Purification and mass spectroscopic analysis of human CB1 cannabinoid receptor functionally expressed using the baculovirus system

Abstract: The cannabinoid receptor 1 (CB1) cannabinoid receptor is an essential component of the cannabinergic system. It has been recognized as a therapeutic target for treating numerous diseases and is currently receiving considerable attention by the pharmaceutical community. Target‐based drug design, utilizing three‐dimensional information of receptor structure and ligand‐binding motifs, requires significant amounts of purified protein. To facilitate the purification of CB1, we have expressed the receptor fused to various epitope tags using the baculovirus expression system. In addition, expression levels and ligand‐binding profiles corresponding to the expressed fusion proteins have been compared. C‐terminal histidine (His)‐tagged CB1 gave a B_max higher than most other systems previously reported in the literature, and was selected for subsequent metal affinity chromatography purification and mass spectroscopic (MS) analysis. Moreover, cells expressing C‐terminal His‐tagged CB1 were shown to inhibit forskolin‐stimulated cyclic adenosine 3′,5′‐monophosphate (cAMP) production in a concentration‐dependent manner in the presence of CP‐55,940, confirming the expressed receptor's functional characteristics. A Western blot analysis of the purified receptor showed several forms of CB1, the most abundant being a 57 kDa monomeric protein. The purified CB1 preparations were subjected to protein digestion followed by MS. Fragments corresponding to >70% of the receptor were identified by this method, confirming the identity and purity of the expressed protein. The work presented here demonstrates that epitope‐tagged CB1 can be expressed in sufficient amounts and purified to homogeneity for MS analysis. Moreover, these results will serve as a basis for future experiments aimed at characterizing the ligand‐binding domains using covalently reacting receptor probes.     

Ligand based structural studies of the CB1 cannabinoid receptor

Abstract: The structural characterization of G‐protein coupled receptors (GPCRs) is quite important as these proteins represent a vast number of therapeutic targets involved in drug discovery. However, solving the three‐dimensional structure of GPCR has been a significant obstacle in structural biology. A variety of reasons, including their large molecular weight, intricate interhelical packing, as well as their membrane‐associated topology, has hindered efforts aimed at their purification. In the absence of pure protein, available in the native conformation, classical methods of structural analysis such as X‐ray crystallography and nuclear magnetic resonance spectroscopy cannot be utilized successfully. Alternative methods must therefore be explored to facilitate the structural features involved in drug–receptor interactions. The methods described herein detail the use of covalent probes, or affinity labels, capable of binding covalently to a target GPCR at its binding site(s). Our approach involves the incorporation of a number of reactive moieties in different regions of the ligand molecule each of which is expected to react with different amino acid residues. Information obtained from such work coupled with computer modeling and validated by the use of site‐directed mutagenesis of GPCRs allows for three‐dimensional mapping of the receptor binding site. It also sheds light on the different possible binding motifs for the various classes of agonists and antagonists and identifies amino acid residues involved with GPCR activation or inactivation.     

Targeting the cannabinoid CB2 receptor: modelling and structural determinants of CB2 selective ligands

Recent developments indicate that CB2 receptor ligands have the potential to become therapeutically important. To explore this potential, it is necessary to develop compounds with high affinity for the CB2 receptor and little affinity for the CB1 receptor. This review will discuss structure-activity relations at both receptors for classical cannabinoids and cannabimimetic indoles. Examples of CB2 selective ligands from both classes of compounds are presented and the structural features leading to selectivity are described. Two approaches, receptor mutations and molecular modelling, have been employed to investigate the interaction of ligands with both cannabinoid receptors. These results obtained from these techniques are discussed.     

Elevated cannabinoid receptor 1 and G protein‐coupled receptor 55 expression in proximal tubule cells and whole kidney exposed to diabetic conditions

Hyperglycaemia increases the risk of developing diabetic nephropathy, with primary targets in the glomerulus and proximal tubule. Importantly, glomerular damage in the kidney leads to elevated albumin levels in the filtrate, which contributes to tubular structural modifications that lead to dysfunction. Diabetes alters the endocannabinoid system in a number of target organs, with previous research characterizing tissue‐specific changes in the expression of the cannabinoid receptor 1 (CB₁) and G protein‐coupled receptor 55 (GPR55), a putative cannabinoid receptor, in diabetes. Although these receptors have a functional role in the cannabinoid system in the kidney, there has been little investigation into changes in the expression of CB₁ and GPR55 in the proximal tubule under diabetic conditions. In this study, CB₁ and GPR55 messenger RNA and protein levels were quantified in cultured human kidney cells and then treated with either elevated glucose, elevated albumin, or a combination of glucose and albumin for 4, 6, 18, or 24 h. In addition, CB₁ and GPR55 protein expression was characterized in whole‐kidney lysate from streptozotocin‐induced diabetic Sprague‐Dawley rats. In vitro exposure to elevated glucose and albumin increased CB₁ and GPR55 messenger RNA and protein expression in proximal tubule cells in a time‐dependant manner. In whole kidney of streptozotocin‐induced diabetic rats, CB₁ protein was upregulated, whereas GPR55 protein concentration was not altered. Thus, expression of CB₁ and GPR55 in proximal tubules is altered in response to elevated levels of glucose and albumin. Further investigations should determine if these receptors are effective physiological targets for the treatment and prevention of diabetic nephropathy.     

Design,expression, and characterization of a synthetic human cannabinoid receptor and cannabinoid receptor/ G‐protein fusion protein

Abstract: We report here the synthesis and characterization of two gene constructs designed to facilitate structure/function studies of the human neuronal cannabinoid receptor, CB1. The first gene, which we call shCB1, is a synthetic gene containing unique restriction sites spaced roughly 50–100 bases apart to facilitate rapid mutagenesis and cloning. A nine amino acid epitope tag (from the rhodopsin C‐terminus) is also present in the shCB1 C‐terminal tail to enable detection and purification using the monoclonal antibody 1D4. We find that that the shCB1 gene can be transiently expressed in COS cells with yield of ～ 10–15 µg receptor per 15 cm plate and is wild type like in its ability to bind cannabinoid ligands. Our confocal microscopy studies indicate shCB1 targets to the membrane of HEK293 cells and is internalized in response to agonist. To facilitate functional studies, we also made a chimera in which the C‐terminus of shCB1 was fused with the N‐terminus of a G‐protein alpha subunit, Gαi. The shCB1/Gαi chimera shows agonist stimulated GTPγS binding, and thus provides a simplified way to measure agonist induced CB1 activation. Taken together, the shCB1 and shCB1/Gαi gene constructs provide useful tools for biochemical and biophysical examinations of CB1 structure, activation and attenuation.     

Biosynthesis and purification of a hydrophobic peptide from transmembrane domains of G‐protein‐coupled CB2 receptor

Abstract: A major challenge for the structural study of the seven‐transmembrane G‐protein‐coupled receptors is to obtain a sufficient amount of purified protein at the milligram level, which is required for either nuclear magnetic resonance (NMR) spectroscopy or X‐ray crystallography. In order to develop a high‐yield and cost‐effective method, and also to obtain preliminary structural information for the computer modeling of the three‐dimensional receptor structural model, a highly hydrophobic peptide from human cannabinoid subtype 2 receptor CB2_65?101, was chosen to develop high‐yield membrane protein expression and purification methods. The peptide included the second transmembrane helix with the associated loop regions of the CB2 receptor. It was over‐expressed in Escherichia coli, with a modified TrpΔ LE1413 (TrpLE) leading fusion sequence and a nine‐histidine tag, and was then separated and purified from the tag in a preparative scale. An experimental protocol for the chemical cleavage of membrane protein fragment was developed using cyanogen bromide to remove the TrpLE tag from the hydrophobic fusion protein. In addition, protein uniformly labeled with isotopic ¹⁵N was obtained by expression in ¹⁵N‐enriched minimum media. The developed and optimized preparation scheme of expression, cleavage, and purification provided a sufficient amount of peptide for NMR structure analysis and other biophysical studies that will be reported elsewhere. The process of fusion protein cleavage following purification was monitored by high‐performance liquid chromatography (HPLC) and mass spectrometry (MS), and the final sample was validated by MS and circular dichroism experiments.     

Biology and therapeutic potential of cannabinoid CB2 receptor inverse agonists

Evidence has emerged suggesting a role for the cannabinoid CB2 receptor in immune cell motility. This provides a rationale for a novel and generalized immunoregulatory role for cannabinoid CB2 receptor-specific compounds. In support of this possibility, we will review the biology of a class of cannabinoid CB2 receptor-specific inverse agonist, the triaryl bis-sulfones. We will show that one candidate, Sch.414319, is potent and selective for the cannabinoid CB2 receptor, based on profiling studies using biochemical assays for 45 enzymes and 80 G-protein coupled receptors and ion channels. We will describe initial mechanistic studies using this optimized triaryl bis-sulfone, showing that the compound exerts a broad effect on cellular protein phosphorylations in human monocytes. This profile includes the down regulation of a required phosphorylation of the monocyte-specific actin bundling protein L-plastin. We suggest that this observation may provide a mechanism for the observed activity of Sch.414319 in vivo. Our continued analysis of the in vivo efficacy of this compound in diverse disease models shows that Sch.414319 is a potent modulator of immune cell mobility in vivo, can modulate bone damage in antigen-induced mono-articular arthritis in the rat, and is uniquely potent at blocking experimental autoimmune encephalomyelitis in the rat.     

Recent advances in cannabinoid receptor agonists and antagonists

This review is an overview of the recent advances in cannabinoid chemistry with a special emphasis on the patent literature. The term cannabinoid includes analogues of the natural components of cannabis, endocannabinoids and a wide array of chemical structures such as 1,5-diarylpyrazoles, indoles, quinolines and arylsulphonamide derivatives capable of acting as cannabinoid receptor agonists and antagonists. These receptors, discovered in the early nineties, seem to be involved in different biochemical processes and thus represent interesting therapeutic targets for drug research.     

CB2 cannabinoid receptors: new vistas

Over the years CB₂ cannabinoid receptors have received much less attention than CB₁ receptors, the latter mediating most of the psychoactive effects of cannabis. Primarily this was due to difficulties in assigning a physiological function to CB₂ receptors. In recent years this situation has changed, and CB₂ receptors have been implicated in processes as diverse as analgesia, hepatic fibrosis, bone growth, and atherosclerosis. This special issue of the British Journal of Pharmacology addresses these and other topics in CB₂ receptor research by compiling a series of reviews and primary research papers stemming from a meeting ‘CB2₂ cannabinoid receptors: New vistas'' that was held in Banff, Canada, from May 31 to June 3, 2007.     

Evidence for both inverse agonism at the cannabinoid CB1 receptor and the lack of an endogenous cannabinoid tone in the rat and guinea-pig isolated ileum myenteric plexus-longitudinal muscle preparation

Background and purpose:

Cannabinoid receptor agonists reduce intestinal propulsion in rodents through the CB₁ receptor. In addition to its antagonistic activity at this receptor, rimonabant (N-(piperidino)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxyamide) alone augments intestinal transit. Using rat and guinea-pig ileum MPLM (myenteric plexus-longitudinal muscle) preparations, we investigated whether the latter effect was through inverse agonism or antagonism of endocannabinoid agonist(s).

Experimental approach:

Inverse agonism was investigated by comparing the maximal enhancement of electrically evoked contractions of the MPLM by two CB₁ receptor antagonists, AM 251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and O-2050 [(6aR,10aR)-3-(1-methanesulphonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6-H-dibenzo[b,d]pyran], with that produced by rimonabant. To reveal ongoing endocannabinoid activity, effects of inhibiting endocannabinoid hydrolysis by fatty acid amide hydrolase (FAAH) using AA-5HT (arachidonyl-5-hydroxytryptamine), PMSF (phenylmethylsulphonyl fluoride) or URB-597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate), or putative uptake using VDM-11 [(5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] was evaluated.

Key results:

The presence of CB₁ receptors was revealed by antagonism of exogenous anandamide, arachidonylethanolamide (AEA) and WIN 55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate] by rimonabant. The rank order of potentiation of contractions was AM 251 > rimonabant > O-2050. Neither the FAAH inhibitors nor VDM-11 affected electrically evoked contractions. Each FAAH inhibitor increased the potency of AEA but not WIN 55,212-2. VDM-11 did not alter the inhibitory effect of AEA.

Conclusions and implications:

The different levels of maximal potentiation of contractions by the CB₁ receptor antagonists suggest inverse agonism. The potentiation of the action of AEA by the FAAH inhibitors showed that FAAH was present. The lack of effect of FAAH inhibitors and VDM-11 alone on electrically evoked contractions, and on the potency of exogenous AEA suggests that pharmacologically active endocannabinoids were not released and the endocannabinoid transporter was absent. Thus, the CB₁ receptor antagonists behave as inverse agonists.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

Ligand Binding Sensitivity of the Extracellular Loop Two of the Cannabinoid Receptor 1

The cannabinoid receptor one (CB1) is a class A G-protein-coupled receptor thought to bind ligands primarily within its helical bundle. Evidence suggests, however, that the extracellular domain may also play a role. We have previously shown that the C-terminus of the extracellular loop 2 of CB1 is important in binding some compounds; receptors with mutations in this region (F268W, P269A, H270A, and I271A) bound some agonists with severely reduced affinity relative to the wild-type receptor. In the present work, we examine the impact of these mutations on binding a chemically diverse set of ligands. The receptors, F268W and I271A, exhibited a greater sensitivity to binding the inverse agonists/antagonists SLV319, AVE1625, NESS0327 relative to P269A and H270A, suggesting that the Pro and His are not involved in binding those compounds. In contrast, binding of the agonists, BAY593074 and WIN55212-2, was diminished in all four receptors, suggesting the conformational unit contributed by all four residues is important. A more marked loss in binding was observed for agonists of the nonclassical (CP55940) and classical (HU-210, JWH061, JWH179) cannabinoid classes and for a silent antagonist derivative (O-2050), pointing to the critical nature of this region for binding both the bicyclic/tricyclic core and the alkyl chain of these derivatives. However, moving the location of the alkyl chain on a series of pyrazole analogues shows it can be better accommodated in certain locations (O-1255) than others (O-1302, O-1690) and underscores the involvement of residues F268 and I271.     

Synthesis and in vitro evaluation of novel 2-oxo-1,2-dihydroquinoline CB2 receptor inverse agonists

Aliphatic amides of 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid 6 were synthesized and evaluated for their CB2 and CB1 receptor activities by a [35S]GTPgammaS membrane-binding assay. Compounds 7-18 produced CB2 inverse agonist activities with a variety of potencies (IC50: 1 nm-1 microm). By contrast, no considerable antagonism of CB1 receptor was observed by 1-10 microm concentrations indicating that synthesized compounds were CB2 receptor selective.     

稳定表达大麻受体2的CHO细胞株的构建

目的建立稳定高效表达人重组大麻受体2(CB2)的中国仓鼠卵巢(CHO-K1)细胞株,为体外高通量筛选CB2激动剂和拮抗剂奠定基础。方法通过脂质体介导的方法将构建的表达载体pcDNA3.1(+)-CB2转染入CHO-K1细胞中,然后用含G418的选择性培养液进行筛选,挑取耐药克隆;培养并收集耐药克隆细胞,用RT-PCR方法做进一步筛选;序列测定鉴定整合基因的序列;筛选的阳性克隆用放射性配体-受体结合实验进行进一步的鉴定和受体活性分析。结果转染细胞在含G418的选择性培养基中生长出28个耐药单克隆,用RT-PCR方法检测出17个CB2mRNA表达量较高的阳性克隆;RT-PCR扩增片段测定鉴定正确;挑选其中最优的克隆进行放射性配体-受体结合实验,结果显示,表达受体具有与CB2激动剂WIN55212-2特异结合的活性,其Kd和Bmax值分别(1.21±0.47)nmol.L-1和(3.12±0.7)nmol.g-1蛋白,这一结果与天然CB2的特性相似。结论建立了稳定高效表达人重组CB2的CHO-K1细胞株。     

内源性大麻素系统与老年性痴呆

老年性痴呆即阿尔茨海默病（Alzheimer’Sdisease,AD）是一种慢性进行性的神经退行性病变,其特征性病理改变为β-淀粉样蛋白（β-amy-loidpeptide,Aβ）沉积形成的细胞外老年斑和tau蛋白过度磷酸化形成的神经细胞内神经原纤维缠结,以及神经元丢失伴随胶质细胞增生等。最近的研究发现内源性大麻素系统对AD的病程有预防治疗作用,主要基于其对神经的保护作用和抗炎功效。本文综述了内源性大麻素对AD的保护作用的几个方面,旨在为AD的治疗开辟新的策略和思路。     

The impact of cannabinoid type 2 receptors (CB2Rs) in neuroprotection against neurological disorders

Cannabinoids have long been used for their psychotropic and possible medical properties of symptom relief. In the past few years, a vast literature shows that cannabinoids are neuroprotective under different pathological situations. Most of the effects of cannabinoids are mediated by the well-characterized cannabinoid receptors, the cannabinoid type 1 receptor (CB1R) and cannabinoid type 2 receptor (CB2R). Even though CB1Rs are highly expressed in the central nervous system (CNS), the adverse central side effects and the development of tolerance resulting from CB1R activation may ultimately limit the clinical utility of CB1R agonists. In contrast to the ubiquitous presence of CB1Rs, CB2Rs are less commonly expressed in the healthy CNS but highly upregulated in glial cells under neuropathological conditions. Experimental studies have provided robust evidence that CB2Rs seem to be involved in the modulation of different neurological disorders. In this paper, we summarize the current knowledge regarding the protective effects of CB2R activation against the development of neurological diseases and provide a perspective on the future of this field. A better understanding of the fundamental pharmacology of CB2R activation is essential for the development of clinical applications and the design of novel therapeutic strategies.     

Direct suppression of autoreactive lymphocytes in the central nervous system via the CB2 receptor

The cannabinoid system is now recognized as a regulator of both the nervous and immune systems. Although marijuana has been used for centuries for the treatment of a variety of disorders, its therapeutic mechanisms are only now being understood. The best-studied plant cannabinoid, delta9-tetrahydrocannabinol (THC), produced by Cannabis sativa and found in marijuana, has shown evidence of being immunosuppressive in both in vivo and in vitro. Since THC binds to at least two receptors that are differentially expressed by the immune and nervous systems, it has not been possible to clearly discriminate the biological effects it exerts in the two systems. In addition, endogenous cannabinoids have also been described that bind to both receptors and exert both neuronal and immune modulatory activity. The generation of mice deficient in specific cannabinoid receptors has facilitated studies to discriminate cannabinoid-specific functions. This review focuses on the function of the cannabinoid receptor 2 (CB2), primarily expressed in the immune system, in regulating T cell effector functions associated with autoimmune inflammation in the central nervous system (CNS).     

Characterization of cannabinoid receptor ligands in tissues natively expressing cannabinoid CB2 receptors

Background and Purpose

Although cannabinoid CB₂ receptor ligands have been widely characterized in recombinant systems in vitro, little pharmacological characterization has been performed in tissues natively expressing CB₂ receptors. The aim of this study was to compare the pharmacology of CB₂ receptor ligands in tissue natively expressing CB₂ receptors (human, rat and mouse spleen) and hCB₂-transfected CHO cells.

Experimental Approach

We tested the ability of well-known cannabinoid CB₂ receptor ligands to stimulate or inhibit [³⁵S]GTPγS binding to mouse, rat and human spleen membranes and to hCB₂-transfected CHO cell membranes. cAMP assays were also performed in hCB₂-CHO cells.

Key Results

The data presented demonstrate that: (i) CP 55,940, WIN 55,212-2 and JWH 133 behave as CB₂ receptor full agonists both in spleen and hCB₂-CHO cells, in both [³⁵S]GTPγS and cAMP assays; (ii) JWH 015 behaves as a low-efficacy agonist in spleen as well as in hCB₂-CHO cells when tested in the [³⁵S]GTPγS assay, while it displays full agonism when tested in the cAMP assay using hCB₂-CHO cells; (iii) (R)-AM 1241 and GW 405833 behave as agonists in the [³⁵S]GTPγS assay using spleen, instead it behaves as a low-efficacy inverse agonist in hCB₂-CHO cells; and (iv) SR 144528, AM 630 and JTE 907 behave as CB₂ receptor inverse agonists in all the tissues.

Conclusion and Implications

Our results demonstrate that CB₂ receptor ligands can display differential pharmacology when assays are conducted in tissues that natively express CB₂ receptors and imply that conclusions from recombinant CB₂ receptors should be treated with caution.     

Real-time characterization of cannabinoid receptor 1 (CB1) allosteric modulators reveals novel mechanism of action

BACKGROUND AND PURPOSE

The cannabinoid receptor type 1 (CB₁) has an allosteric binding site. The drugs {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593"}}ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB₁, we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators.

EXPERIMENTAL APPROACH

Studies of CB₁ signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB₁. We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB₁ internalization.

KEY RESULTS

{"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593"}}ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB₁-mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization.

CONCLUSIONS AND IMPLICATIONS

Contrary to previous studies characterizing allosteric modulators at CB_1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.     

Positive allosteric modulation of the human cannabinoid (CB1) receptor by RTI-371, a selective inhibitor of the dopamine transporter

Background and purpose

In our search for an indirect dopamine agonist as therapy for cocaine addiction, several selective inhibitors of the dopamine transporter (DAT), which are 3-phenyltropane analogues, were assayed for their effect on locomotor activity in mice. Interestingly, several of the compounds showed a poor correlation between stimulation of locomotion and DAT inhibition. One of the compounds, 3β-(4-methylphenyl)-2β-[3-(4-chlorophenyl)isoxazol-5-yl]tropane (RTI-371), was shown to cross the blood-brain barrier, by binding studies in vivo, and block cocaine-induced locomotor stimulation. As poor pharmacokinetics could not explain the behavioural effects of RTI-371, this compound was screened through our functional assays for activity at other CNS receptors. Initial screening identified RTI-371 as a positive allosteric modulator of the human CB₁ (hCB₁) receptor.

Experimental approach

The effect of RTI-371 and other DAT-selective inhibitors on CP55940-stimulated calcium mobilization was characterized in a calcium mobilization-based functional assay for the hCB₁ receptor. Selected compounds were also characterized in a similar assay for human µ opioid receptor activation to assess the specificity of their effects.

Key results

RTI-371 and several other DAT-selective inhibitors with atypical actions on locomotor behaviour increased the efficacy of CP55940 in a concentration-dependent manner.

Conclusions and implications

These results suggest that the lack of correlation between the DAT-binding affinity and locomotor stimulation of RTI-371 could be due at least in part to its activity as a positive modulator of the hCB₁ receptor.