Bias Factor and Therapeutic Window Correlate to Predict Safer Opioid Analgesics.

Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit ßarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that ßarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.

A µ-opioid receptor modulator that works cooperatively with naloxone.

The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Molecular insights into the biased signaling mechanism of the µ-opioid receptor.

GPCR functional selectivity opens new opportunities for the design of safer drugs. Ligands orchestrate GPCR signaling cascades by modulating the receptor conformational landscape. Our study provides insights into the dynamic mechanism enabling opioid ligands to preferentially activate the G protein over the ß-arrestin pathways through the µ-opioid receptor (µOR). We combine functional assays in living cells, solution NMR spectroscopy, and enhanced-sampling molecular dynamic simulations to identify the specific µOR conformations induced by G protein-biased agonists. In particular, we describe the dynamic and allosteric communications between the ligand-binding pocket and the receptor intracellular domains, through conserved motifs in class A GPCRs. Most strikingly, the biased agonists trigger µOR conformational changes in the intracellular loop 1 and helix 8 domains, which may impair ß-arrestin binding or signaling. The findings may apply to other GPCR families and provide key molecular information that could facilitate the design of biased ligands.

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

In vivo photopharmacology with a caged mu opioid receptor agonist drives rapid changes in behavior.

Photoactivatable drugs and peptides can drive quantitative studies into receptor signaling with high spatiotemporal precision, yet few are compatible with behavioral studies in mammals. We developed CNV-Y-DAMGO-a caged derivative of the mu opioid receptor-selective peptide agonist DAMGO. Photoactivation in the mouse ventral tegmental area produced an opioid-dependent increase in locomotion within seconds of illumination. These results demonstrate the power of in vivo photopharmacology for dynamic studies into animal behavior.

Chronic Morphine Induces Adaptations in Opioid Receptor Signaling in a Thalamostriatal Circuit That Are Location Dependent, Sex Specific, and Regulated by µ-Opioid Receptor Phosphorylation.

Chronic opioid exposure induces tolerance to the pain-relieving effects of opioids but sensitization to some other effects. While the occurrence of these adaptations is well understood, the underlying cellular mechanisms are less clear. This study aimed to determine how chronic treatment with morphine, a prototypical opioid agonist, induced adaptations to subsequent morphine signaling in different subcellular contexts. Opioids acutely inhibit glutamatergic transmission from medial thalamic (MThal) inputs to the dorsomedial striatum (DMS) via activity at µ-opioid receptors (MORs). MORs are present in somatic and presynaptic compartments of MThal neurons terminating in the DMS. We investigated the effects of chronic morphine treatment on subsequent morphine signaling at MThal-DMS synapses and MThal cell bodies in male and female mice. Surprisingly, chronic morphine treatment increased subsequent morphine inhibition of MThal-DMS synaptic transmission (morphine facilitation) in male, but not female, mice. At MThal cell bodies, chronic morphine treatment decreased subsequent morphine activation of potassium conductance (morphine tolerance) in both male and female mice. In knock-in mice expressing phosphorylation-deficient MORs, chronic morphine treatment resulted in tolerance to, rather than facilitation of, subsequent morphine signaling at MThal-DMS terminals, suggesting phosphorylation deficiency unmasks adaptations that counter the facilitation observed at presynaptic terminals in wild-type mice. The results of this study suggest that the effects of chronic morphine exposure are not ubiquitous; rather adaptations in MOR function may be determined by multiple factors such as subcellular receptor distribution, influence of local circuitry, and sex.

Activation mechanism of the µ-opioid receptor by an allosteric modulator.

Allosteric modulators of G-protein-coupled receptors (GPCRs) enhance signaling by binding to GPCRs concurrently with their orthosteric ligands, offering a novel approach to overcome the efficacy limitations of conventional orthosteric ligands. However, the structural mechanism by which allosteric modulators mediate GPCR signaling remains largely unknown. Here, to elucidate the mechanism of µ-opioid receptor (MOR) activation by allosteric modulators, we conducted solution NMR analyses of MOR by monitoring the signals from methionine methyl groups. We found that the intracellular side of MOR exists in an equilibrium between three conformations with different activities. Interestingly, the populations in the equilibrium determine the apparent signaling activity of MOR. Our analyses also revealed that the equilibrium is not fully shifted to the conformation with the highest activity even in the full agonist-bound state, where the intracellular half of TM6 is outward-shifted. Surprisingly, an allosteric modulator for MOR, BMS-986122, shifted the equilibrium toward the conformation with the highest activity, leading to the increased activity of MOR in the full agonist-bound state. We also determined that BMS-986122 binds to a cleft in the transmembrane region around T162 on TM3. Together, these results suggest that BMS-986122 binding to TM3 increases the activity of MOR by rearranging the direct interactions of TM3 and TM6, thus stabilizing TM6 in the outward-shifted position which is favorable for G-protein binding. These findings shed light on the rational developments of novel allosteric modulators that activate GPCRs further than orthosteric ligands alone and pave the way for next-generation GPCR-targeting therapeutics.

Biasing Gßγ Downstream Signaling with Gallein Inhibits Development of Morphine Tolerance and Potentiates Morphine-Induced Nociception in a Tolerant State.

Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gßγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gßγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gßγ and inhibit interactions of Gßγ with phospholipase-Cß3 (PLCß3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCß3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gßγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gßγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cß is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.

The Effects of Chronic Naltrexone on Reinstatement of Opioid-Induced Drug-Seeking Behavior and Antinociception.

Opioid addiction is a chronic relapsing disorder in which drug-seeking behavior during abstinence can be provoked by exposure to a µ-opioid receptor (MOR) agonist or opioid-associated cues. Opioid self-administration behavior in laboratory subjects can be reinstated by priming with MOR agonists or agonist-related stimuli, providing a procedure suitable for relapse-related studies. The opioid antagonist naltrexone has been forwarded as a medication that can forestall relapse and, in an extended-release formulation, has demonstrated some treatment success. However, chronic naltrexone treatment has not been extensively investigated in nonhuman subjects and aspects of its pharmacology remain uncertain. For example, the relative effectiveness of naltrexone in reducing the priming strength of opioid agonists differing in efficacy is not well understood. Here, using intravenous self-administration and warm-water tail withdrawal procedures, we investigated changes in the direct reinforcing effects of oxycodone and in the priming strength and antinociceptive effects of opioid agonists in squirrel monkeys (n = 4) during chronic treatment with naltrexone (0.2 mg/kg/d). Results show that naltrexone produced: 1) a 10-fold rightward shift in the dose-response function for the reinforcing effects of oxycodone, and 2) in reinstatement and antinociception experiments, comparable rightward shifts in the dose-response functions for higher-efficacy MOR agonists (methadone, heroin, and oxycodone) but rightward and downward shifts in the dose-response functions for lower-efficacy MOR agonists (buprenorphine, nalbuphine, and butorphanol). These results suggest that, although chronic naltrexone should be effective in forestalling relapse following exposure to lower- and higher-efficacy agonists, the inability of lower-efficacy agonists to surmount naltrexone antagonism may complicate the prescription of opioids for pain. SIGNIFICANCE STATEMENT: Although naltrexone is commonly used in the treatment of opioid use disorder, its ability to reduce the priming strength of opioid agonists has not been extensively investigated. This study shows that chronic naltrexone treatment induces rightward shifts in the reinstatement and antinociceptive properties of higher efficacy opioid agonists, but rightward and downward shifts for lower efficacy opioid agonists, suggesting lower efficacy agonists may not be able to surmount naltrexone-induced antagonism of these two effects, and perhaps naltrexone offers greater protection against lower efficacy agonists.

Epidural administration of large dose of opioid µ receptor agonist may impair cardiac functions and myocardial viability via desensitizing transient receptor potential vanilloid 1.

The incidence of postoperative myocardial injury remains high as the underlying pathogenesis is still unknown. The dorsal root ganglion (DRG) neurons express transient receptor potential vanilloid 1 (TRPV1) and its downstream effector, calcitonin gene-related peptide (CGRP) participating in transmitting pain signals and cardiac protection. Opioids remain a mainstay therapeutic option for moderate-to-severe pain relief clinically, as a critical component of multimodal postoperative analgesia via intravenous and epidural delivery. Evidence indicates the interaction of opioids and TRPV1 activities in DRG neurons. Here, we verify the potential impairment of myocardial viability by epidural usage of opioids in postoperative analgesia. We found that large dose of epidural morphine (50 µg) significantly worsened the cardiac performance (+dP/dtmax reduction by 11% and -dP/dtmax elevation by 24%, all P < 0.001), the myocardial infarct size (morphine vs Control, 0.54 ± 0.09 IS/AAR vs. 0.23 ± 0.06 IS/AAR, P < 0.001) and reduced CGRP in the myocardium (morphine vs. Control, 9.34 ± 2.24 pg/mg vs. 21.23 ± 4.32 pg/mg, P < 0.001), while induced definite suppression of nociception in the postoperative animals. It was demonstrated that activation of µ-opioid receptor (µ-OPR) induced desensitization of TRPV1 by attenuating phosphorylation of the channel in the dorsal root ganglion neurons, via inhibiting the accumulation of cAMP. CGRP may attenuated the buildup of ROS and the reduction of mitochondrial membrane potential in cardiomyocytes induced by hypoxia/reoxygenation. The findings of this study indicate that epidurally giving large dose of µ-OPR agonist may aggravate myocardial injury by inhibiting the activity of TRPV1/CGRP pathway.

IUPHAR review: Recent progress in the development of Mu opioid receptor modulators to treat opioid use disorders.

Opioid Use Disorder (OUD) can be described as intense preoccupation with using or obtaining opioids despite the negative consequences associated with their use. As the number of OUD cases in the U.S. increase, so do the number of opioid-related overdose deaths. In 2022, opioid-related overdose became the No. 1 cause of death for individuals in the U.S. between the ages of 25 and 64 years of age. Because of the introduction of highly potent synthetic opioids (e.g. fentanyl) to the illicit drug market, there is an urgent need for therapeutics that successfully reduce the number of overdoses and can help OUD patients maintain sobriety. Most abused opioids stimulate the mu-opioid receptor (MOR) and activation of this receptor can lead to positive (e.g., euphoria) consequences. However, the negative side effects of MOR stimulation can be fatal (e.g., sedation, respiratory depression). Therefore, the MOR is an attractive target for developing medications to treat OUD. Current FDA drugs include MOR agonists that aid in detoxification and relapse prevention, and MOR antagonists that also serve as maintenance therapies or reverse overdose. These medications are limited by their abuse potential, adverse effects, or pharmacological profiles which leaves ample room for research into designing new chemical entities with optimal physiological effects. These includes, orthosteric ligands that target the primary binding site of the MOR, allosteric ligands that positively, negatively, or "silently" modulate receptor function, and lastly, bitopic ligands target both the orthosteric and allosteric sites simultaneously.

Mu and delta opioid receptors diverge.

Contrary to current models, Scherrer et al. (2009) provide evidence that mu and delta opioid receptors are not expressed by the same pain-sensing neurons. In mice, agonists for these receptors produce analgesia restricted to either noxious heat or mechanical stimuli, implying that the receptors act on distinct fibers to mediate completely different types of pain relief.

Dissociation of the opioid receptor mechanisms that control mechanical and heat pain.

Delta and mu opioid receptors (DORs and MORs) are inhibitory G protein-coupled receptors that reportedly cooperatively regulate the transmission of pain messages by substance P and TRPV1-expressing pain fibers. Using a DOReGFP reporter mouse we now show that the DOR and MOR are, in fact, expressed by different subsets of primary afferents. The MOR is expressed in peptidergic pain fibers, the DOR in myelinated and nonpeptidergic afferents. Contrary to the prevailing view, we demonstrate that the DOR is trafficked to the cell surface under resting conditions, independently of substance P, and internalized following activation by DOR agonists. Finally, we show that the segregated DOR and MOR distribution is paralleled by a remarkably selective functional contribution of the two receptors to the control of mechanical and heat pain, respectively. These results demonstrate that behaviorally relevant pain modalities can be selectively regulated through the targeting of distinct subsets of primary afferent pain fibers.

Strategies for developing µ opioid receptor agonists with reduced adverse effects.

Opioids are currently the most effective and widely used painkillers in the world. Unfortunately, the clinical use of opioid analgesics is limited by serious adverse effects. Many researchers have been working on designing and optimizing structures in search of novel µ opioid receptor(MOR) agonists with improved analgesic activity and reduced incidence of adverse effects. There are many strategies to develop MOR drugs, mainly focusing on new low efficacy agonists (potentially G protein biased agonists), MOR agonists acting on different Gα subtype, targeting opioid receptors in the periphery, acting on multiple opioid receptor, and targeting allosteric sites of opioid receptors, and others. This review summarizes the design methods, clinical applications, and structure-activity relationships of small-molecule agonists for MOR based on these different design strategies, providing ideas for the development of safer novel opioid ligands with therapeutic potential.

Structure of the µ-opioid receptor-Gi protein complex.

The µ-opioid receptor (µOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by µOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the µOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the µOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR.

In vitro structure-activity relationships and forensic case series of emerging 2-benzylbenzimidazole 'nitazene' opioids.

2-Benzylbenzimidazole 'nitazene' opioids are presenting a growing threat to public health. Although various nitazenes were previously studied, systematic comparisons of the effects of different structural modifications to the 2-benzylbenzimidazole core structure on µ-opioid receptor (MOR) activity are limited. Here, we assessed in vitro structure-activity relationships of 9 previously uncharacterized nitazenes alongside known structural analogues. Specifically, we focused on MOR activation by 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl modifications), 'desnitazene' analogues (lacking the 5-nitro group), and N-desethyl analogues. The results from two in vitro MOR activation assays (ß-arrestin 2 recruitment and inhibition of cAMP accumulation) showed that 'ring' modifications overall yield highly active drugs. With the exception of 4'-OH analogues (which are metabolites), N-pyrrolidino substitutions were generally more favorable for MOR activation than N-piperidine substitutions. Furthermore, removal of the 5-nitro group on the benzimidazole ring consistently caused a pronounced decrease in potency. The N-desethyl modifications showed important MOR activity, and generally resulted in a slightly lowered potency than comparator nitazenes. Intriguingly, N-desethyl isotonitazene was the exception and was consistently more potent than isotonitazene. Complementing the in vitro findings and demonstrating the high harm potential associated with many of these compounds, we describe 85 forensic cases from North America and the United Kingdom involving etodesnitazene, N-desethyl etonitazene, N-desethyl isotonitazene, N-pyrrolidino metonitazene, and N-pyrrolidino protonitazene. The low-to-sub ng/mL blood concentrations observed in most cases underscore the drugs' high potencies. Taken together, by bridging pharmacology and case data, this study may aid to increase awareness and guide legislative and public health efforts.

G protein signaling-biased mu opioid receptor agonists that produce sustained G protein activation are noncompetitive agonists.

The ability of a ligand to preferentially promote engagement of one signaling pathway over another downstream of GPCR activation has been referred to as signaling bias, functional selectivity, and biased agonism. The presentation of ligand bias reflects selectivity between active states of the receptor, which may result in the display of preferential engagement with one signaling pathway over another. In this study, we provide evidence that the G protein-biased mu opioid receptor (MOR) agonists SR-17018 and SR-14968 stabilize the MOR in a wash-resistant yet antagonist-reversible G protein-signaling state. Furthermore, we demonstrate that these structurally related biased agonists are noncompetitive for radiolabeled MOR antagonist binding, and while they stimulate G protein signaling in mouse brains, partial agonists of this class do not compete with full agonist activation. Importantly, opioid antagonists can readily reverse their effects in vivo. Given that chronic treatment with SR-17018 does not lead to tolerance in several mouse pain models, this feature may be desirable for the development of long-lasting opioid analgesics that remain sensitive to antagonist reversal of respiratory suppression.

Structure-Activity Relationship Study of CYM51010, an agonist for the µ-δ Opioid Receptor Heterodimer.

Although opioid analgesics are indispensable in treating pain, these drugs are accompanied by life-threatening side effects. While clinically relevant opioid drugs target the µ opioid receptor (MOR), a heterodimer between the MOR and the δ opioid receptor (DOR) has emerged as another target to develop safer analgesics. Although some heterodimer-preferring agonists have been reported so far, it is still difficult to activate the MOR/DOR heterodimer selectively in the presence of MOR or DOR monomers/homodimers. To gain insights to develop selective agonists for MOR/DOR, herein we prepared analogs of CYM51010, one of the reported heterodimer-preferring agonists, and collected structure-activity relationship information. We found that the ethoxycarbonyl group was needed for the activity for the heterodimer, although this group could be substituted with functional groups with similar sizes, such as an ethoxycarbonyl group. As for the acetylaminophenyl group, not a type of substituent, but rather a substituent located at a specific position (para-position) was essential for the activity. Changing the linker length between the acetylaminophenyl group and the piperidine moiety also had deleterious effects on the activity. On the other hand, the substitution of the acetylamino group with a trifluoroacetylamino group and the substitution of the phenethyl group with a benzyl group diminished the activities for the monomers/homodimers while keeping the activity for MOR/DOR, which enhanced the selectivity. Our findings herein will play an important role in developing selective agonists for MOR/DOR and for elucidating the physiological roles of this heterodimer in analgesic processes and in the establishment of side effects.

CBD Versus CBDP: Comparing In Vitro Receptor-Binding Activities.

Phytocannabinoids with seven-carbon alkyl chains (phorols) have gained a lot of attention, as they are commonly believed to be more potent versions of typical cannabinoids with shorter alkyl chains. At the time of this article, cannabidiphorol (CBDP) and tetrahydrocannabiphorol (THCP) can both be purchased in the North American market, even though their biological activities are nearly unknown. To investigate their relative potency, we conducted in vitro receptor-binding experiments with CBDP (cannabinoid CB1/CB2 receptor antagonism, serotonin 5HT-1A agonism, dopamine D2S (short form) agonism, and mu-opioid negative allosteric modulation) and compared the observed activity with that of CBD. To our knowledge, this is the first publication to investigate CBDP's receptor activity in vitro. A similar activity profile was observed for both CBD and CBDP, with the only notable difference at the CB2 receptor. Contrary to common expectations, CBD was found to be a slightly more potent CB2 antagonist than CBDP (p < 0.05). At the highest tested concentration, CBD demonstrated antagonist activity with a 33% maximum response of SR144528 (selective CB2 antagonist/inverse agonist). CBDP at the same concentration produced a weaker antagonist activity. A radioligand binding assay revealed that among cannabinoid and serotonin receptors, CB2 is likely the main biological target of CBDP. However, both CBD and CBDP were found to be significantly less potent than SR144528. The interaction of CBDP with the mu-opioid receptor (MOR) produced unexpected results. Although the cannabidiol family is considered to be a set of negative allosteric modulators (NAMs) of opioid receptors, we observed a significant increase in met-enkephalin-induced mu-opioid internalization when cells were incubated with 3 µM of CBDP and 1 µM met-enkephalin, a type of activity expected from positive allosteric modulators (PAMs). To provide a structural explanation for the observed PAM effect, we conducted molecular docking simulations. These simulations revealed the co-binding potential of CBDP (or CBD) and met-enkephalin to the MOR.