Biased agonism at G protein–coupled receptors describes the phenomenon whereby some drugs can activate some downstream signaling activities to the relative exclusion of others. Descriptions of biased agonism focusing on the differential engagement of G proteins versus β-arrestins are commonly limited by the small response windows obtained in pathways that are not amplified or are less effectively coupled to receptor engagement, such as β-arrestin recruitment. At the μ-opioid receptor (MOR), G protein–biased ligands have been proposed to induce less constipation and respiratory depressant side effects than opioids commonly used to treat pain. However, it is unclear whether these improved safety profiles are due to a reduction in β-arrestin–mediated signaling or, alternatively, to their low intrinsic efficacy in all signaling pathways. Here, we systematically evaluated the most recent and promising MOR-biased ligands and assessed their pharmacological profile against existing opioid analgesics in assays not confounded by limited signal windows. We found that oliceridine, PZM21, and SR-17018 had low intrinsic efficacy. We also demonstrated a strong correlation between measures of efficacy for receptor activation, G protein coupling, and β-arrestin recruitment for all tested ligands. By measuring the antinociceptive and respiratory depressant effects of these ligands, we showed that the low intrinsic efficacy of opioid ligands can explain an improved side effect profile. Our results suggest a possible alternative mechanism underlying the improved therapeutic windows described for new opioid ligands, which should be taken into account for future descriptions of ligand action at this important therapeutic target.

Indole and indazole synthetic cannabinoids (SCs) featuring l-valinate or l-tert-leucinate pendant group have recently emerged as prevalent recreational drugs, and their use has been associated with serious adverse health effects. Due to the limited pharmacological data available for these compounds, 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and their analogues were synthesized and assessed for cannabimimetic activity in vitro and in vivo. All SCs acted as potent, highly efficacious agonists at CB1 (EC50 = 0.45-36 nM) and CB2 (EC50 = 4.6-128 nM) receptors in a fluorometric assay of membrane potential, with a general preference for CB1 activation. The cannabimimetic properties of two prevalent compounds with confirmed toxicity in humans, 5F-AMB and MDMB-FUBINACA, were demonstrated in vivo using biotelemetry in rats. Bradycardia and hypothermia were induced by 5F-AMB and MDMB-FUBINACA doses of 0.1-1 mg/kg (and 3 mg/kg for 5F-AMB), with MDMB-FUBINACA showing the most dramatic hypothermic response recorded in our laboratory for any SC (>3 °C at 0.3 mg/kg). Reversal of hypothermia by pretreatment with a CB1, but not CB2, antagonist was demonstrated for 5F-AMB and MDMB-FUBINACA, consistent with CB1-mediated effects in vivo. The in vitro and in vivo data indicate that these SCs act as highly efficacious CB receptor agonists with greater potency than Δ(9)-THC and earlier generations of SCs.

Opioids are widely prescribed analgesics, but their use is limited due to development of tolerance and addiction, as well as high variability in individual response. The development of improved opioid analgesics requires high-throughput functional assays to assess large numbers of potential opioid ligands. In this study, we assessed the ability of a proprietary "no-wash" fluorescent membrane potential dye to act as a reporter of µ-opioid receptor (MOR) activation and desensitization via activation of G-protein-coupled inwardly rectifying potassium channels. AtT-20 cells stably expressing mouse MOR were assayed in 96-well plates using the Molecular Devices FLIPR membrane potential dye. Dye emission intensity decreased upon membrane hyperpolarization. Fluorescence decreased in a concentration-dependent manner upon application of a range of opioid ligands to the cells, with high-efficacy agonists producing a decrease of 35% to 40% in total fluorescence. The maximum effect of morphine faded in the continued presence of agonist, reflecting receptor desensitization. The effects of opioids were prevented by prior treatment with pertussis toxin and blocked by naloxone. We have demonstrated this assay to be an effective method for assessing ligand signaling at MOR, which may potentially be scaled up as an additional high-throughput screening technique for characterizing novel opioid ligands.

Introduction: Compounds present in Cannabis sativa such as phytocannabinoids and terpenoids may act in concert to elicit therapeutic effects. Cannabinoids such as Δ9-tetrahydrocannabinol (Δ9-THC) directly activate cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2); however, it is not known if terpenoids present in Cannabis also affect cannabinoid receptor signaling. Therefore, we examined six common terpenoids alone, and in combination with cannabinoid receptor agonists, on CB1 and CB2 signaling in vitro.Materials and Methods: Potassium channel activity in AtT20 FlpIn cells transfected with human CB1 or CB2 receptors was measured in real time using FLIPR® membrane potential dye in a FlexStation 3 plate reader. Terpenoids were tested individually and in combination for periods up to 30 min. Endogenous somatostatin receptors served as a control for direct effects of drugs on potassium channels.Results: α-Pinene, β-pinene, β-caryophyllene, linalool, limonene, and β-myrcene (up to 30–100 μM) did not change membrane potential in AtT20 cells expressing CB1 or CB2, or affect the response to a maximally effective concentration of the synthetic cannabinoid CP55,940. The presence of individual or a combination of terpenoids did not affect the hyperpolarization produced by Δ9-THC (10 μM): (CB1: control, 59%±7%; with terpenoids (10 μM each) 55%±4%; CB2: Δ9-THC 16%±5%, with terpenoids (10 μM each) 17%±4%). To investigate possible effect on desensitization of CB1 responses, all six terpenoids were added together with Δ9-THC and signaling measured continuously over 30 min. Terpenoids did not affect desensitization, after 30 min the control hyperpolarization recovered by 63%±6% in the presence of the terpenoids recovery was 61%±5%.Discussion: None of the six of the most common terpenoids in Cannabis directly activated CB1 or CB2, or modulated the signaling of the phytocannabinoid agonist Δ9-THC. These results suggest that if a phytocannabinoid–terpenoid entourage effect exists, it is not at the CB1 or CB2 receptor level. It remains possible that terpenoids activate CB1 and CB2 signaling pathways that do not involve potassium channels; however, it seems more likely that they may act at different molecular target(s) in the neuronal circuits important for the behavioral effect of Cannabis.

Background and Purpose: Cannabichromene (CBC) is one of the most abundant phytocannabinoids in Cannabis spp. It has modest antinociceptive and antiinflammatory effects and potentiates some effects of Δ 9-tetrahydrocannabinol in vivo. How CBC exerts these effects is poorly defined and there is little information about its efficacy at cannabinoid receptors. We sought to determine the functional activity of CBC at cannabinoid CB 1 and CB 2 receptors. Experimental Approach: AtT20 cells stably expressing haemagglutinin-tagged human CB 1 and CB 2 receptors were used. Assays of cellular membrane potential and loss of cell surface receptors were performed. Key Results: CBC activated CB 2 but not CB 1 receptors to produce hyperpolarization of AtT20 cells. This activation was inhibited by a CB 2 receptor antagonist AM630, and sensitive to Pertussis toxin. Application of CBC reduced activation of CB 2 , but not CB 1 , receptors by subsequent co-application of CP55,940, an efficacious CB 1 and CB 2 receptor agonist. Continuous CBC application induced loss of cell surface CB 2 receptors and desensitization of the CB 2 receptor-induced hyperpolarization. Conclusions and Implications: CBC is a selective CB 2 receptor agonist displaying higher efficacy than tetrahydrocannabinol in hyperpolarizing AtT20 cells. CBC can also recruit CB 2 receptor regulatory mechanisms. CBC may contribute to the potential therapeutic effectiveness of some cannabis preparations, potentially through CB 2 receptor-mediated modulation of inflammation. 1 | INTRODUCTION Cannabichromene (CBC) is one of over 100 phytochemicals (collectively referred to as phytocannabinoids) that are found in Cannabis spp (ElSohly & Gul, 2014). CBC was identified in 1966 and is one of the most abundant phytocannabinoids alongside Δ 9-tetrahydrocannabinol (THC),

Background and Purpose: The morbidity and mortality associated with recreational use of synthetic cannabinoid receptor agonists (SCRAs) may reflect strong activation of CB 1 receptors and is a major health concern. The properties of SCRA at CB 1 receptors are not well defined. Here we have developed an assay to determine acute CB 1 receptor efficacy using receptor depletion with the irreversible CB 1 receptor antagonist AM6544, with application of the Black and Leff operational model to calculate efficacy.Experimental Approach: Receptor depletion in mouse AtT-20 pituitary adenoma cells stably expressing human CB 1 receptors was achieved by pretreatment of cells with AM6544 (10 μM, 60 min). The CB 1 receptor-mediated hyperpolarisation of AtT-20 cells was measured using fluorescence-based membrane potential dye. From data fit to the operational model, the efficacy (τ) and affinity (K A ) parameters were obtained for each drug.Key Results: AM6544 did not affect the potency or maximal effect of native somatostatin receptor-induced hyperpolarization. The τ value of Δ 9 -THC was 80-fold less than the reference CB receptor agonist CP55940 and 260-fold less than the highest efficacy SCRA, 5F-MDMB-PICA. The operational efficacy of SCRAs ranged from 233 (5F-MDMB-PICA) to 28 (AB-PINACA), with CP55940 in the middle of the efficacy rank order. There was no correlation between the τ and K A values.Conclusions and Implications: All SCRAs tested showed substantially higher efficacy at CB 1 receptors than Δ 9 -THC, which may contribute to the adverse effects seen with these drugs but not Δ 9 -THC.

BACKGROUND AND PURPOSEThere is significant variation in individual response to opioid drugs, which may result in inappropriate opioid therapy. Polymorphisms of the μ opioid receptor (MOP receptor) may contribute to individual variation in opioid response by affecting receptor function, and the effect may be ligand-specific. We sought to determine functional differences in MOP receptor signalling at several signalling pathways using a range of structurally distinct opioid ligands in cells expressing wild-type MOP receptors (MOPr-WT) and the commonly occurring MOP receptor variant, N40D. EXPERIMENTAL APPROACHMOPr-WT and MOPr-N40D were stably expressed in CHO cells and in AtT-20 cells. Assays of AC inhibition and ERK1/2 phosphorylation were performed on CHO cells, and assays of K activation were performed on AtT-20 cells. Signalling profiles for each ligand were compared between variants. KEY RESULTSBuprenorphine efficacy was reduced by over 50% at MOPr-N40D for AC inhibition and ERK1/2 phosphorylation. Buprenorphine potency was reduced threefold at MOPr-N40D for K channel activation. Pentazocine efficacy was reduced by 50% for G-protein-gated inwardly rectifying K channel activation at MOPr-N40D. No other differences were observed for any other ligands tested. CONCLUSIONS AND IMPLICATIONSThe N40D variant is present in 10-50% of the population. Buprenorphine is a commonly prescribed opioid analgesic, and many individuals do not respond to buprenorphine therapy. This study demonstrates that buprenorphine signalling to several effectors via the N40D variant of MOP receptors is impaired, and this may have important consequences in a clinical setting for individuals carrying the N40D allele.

Synthetic cannabinoid receptor agonists (SCRAs) represent the most rapidly expanding class of new psychoactive substances (NPSs). Despite the prevalence and potency of recent chiral indole-3-carboxamide SCRAs, few pharmacological data are available regarding the enantiomeric bias of these NPSs toward human CB1 and CB2 receptors. A series of homochiral indole-3-carboxamides derived from (S)- and (R)-α-methylbenzylamine and featuring variation of the 1-alkyl substituent were prepared, pharmacologically evaluated, and compared to related achiral congeners derived from cumyl- and benzylamine. Competitive binding assays demonstrated that all analogues derived from either enantiomer of α-methylbenzylamine (14–17) showed affinities for CB1 (K i = 47.9–813 nM) and CB2 (K i = 47.9–347 nM) that were intermediate to that of the corresponding benzylic (10–13, CB1 K i = 550 nM to >10 μM; CB2 K i = 61.7 nM to >10 μM) and cumyl derivatives (6–9, CB1 K i = 12.6–21.4 nM; CB2 K i = 2.95–24.5 nM). In a fluorometric membrane potential assay, all α-methylbenzyl analogues (excluding 17) were potent, efficacious agonists of CB1 (EC50 = 32–464 nM; E max = 89–104%) and low efficacy agonists of CB2 (EC50 = 54–500 nM; E max = 52–77%), with comparable or greater potency than the benzyl analogues and much lower potency than the cumyl derivatives, consistent with binding trends. The relatively greater affinity and potency of (S)-14–17 compared to (R)-14–17 analogues at CB1 highlighted an enantiomeric bias for this series of SCRAs. Molecular dynamics simulations provided a conformational basis for the observed differences in agonist potency at CB1 pending benzylic substitution.