Low-efficacy Agonist Research Articles

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.

Interaction between buprenorphine and norbuprenorphine in neonatal opioid withdrawal syndrome

Buprenorphine (BUP) is the preferred treatment for opioid use disorder during pregnancy but can cause neonatal opioid withdrawal syndrome (NOWS). Norbuprenorphine (NorBUP), an active metabolite of BUP, is implicated in BUP-associated NOWS. We hypothesized that BUP, a low-efficacy agonist of mu opioid receptors, will not antagonize NorBUP, a high-efficacy agonist of mu opioid receptors, in producing NOWS. To test this hypothesis, we treated pregnant Long-Evans rats with BUP (0, 0.01, 0.1 or 1mg/kg/day) ± NorBUP (1mg/kg/day) from gestation day 9 until pup delivery, and tested pups for opioid dependence using our established NOWS model. We used LC-MS-MS to quantify brain concentrations of BUP, NorBUP, and their glucuronide conjugates. BUP had little effect on NorBUP-induced NOWS, with the exception of 1mg/kg/day BUP significantly increasing NorBUP-induced NOWS by 58% in females. BUP and NorBUP brain concentrations predicted NOWS in multiple linear regression models. Interestingly, NorBUP contributed more to NOWS in females (βNorBUP = 51.34, p = 0.0001) than in males (βNorBUP = 19.21, P = 0.093), while BUP was similar for females (βBUP = 10.62, P = 0.0017) and males (βBUP = 11.38, P = 0.009). We are the first to report that NorBUP induces NOWS in the presence of BUP and it is more influential in females than males in the contribution of NorBUP to BUP-associated NOWS. These findings suggest that females are more susceptible to NorBUP-induced NOWS, and that treatment strategies that reduce prenatal NorBUP exposure may be more effective for females than males.

OREX-1038: a potential new treatment for pain with low abuse liability and limited adverse effects.

Drugs targeting mu opioid receptors are the mainstay of clinical practice for treating moderate-to-severe pain. While they can offer excellent analgesia, their use can be limited by adverse effects, including constipation, respiratory depression, tolerance, and abuse liability. Multifunctional ligands acting at mu opioid and nociceptin/orphanin FQ peptide receptors might provide antinociception with substantially improved adverse-effect profiles. This study explored one of these ligands, OREX-1038 (BU10038), in several assays in rodents and nonhuman primates. Binding and functional studies confirmed OREX-1038 to be a low-efficacy agonist at mu opioid and nociceptin/orphanin FQ peptide receptors and an antagonist at delta and kappa opioid receptors with selectivity for opioid receptors over other proteins. OREX-1038 had long-acting antinociceptive effects in postsurgical and complete Freund's adjuvant (CFA)-induced thermal hyperalgesia assays in rats and a warm water tail-withdrawal assay in monkeys. OREX-1038 was active for at least 24 h in each antinociception assay, and its effects in monkeys did not diminish over 22 days of daily administration. This activity was coupled with limited effects on physiological signs (arterial pressure, heart rate, and body temperature) and no evidence of withdrawal after administration of naltrexone or discontinuation of treatment in monkeys receiving OREX-1038 daily. Over a range of doses, OREX-1038 was only transiently self-administered, which diminished rapidly to nonsignificant levels; overall, both OREX-1038 and buprenorphine maintained less responding than remifentanil. These results support the concept of dual mu and nociceptin/orphanin FQ peptide receptor partial agonists having improved pharmacological profiles compared with opioids currently used to treat pain.

In vitro and in vivo pharmacology of kratom.

Kratom products have been historically and anecdotally used in south Asian countries for centuries to manage pain and opioid withdrawal. The use of kratom products has dramatically increased in the United States. More than 45 kratom alkaloids have been isolated, yet the overall pharmacology of the individual alkaloids is still not well characterized. The purpose of this chapter is to summarize in vitro and in vivo opioid activities of the primary kratom alkaloid mitragynine and its more potent metabolite 7-hydroxymitragynine. Following are experimental procedures described to characterize opioid receptor activity; receptor binding and functional assays, antinociceptive assays, operant conditioning assays, and respiratory plethysmography. The capacity of kratom alkaloids to confer tolerance and physical dependence as well as their pharmacokinetic properties are also summarized. The data reviewed here suggest that kratom products and mitragynine possess low efficacy agonist activity at the mu-opioid receptor in vivo. In addition, kratom products and mitragynine have been demonstrated to antagonize the effects of high efficacy mu-opioid agonists. The data further suggest that 7-hydroxymitragynine formed in vivo by metabolism of mitragynine may be minimally involved in the overall behavioral profile of mitragynine and kratom, whereas 7-hydroxymitragynine itself, at sufficiently high doses administered exogenously, shares many of the same abuse- and dependence-related behavioral effects associated with traditional opioid agonists. The apparent low efficacy of kratom products and mitragynine at mu-opioid receptors supports the development of these ligands as effective and potentially safe medications for opioid use disorder.

Effects of Dopamine D1-Like Receptor Ligands on Food-Cocaine Choice in Socially Housed Male Cynomolgus Monkeys

Although dopamine plays a prominent role in mediating cocaine's abuse-related effects, the specific roles of dopamine receptor subtypes are not fully understood. Whereas the effects of drugs acting at dopamine D2-like receptors (D2Rs) have been characterized, less is known about dopamine D1-like receptors (D1Rs). The present experiments examined the effects of drugs with varying intrinsic efficacy at D1R on the relative reinforcing strength of cocaine in male cynomolgus monkeys. Use of socially housed monkeys permitted the assessment of whether social status influenced the behavioral effects of D1R-acting drugs. The high-efficacy D1R agonist SKF 81297, low-efficacy D1R agonist SKF 38393, and D1R antagonist SCH 23390 were administered acutely to monkeys self-administering cocaine under a food-cocaine choice procedure in which a cocaine-choice dose-effect curve was determined daily. To assess selectivity of behavioral effects on cocaine choice, effects of doses that did not disrupt responding (indicated by a ≥35% decrease in total reinforcers delivered) were analyzed. Neither SKF 81297 nor SCH 23390 affected cocaine choice in dominant or subordinate monkeys. However, the low-efficacy agonist SKF 38393 selectively decreased cocaine choice; this effect was larger and only reached statistical significance in subordinate monkeys. Increasing the time between D1-acting drug administration and the cocaine choice session did not affect these results. The results indicate that, like D2R-acting drugs, the behavioral effects of D1R-acting drugs on cocaine choice can vary according to intrinsic efficacy and social status. Moreover, they demonstrate that D1R-acting drugs affect behavior under a narrower range of conditions than D2R-acting drugs. SIGNIFICANCE STATEMENT: Cocaine use disorder represents an insidious public health concern with no Food and Drug Administration-approved medications. Although dopamine receptors have been strongly implicated in mediating the abuse-related effects of cocaine, the roles of dopamine receptor subtypes are incompletely understood. The present study in nonhuman primates found that cocaine choice was decreased only by a low-efficacy D1R agonist, and that this effect depended on the social status of the monkey.

Site selective C\u2013H functionalization of Mitragyna alkaloids reveals a molecular switch for tuning opioid receptor signaling efficacy

Mitragynine (MG) is the most abundant alkaloid component of the psychoactive plant material “kratom”, which according to numerous anecdotal reports shows efficacy in self-medication for pain syndromes, depression, anxiety, and substance use disorders. We have developed a synthetic method for selective functionalization of the unexplored C11 position of the MG scaffold (C6 position in indole numbering) via the use of an indole-ethylene glycol adduct and subsequent iridium-catalyzed borylation. Through this work we discover that C11 represents a key locant for fine-tuning opioid receptor signaling efficacy. 7-Hydroxymitragynine (7OH), the parent compound with low efficacy on par with buprenorphine, is transformed to an even lower efficacy agonist by introducing a fluorine substituent in this position (11-F-7OH), as demonstrated in vitro at both mouse and human mu opioid receptors (mMOR/hMOR) and in vivo in mouse analgesia tests. Low efficacy opioid agonists are of high interest as candidates for generating safer opioid medications with mitigated adverse effects.

Exploring Stereochemical and Conformational Requirements at Cannabinoid Receptors for Synthetic Cannabinoids Related to SDB-006, 5F-SDB-006, CUMYL-PICA, and 5F-CUMYL-PICA.

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 (Ki = 47.9-813 nM) and CB2 (Ki = 47.9-347 nM) that were intermediate to that of the corresponding benzylic (10-13, CB1 Ki = 550 nM to >10 μM; CB2 Ki = 61.7 nM to >10 μM) and cumyl derivatives (6-9, CB1 Ki = 12.6-21.4 nM; CB2 Ki = 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; Emax = 89-104%) and low efficacy agonists of CB2 (EC50 = 54-500 nM; Emax = 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.

D1 receptors in the anterior cingulate cortex modulate basal mechanical sensitivity threshold and glutamatergic synaptic transmission

The release of dopamine (DA) into target brain areas is considered an essential event for the modulation of many physiological effects. While the anterior cingulate cortex (ACC) has been implicated in pain related behavioral processes, DA modulation of synaptic transmission within the ACC and pain related phenotypes remains unclear. Here we characterized a Crispr/Cas9 mediated somatic knockout of the D1 receptor (D1R) in all neuronal subtypes of the ACC and find reduced mechanical thresholds, without affecting locomotion and anxiety. Further, the D1R high-efficacy agonist SKF 81297 and low efficacy agonist (±)-SKF-38393 inhibit α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) currents in the ACC. Paradoxically, the D1R antagonists SCH-23390 and SCH 33961 when co-applied with D1R agonists produced a robust short-term synergistic depression of AMPAR currents in the ACC, demonstrating an overall inhibitory role for D1R ligands. Overall, our data indicate that absence of D1Rs in the ACC enhanced peripheral sensitivity to mechanical stimuli and D1R activation decreased glutamatergic synaptic transmission in ACC neurons.

HIV Tat Protein Selectively Impairs CB1 Receptor-Mediated Presynaptic Inhibition at Excitatory But Not Inhibitory Synapses.

Despite the success of antiretroviral therapy in suppressing viral load, nearly half of the 37 million people infected with HIV experience cognitive and motor impairments, collectively classified as HIV-associated neurocognitive disorders (HAND). In the CNS, HIV-infected microglia release neurotoxic agents that act indirectly to elicit excitotoxic synaptic injury. HIV trans-activator of transcription (Tat) protein is one such neurotoxin that is thought to play a major role in the neuropathogenesis of HAND. The endocannabinoid (eCB) system provides on-demand neuroprotection against excitotoxicity, and exogenous cannabinoids attenuate neurotoxicity in animal models of HAND. Whether this neuroprotective system is altered in the presence of HIV is unknown. Here, we examined the effects of Tat on the eCB system in rat primary hippocampal cultures. Using whole-cell patch-clamp electrophysiology, we measured changes in retrograde eCB signaling following exposure to Tat. Treatment with Tat significantly reduced the magnitude of depolarization-induced suppression of excitation (DSE) in a graded manner over the course of 48 h. Interestingly, Tat did not alter this form of short-term synaptic plasticity at inhibitory terminals. The Tat-induced decrease in eCB signaling resulted from impaired CB1 receptor (CB1R)-mediated presynaptic inhibition of glutamate release. This novel loss-of-function was particularly dramatic for low-efficacy agonists such as the eCB 2-arachidonoylglycerol (2-AG) and Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive ingredient in marijuana. Our observation that HIV Tat decreases CB1R function in vitro suggests that eCB-mediated neuroprotection may be reduced in vivo; this effect of Tat may contribute to synaptodendritic injury in HAND.

Neurochemical pharmacology of psychoactive substituted N-benzylphenethylamines: High potency agonists at 5-HT2A receptors

The use of new psychoactive substituted 2,5-dimethoxy-N-benzylphenethylamines is associated with abuse and toxicity in the United States and elsewhere and their pharmacology is not well known. This study compares the mechanisms of action of 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe), 2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25E-NBOMe), 2-(2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25H-NBOMe), 2-(((4-iodo-2,5-dimethoxyphenethyl)amino)methyl)phenol (25I-NBOH); and 2-(2,5-dimethoxy-4-nitrophenyl)-N-(2-methoxybenzyl)ethanamine) (25N-NBOMe) with hallucinogens and stimulants. Mammalian cells heterologously expressing 5-HT1A, 5-HT2A, 5-HT2B or 5-HT2C receptors, or dopamine, serotonin or norepinephrine transporters (DAT, SERT and NET, respectively) were used to assess drug affinities at radioligand binding sites. Potencies and efficacies were determined using [35S]GTPγS binding assays (5-HT1A), inositol-phosphate accumulation assays (5-HT2A, 5-HT2B and 5-HT2C), and uptake and release assays (transporters). The substituted phenethylamines were very low potency and low efficacy agonists at the 5-HT1A receptor. 25D-NBOMe, 25E-NBOMe, 25H-NBOMe, 25I-NBOH and 25N-NBOMe had very high affinity for, and full efficacy at, 5-HT2A and 5-HT2C receptors. In the 5-HT2A receptor functional assay, 25D-NBOMe, 25E-NBOMe, 25I-NBOH and 25N-NBOMe had subnanomolar to low nanomolar potencies similar to (+)lysergic acid diethylamide (LSD) while 25H-NBOMe had lower potency, similar to serotonin. At the 5-HT2C receptor, four had very high potencies, similar to LSD and serotonin, while 25H-NBOMe had lower potency. At the 5-HT2B receptor, the compounds had lower affinity, potency and efficacy compared to 5-HT2A or 5-HT2C. The phenethylamines had low to mid micromolar affinities and potencies at the transporters. These results demonstrate that these –NBOMe and –NBOH substituted phenethylamines have a biochemical pharmacology consistent with hallucinogenic activity, with little psychostimulant activity.

The novel μ-opioid receptor agonist PZM21 depresses respiration and induces tolerance to antinociception.

Background and PurposePZM21 is a novel μ‐opioid receptor ligand that has been reported to induce minimal arrestin recruitment and be devoid of the respiratory depressant effects characteristic of classical μ receptor ligands such as morphine. We have re‐examined the signalling profile of PZM21 and its ability to depress respiration.Experimental ApproachG protein (Gi) activation and arrestin‐3 translocation were measured in vitro, using BRET assays, in HEK 293 cells expressing μ receptors. Respiration (rate and tidal volume) was measured in awake, freely moving mice by whole‐body plethysmography, and antinociception was measured by the hot plate test.Key ResultsPZM21 (10−9 – 3 × 10−5 M) produced concentration‐dependent Gi activation and arrestin‐3 translocation. Comparison with responses evoked by morphine and DAMGO revealed that PZM21 was a low efficacy agonist in both signalling assays. PZM21 (10–80 mg·kg−1) depressed respiration in a dose‐dependent manner. The respiratory depression was due to a decrease in the rate of breathing not a decrease in tidal volume. On repeated daily administration of PZM21 (twice daily doses of 40 mg·kg−1), complete tolerance developed to the antinociceptive effect of PZM21 over 3 days but no tolerance developed to its respiratory depressant effect.Conclusion and ImplicationsThese data demonstrate that PZM21 is a low efficacy μ receptor agonist for both G protein and arrestin signalling. Contrary to a previous report, PZM21 depresses respiration in a manner similar to morphine, the classical opioid receptor agonist.

Assessment of the Molecular Mechanisms of Action of Novel 4-Phenylpyridine-2-One and 6-Phenylpyrimidin-4-One Allosteric Modulators at the M1 Muscarinic Acetylcholine Receptors.

Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism but whose molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, benzyl quinolone carboxylic acid (BQCA), in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signal-regulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and β-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated.

Drug Binding Poses Relate Structure with Efficacy in the μ Opioid Receptor

The μ-opioid receptor (MOPr) is a clinically important G protein-coupled receptor that couples to Gi/o proteins and arrestins. At present, the receptor conformational changes that occur following agonist binding and activation are poorly understood. This study has employed molecular dynamics simulations to investigate the binding mode and receptor conformational changes induced by structurally similar opioid ligands of widely differing intrinsic agonist efficacy, norbuprenorphine, buprenorphine, and diprenorphine. Bioluminescence resonance energy transfer assays for Gi activation and arrestin-3 recruitment in human embryonic kidney 293 cells confirmed that norbuprenorphine is a high efficacy agonist, buprenorphine a low efficacy agonist, and diprenorphine an antagonist at the MOPr. Molecular dynamics simulations revealed that these ligands adopt distinct binding poses and engage different subsets of residues, despite sharing a common morphinan scaffold. Notably, norbuprenorphine interacted with sodium ion-coordinating residues W2936.48 and N1503.35, whilst buprenorphine and diprenorphine did not. Principal component analysis of the movements of the receptor transmembrane domains showed that the buprenorphine-bound receptor occupied a distinct set of conformations to the norbuprenorphine-bound receptor. Addition of an allosteric sodium ion caused the receptor and ligand to adopt an inactive conformation. The differences in ligand–residue interactions and receptor conformations observed here may underlie the differing efficacies for cellular signalling outputs for these ligands.

Two Janus Cannabinoids That Are Both CB2 Agonists and CB1 Antagonists.

The cannabinoid signaling system includes two G protein-coupled receptors, CB1 and CB2 These receptors are widely distributed throughout the body and have each been implicated in many physiologically important processes. Although the cannabinoid signaling system has therapeutic potential, the development of receptor-selective ligands remains a persistent hurdle. Because CB1 and CB2 are involved in diverse processes, it would be advantageous to develop ligands that differentially engage CB1 and CB2 We now report that GW405833 [1-(2,3-dichlorobenzoyl)-5-methoxy-2-methyl-3-[2-(4-morpholinyl)ethyl]-1H-indole] and AM1710 [1-hydroxy-9-methoxy-3-(2-methyloctan-2-yl)benzo[c]chromen-6-one], described as selective CB2 agonists, can antagonize CB1 receptor signaling. In autaptic hippocampal neurons, GW405833 and AM1710 both interfered with CB1-mediated depolarization-induced suppression of excitation, with GW405833 being more potent. In addition, in CB1-expressing human embryonic kidney 293 cells, GW405833 noncompetitively antagonized adenylyl cyclase activity, extracellular signal-regulated kinase 1/2 phosphorylation, phosphatidylinositol 4,5-bisphosphate signaling, and CB1 internalization by CP55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol). In contrast, AM1710 behaved as a low-potency competitive antagonist/inverse agonist in these signaling pathways. GW405833 interactions with CB1/arrestin signaling were complex: GW405833 differentially modulated arrestin recruitment in a time-dependent fashion, with an initial modest potentiation at 20 minutes followed by antagonism starting at 1 hour. AM1710 acted as a low-efficacy agonist in arrestin signaling at the CB1 receptor, with no evident time dependence. In summary, we determined that GW405833 and AM1710 are not only CB2 agonists but also CB1 antagonists, with distinctive and complex signaling properties. Thus, experiments using these compounds must take into account their potential activity at CB1 receptors.

Functional Selectivity of CB2 Cannabinoid Receptor Ligands at a Canonical and Noncanonical Pathway.

The CB2 cannabinoid receptor (CB2) remains a tantalizing, but unrealized therapeutic target. CB2 receptor ligands belong to varied structural classes and display extreme functional selectivity. Here, we have screened diverse CB2 receptor ligands at canonical (inhibition of adenylyl cyclase) and noncanonical (arrestin recruitment) pathways. The nonclassic cannabinoid (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) was the most potent agonist for both pathways, while the classic cannabinoid ligand (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran JWH133) was the most efficacious agonist among all the ligands profiled in cyclase assays. In the cyclase assay, other classic cannabinoids showed little [(-)-trans-Δ(9)-tetrahydrocannabinol and (-)-(6aR,7,10,10aR)-tetrahydro-6,6,9-trimethyl-3-(1-methyl-1-phenylethyl)-6H-dibenzo[b,d]pyran-1-ol] (KM233) to no efficacy [(6aR,10aR)-1-methoxy-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromene(L759633) and (6aR,10aR)-3-(1,1-dimethylheptyl)-6a,7,8,9,10,10a-hexahydro-1-methoxy-6,6-dimethyl-9-methylene-6H-dibenzo[b,d]pyran]L759656. Most aminoalkylindoles, including [(3R)-​2,​3-​dihydro-​5-​methyl-​3-​(4-​morpholinylmethyl)pyrrolo[1,​2,​3-​de]-​1,​4-​benzoxazin-​6-​yl]-​1-​naphthalenyl-​methanone,​ monomethanesulfonate (WIN55212-2), were moderate efficacy agonists. The cannabilactone 3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c)chromen-6-one (AM1710) was equiefficacious to CP55940 to inhibit adenylyl cyclase, albeit with lower potency. In the arrestin recruitment assays, all classic cannabinoid ligands failed to recruit arrestins, indicating a bias toward G-protein coupling for this class of compound. All aminoalkylindoles tested, except for WIN55212-2 and (1-​pentyl-​1H-​indol-​3-​yl)(2,​2,​3,​3-​tetramethylcyclopropyl)-​methanone (UR144), failed to recruit arrestin. WIN55212-2 was a low efficacy agonist for arrestin recruitment, while UR144 was arrestin biased with no significant inhibition of cyclase. Endocannabinoids were G-protein biased with no arrestin recruitment. The diarylpyrazole antagonist 5-​(4-​chloro-​3-​methylphenyl)-​1-​[(4-​methylphenyl)methyl]-​N-​[(1S,​2S,​4R)-​1,​3,​3-​trimethylbicyclo[2.2.1]hept-​2-​yl]-​1H-​pyrazole-​3-​carboxamide (SR144258) was an inverse agonist in cyclase and arrestin recruitment assays while the aminoalkylindole 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630) and carboxamide N-(1,3-benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinolinecarboxamide (JTE907) were inverse agonists in cyclase but low efficacy agonists in arrestin recruitment assays. Thus, CB2 receptor ligands display strong and varied functional selectivity at both pathways. Therefore, extreme care must be exercised when using these compounds to infer the role of CB2 receptors in vivo.

Bifunctional compounds targeting both D2 and non-α7 nACh receptors: Design, synthesis and pharmacological characterization

We designed, prepared and tested a set of structural analogs 1–4 as new hybrid compounds by incorporating, through a common alkyl chain of variable length, the pharmacophoric elements of N-n-alkyl nicotinium salts (non-α7 nicotinic acetylcholine receptors antagonists) and of 7-hydroxy-2-(aminomethyl)chromanes (dopaminergic D2 receptor agonists). The target compounds, which were assayed in binding experiments and electrophysiological, functional and Erk1/2 activation tests, essentially combined the pharmacological profiles of their individual receptor ligands. Among the studied derivatives, hybrid 2, one of the shortest homologs, in addition to the antagonist nicotinic profile similar to the other three congeners, behaved as a high affinity ligand at the investigated heteromeric nAChRs and as a low efficacy agonist at D2Rs. These bifunctional derivatives represent novel pharmacological tools in the study of nicotine addiction.

Effects of receptor tyrosine kinase inhibitors on VEGF165 a- and VEGF165 b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2.

Background and PurposeReceptor tyrosine kinase inhibitors (RTKIs) targeted at VEGF receptor 2 (VEGFR2) have proved to be attractive approaches to cancer therapy based on their ability to reduce angiogenesis. Here we have undertaken a quantitative analysis of the interaction of RTKIs and two VEGF splice variants, VEGF165a and VEGF165b, with VEGFR2 by studying nuclear factor of activated T-cells (NFAT) reporter gene activity in live HEK-293 cells.Experimental ApproachHEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF165a- and VEGF165b-stimulated luciferase gene expression.Key ResultsVEGF165a produced a concentration-dependent activation of the NFAT-luciferase reporter gene in living cells that was inhibited in a non-competitive fashion by four different RTKIs (cediranib, pazopanib, sorafenib and vandetanib). The potency obtained for each RTKI from this analysis was similar to those obtained in binding studies using purified VEGFR2 kinase domains. VEGF165b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF165a. Analysis of the concentration–response data using the operational model of agonism indicated that both VEGF165 isoforms had similar affinity for VEGFR2.Conclusions and ImplicationsQuantitative pharmacological analysis of the interaction of VEGF165 isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF165a and VEGF165b for activation of the calcineurin- NFAT signalling pathway by this tyrosine kinase receptor.

Allosteric Modulation of the Mu‐Opioid Receptor: Probe Dependence and Role of Na + Ions

The mu-opioid receptor (MOPr), a G protein-coupled receptor (GPCR), is the site of action of opioid analgesics like morphine and oxycodone. All clinically used MOPr ligands bind to the orthosteric site on MOPr, and compete with the endogenous opioid peptides for binding. We recently discovered the first positive allosteric modulators of MOPr (mu-PAMs), with BMS-986122 representing the prototype. We propose that mu-PAMs could represent a novel method for pain management. Here we characterize the effects of BMS-986122 in C6 rat glioma cell membranes expressing MOPr using radioligand binding and G protein activation as measured by GTPγ35S binding to determine orthosteric agonist affinity, potency, and maximal response. BMS-986122 had no effect on antagonist binding to MOPr. In contrast, BMS-986122 enhanced the affinity and/or efficacy of several orthosteric agonists depending on their efficacy. BMS-986122 enhanced the affinity and potency of high efficacy ligands while having no effect on maximal activity. For lower efficacy agonists, BMS-986122 did not alter ligand affinity, but caused an increase in maximal G protein activation. Na+ ions are known to act as negative modulators of MOPr, reducing the affinity and efficacy of orthosteric agonists. We found that there was a strong negative correlation between the effect of Na+ ions and of BMS-986122 on orthosteric ligand affinity. We also show that the presence of BMS-986122 allosterically inhibits the ability of Na+ to alter receptor state. These findings suggest that BMS-986122 has PAM activity at MOPr by allosterically disrupting the ability of Na+ to stabilize the inactive receptor state. This may represent a common mechanism for allostery at Class A GPCRs that are sensitive to Na+ ions. Supported by T32 DA007267 and DA035316.

Disruption of the Na+ ion binding site as a mechanism for positive allosteric modulation of the mu-opioid receptor.

Positive allosteric modulation of the mu-opioid receptor (MOPr), the site of action of all clinically used opioids, represents a potential approach for the management of pain. We recently reported on positive allosteric modulators of MOPr (mu-PAMs), a class A G protein coupled receptor (GPCR). This study was designed to examine the mechanism of allostery by comparing the degree to which opioid ligand structure governs modulation. To do this we examined the interaction of the mu-PAM, BMS-986122, with a chemically diverse range of MOPr orthosteric ligands. Generally, for full agonists BMS-986122 enhanced the binding affinity and potency to activate G protein with no alteration in the maximal effect. In contrast, lower efficacy agonists including morphine were insensitive to alterations in binding affinity and showed little to no change in potency to stimulate G protein. Instead, there was an increase in maximal G protein stimulation. Antagonists were unresponsive to the modulatory effects of BMS-986122. Sodium is a known endogenous allosteric modulator of MOPr and alters orthosteric agonist affinity and efficacy. The sensitivity of an orthosteric ligand to BMS-986122 was strongly correlated with its sensitivity to NaCl. In addition, BMS-986122 decreased the ability of NaCl to modulate agonist binding in an allosteric fashion. Overall, BMS-986122 displayed marked probe dependence that was based upon the efficacy of the orthosteric ligand and can be explained using the Monod-Wyman-Changeux two-state model of allostery. Furthermore, disruption of the Na(+) ion binding site may represent a common mechanism for allosteric modulation of class A GPCRs.

Effects of μ-opioid receptor agonists in assays of acute pain-stimulated and pain-depressed behavior in male rats: role of μ-agonist efficacy and noxious stimulus intensity.

Pain is associated with stimulation of some behaviors and depression of others, and μ-opioid receptor agonists are among the most widely used analgesics. This study used parallel assays of pain-stimulated and pain-depressed behavior in male Sprague-Dawley rats to compare antinociception profiles for six μ-agonists that varied in efficacy at μ-opioid receptors (from highest to lowest: methadone, fentanyl, morphine, hydrocodone, buprenorphine, and nalbuphine). Intraperitoneal injection of diluted lactic acid served as an acute noxious stimulus to either stimulate stretching or depress operant responding maintained by electrical stimulation in an intracranial self-stimulation (ICSS). All μ-agonists blocked both stimulation of stretching and depression of ICSS produced by 1.8% lactic acid. The high-efficacy agonists methadone and fentanyl were more potent at blocking acid-induced depression of ICSS than acid-stimulated stretching, whereas lower-efficacy agonists displayed similar potency across assays. All μ-agonists except morphine also facilitated ICSS in the absence of the noxious stimulus at doses similar to those that blocked acid-induced depression of ICSS. The potency of the low-efficacy μ-agonist nalbuphine, but not the high-efficacy μ-agonist methadone, to block acid-induced depression of ICSS was significantly reduced by increasing the intensity of the noxious stimulus to 5.6% acid. These results demonstrate sensitivity of acid-induced depression of ICSS to a range of clinically effective μ-opioid analgesics and reveal distinctions between opioids based on efficacy at the μ-receptor. These results also support the use of parallel assays of pain-stimulated and -depressed behaviors to evaluate analgesic efficacy of candidate drugs.