explored; however, success in the clinic has been limited by the psychoactive side effects associated with modulation of the neuronally expressed CB1 that are enriched in the CNS. CB2, however, are expressed in peripheral tissues, primarily in immune cells, and thus development of CB2-selective drugs holds the potential to modulate pain among other indications without eliciting anxiety and other undesirable side effects associated with CB1 activation. As part of a collaborative effort among industry and academic laboratories, we performed a high-throughput screen designed to discover selective agonists or positive allosteric modulators (PAMs) of CB2. Although no CB2 PAMs were identified, 167 CB2 agonists were discovered here, and further characterization of four select compounds revealed two with high selectivity for CB2 versus CB1. These results broaden drug discovery efforts aimed at the ECS and may lead to the development of novel therapies for immune modulation and pain management with improved side effect profiles.

Retromer stops beta-arrestin 1-mediated signaling from internalized cannabinoid 2 receptors.

G protein-coupled receptors mediate their complex functions through activation of signaling cascades from receptors localized at the cell surface and endosomal compartments. These signaling pathways are modulated by heterotrimeric G proteins and the scaffold proteins beta-arrestin 1 and 2. However, in contrast to the events occurring at the cell surface, our knowledge of the mechanisms controlling signaling from receptors localized at intracellular compartments is still very limited. Here we sought to investigate the intracellular signaling from cannabinoid 2 receptor (CB2R). First, we show that receptor internalization is required for agonist-induced phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Then we demonstrate that ERK1/2 activation is mediated by beta-arrestin 1 from receptors localized exclusively at Rab4/5 compartments. Finally, we identify the retromer complex as a gatekeeper, terminating beta-arrestin 1-mediated ERK phosphorylation. These findings extend our understanding of the events controlling signaling from endocytosed receptors and identify the retromer as a modulator of beta-arrestin-mediated signaling from CB2R.

Assessing Allosteric Modulation of CB1 at the Receptor and Cellular Levels.

The cannabinoid CB1 receptor is abundant in the central nervous system and regulates neuronal transmission and other key physiological processes including those leading to pain, inflammation, memory, and feeding behavior. CB1 is activated by the endogenous ligands, arachidonoyl ethanolamine and 2-arachidonoyl glycerol, by various synthetic ligands (e.g., CP55940), and by Δ9-tetrahydrocannabinol, the psychoactive component of Cannabis sativa. These CB1 ligands are orthosteric and transduce downstream signals by binding CB1 and primarily inducing Gi coupling, but Gs and ß-arrestin coupling are also possible. Recently, allosteric modulators for CB1 were discovered that bind to topographically distinct sites and can noncompetitively impact the potency and efficacy of orthosteric compounds. These offer the exciting potential for mechanistic analyses and for developing therapeutics. Yet, it is critical to elucidate whether a compound is a positive allosteric modulator or a negative allosteric modulator of orthosteric ligand-induced CB1 profiles to understand pathway specificity and ameliorate diseases. In this chapter, we present equilibrium and kinetic binding analysis to reveal the impact of allosteric modulators on CB1. Also described are activities consistent with CB1 activation (or inactivation) and include cellular internalization of CB1 and downstream signaling patterns. Since many CB1 allosteric modulators do not enhance G protein coupling, it is critical to distinguish CB1 activation and biased signaling patterns via ß-arrestin from CB1 inactivation. These strategies can illuminate pathway specificity and are valuable for the fine-tuning of CB1 function.

Modulation of CB1 cannabinoid receptor by allosteric ligands: Pharmacology and therapeutic opportunities.

Cannabinoid pharmacology has been intensely studied because of cannabis' pervasive medicinal and non-medicinal uses as well as for the therapeutic potential of cannabinoid-based drugs for the treatment of pain, anxiety, substance abuse, obesity, cancer and neurodegenerative disorders. The identification of allosteric modulators of the cannabinoid receptor 1 (CB1) has given a new direction to the development of cannabinoid-based therapeutics due to the many advantages offered by targeting allosteric site(s). Allosteric receptor modulators hold potential to develop subtype-specific and pathway-specific therapeutics. Here we briefly discuss the first-generation of allosteric modulators of CB1 receptor, their structure-activity relationships, signaling pathways and the allosteric binding site(s) on the CB1 receptor. This article is part of the Special Issue entitled "A New Dawn in Cannabinoid Neurobiology".

Pyrimidinyl Biphenylureas: Identification of New Lead Compounds as Allosteric Modulators of the Cannabinoid Receptor CB1.

The allosteric modulator 1-(4-chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea (PSNCBAM-1, 2) bound the cannabinoid receptor 1 (CB1) and antagonized G protein coupling. This compound demonstrated potent anorectic effects similar to the CB1 antagonist rimonabant that once was marketed for the treatment of obesity, suggesting a new chemical entity for the discovery of antiobesity drugs. To increase structural diversity of this class of CB1 ligands, we designed and synthesized two classes of novel analogues, in which the pyridine ring of 2 was replaced by a pyrimidine ring. These positively modulate the binding of the CB1 orthosteric agonist CP55,940 while exhibiting an antagonism of G-protein coupling activity. Interestingly, compounds 7d and 8d demonstrated ERK1/2 phosphorylation mediated via ß-arrestin unlike the orthosteric CP55,940 that does so in a G protein-dependent manner. These can serve as new lead compounds for the future development of CB1 allosteric modulators that show biased agonism and potentially antiobesity behavior via a new mechanism.

Synthesis and biological evaluation of indole-2-carboxamides bearing photoactivatable functionalities as novel allosteric modulators for the cannabinoid CB1 receptor.

5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ORG27569, 1) is a prototypical allosteric modulator for the cannabinoid CB1 receptor. Based on this indole-2-carboxamide scaffold, we designed and synthesized novel CB1 allosteric modulators that possess photoactivatable functionalities, which include benzophenone, phenyl azide, aliphatic azide and phenyltrifluoromethyldiazrine. To assess their allosteric effects, the dissociation constant (KB) and allosteric binding cooperativity factor (α) were determined and compared to their parent compounds. Within this series, benzophenone-containing compounds 26 and 27, phenylazide-containing compound 28, and the aliphatic azide containing compound 36b showed allosteric binding parameters (KB and α) comparable to their parent compound 1, 7, 8, and 9, respectively. We further assessed these modulators for their impact on G-protein coupling activity. Interestingly, these compounds exhibited negative allosteric modulator properties in a manner similar to their parent compounds, which antagonize agonist-induced G-protein coupling. These novel CB1 allosteric modulators, possessing photoactivatable functionalities, provide valuable tools for future photo-affinity labeling and mapping the CB1 allosteric binding site(s).