Discovery and structure-activity relationships (SAR) of a novel class of 2-substituted N-piperidinyl indole-based nociceptin opioid receptor ligands.

The development of SAR around substituted N-piperidinyl indole-based nociceptin opioid receptor (NOP) ligands led to the discovery of a novel series of 2-substituted N-piperidinyl indoles that provide both selective NOP full agonists and bifunctional NOP full agonists-µ opioid (MOP) receptor partial agonists. 2-substituted N-piperidinyl indoles have improved potency at the NOP receptor and are NOP full agonists, compared to our previously reported 3-substituted N-piperidinyl indoles that are selective NOP partial agonists. SAR in this series of 2-substituted N-piperidinyl indoles shows that 2-substitution versus 3-substitution on the indole moiety affects their intrinsic activity and opioid receptor selectivity. Molecular docking of these 2-substituted N-piperidinyl indoles in an active-state NOP homology model and MOP receptor structures provides a rationale for the differences observed in the binding, functional profiles and selectivity of 2-substituted versus 3-substituted N-piperidinyl indoles.

Attenuated G protein signaling and minimal receptor phosphorylation as a biochemical signature of low side-effect opioid analgesics.

Multi-receptor targeting has been proposed as a promising strategy for the development of opioid analgesics with fewer side effects. Cebranopadol and AT-121 are prototypical bifunctional ligands targeting the nociceptin/orphanin FQ peptide receptor (NOP) and µ-opioid receptor (MOP) that elicit potent analgesia in humans and nonhuman primates, respectively. Cebranopadol was reported to produce typical MOP-related side effects such as respiratory depression and reward, whereas AT-121 appeared to be devoid of these liabilities. However, the molecular basis underlying different side effect profiles in opioid analgesics remains unknown. Here, we examine agonist-induced receptor phosphorylation and G protein signaling profiles of a series of chemically diverse mixed MOP/NOP agonists, including cebranopadol and AT-121. We found that these compounds produce strikingly different MOP phosphorylation profiles. Cebranopadol, AT-034 and AT-324 stimulated extensive MOP phosphorylation, whereas AT-201 induced selective phosphorylation at S375 only. AT-121, on the other hand, did not promote any detectable MOP phosphorylation. Conversely, none of these compounds was able to elicit strong NOP phosphorylation and low NOP receptor phosphorylation correlated with partial agonism in a GIRK-channel assay. Our results suggest a close correlation between MOP receptor phosphorylation and side effect profile. Thus, bifunctional MOP/NOP opioid ligands combining low efficacy G protein signaling at both NOP and MOP with no detectable receptor phosphorylation appear to be devoid of side-effects such as respiratory depression, abuse liability or tolerance development, as with AT-121.

Differential In Vitro Pharmacological Profiles of Structurally Diverse Nociceptin Receptor Agonists in Activating G Protein and Beta-Arrestin Signaling at the Human Nociceptin Opioid Receptor.

Agonists at the nociceptin opioid peptide receptor (NOP) are under investigation as therapeutics for nonaddicting analgesia, opioid use disorder, Parkinson's disease, and other indications. NOP full and partial agonists have both been of interest, particularly since NOP partial agonists show a reduced propensity for behavioral disruption than NOP full agonists. Here, we investigated the in vitro pharmacological properties of chemically diverse NOP receptor agonists in assays measuring functional activation of the NOP receptor such as guanosine 5'-O-[gamma-thio]triphosphate (GTPγS) binding, cAMP inhibition, G protein-coupled inwardly rectifying potassium (GIRK) channel activation, phosphorylation, ß-arrestin recruitment and receptor internalization. When normalized to the efficacy of the natural agonist nociceptin/orphanin FQ (N/OFQ), we found that different functional assays that measure intrinsic activity produce inconsistent levels of agonist efficacy, particularly for ligands that were partial agonists. Agonist efficacy obtained in the GTPγS assay tended to be lower than that in the cAMP and GIRK assays. These structurally diverse NOP agonists also showed differential receptor phosphorylation profiles at the phosphosites we examined and induced varying levels of receptor internalization. Interestingly, although the rank order for ß-arrestin recruitment by these NOP agonists was consistent with their ability to induce receptor internalization, their phosphorylation signatures at the time point we investigated were not indicative of the levels of ß-arrestin recruitment or internalization induced by these agonists. It is possible that other phosphorylation sites, yet to be identified, drive the recruitment of NOP receptor ensembles and subsequent receptor trafficking by some nonpeptide NOP agonists. These findings potentially help understand NOP agonist pharmacology in the context of ligand-activated receptor trafficking. SIGNIFICANCE STATEMENT: Chemically diverse agonist ligands at the nociceptin opioid receptor G protein-coupled receptor showed differential efficacy for activating downstream events after receptor binding, in a suite of functional assays measuring guanosine 5'-O-[gamma-thio]triphosphate binding, cAMP inhibition, G protein-coupled inwardly rectifying protein channel activation, ß-arrestin recruitment, receptor internalization and receptor phosphorylation. These analyses provide a context for understanding nociceptin opioid peptide receptor (NOP) agonist pharmacology driven by ligand-induced differential NOP receptor signaling.

Discovery and Structure-Activity Relationships of Nociceptin Receptor Partial Agonists That Afford Symptom Ablation in Parkinson's Disease Models.

A novel series of C(3)-substituted piperdinylindoles were developed as nociceptin opioid receptor (NOP) partial agonists to explore a pharmacological hypothesis that NOP partial agonists would afford a dual pharmacological action of attenuating Parkinson's disease (PD) motor symptoms and development of levodopa-induced dyskinesias. SAR around the C-3 substituents investigated effects on NOP binding, intrinsic activity, and selectivity and showed that while the C(3)-substituted indoles are selective, high affinity NOP ligands, the steric, polar, and cationic nature of the C-3 substituents affected intrinsic activity to afford partial agonists with a range of efficacies. Compounds 4, 5, and 9 with agonist efficacies between 25% and 35% significantly attenuated motor deficits in the 6-OHDA-hemilesioned rat model of PD. Further, unlike NOP antagonists, which appear to worsen dyskinesia expression, these NOP partial agonists did not attenuate or worsen dyskinesia expression. The NOP partial agonists and their SAR reported here may be useful to develop nondopaminergic treatments for PD.

Synthesis, Biological Evaluation, and SAR Studies of 14ß-phenylacetyl Substituted 17-cyclopropylmethyl-7, 8-dihydronoroxymorphinones Derivatives: Ligands With Mixed NOP and Opioid Receptor Profile.

A series of 14ß-acyl substituted 17-cyclopropylmethyl-7,8-dihydronoroxymorphinone compounds has been synthesized and evaluated for affinity and efficacy for mu (MOP), kappa (KOP), and delta (DOP) opioid receptors and nociceptin/orphanin FQ peptide (NOP) receptors. The majority of the new ligands displayed high binding affinities for the three opioid receptors, and moderate affinity for NOP receptors. The affinities for NOP receptors are of particular interest as most classical opioid ligands do not bind to NOP receptors. The predominant activity in the [35S]GTPγS assay was partial agonism at each receptor. The results are consistent with our prediction that an appropriate 14ß side chain would access a binding site within the NOP receptor and result in substantially higher affinity than displayed by the parent compound naltrexone. Molecular modeling studies, utilizing the recently reported structure of the NOP receptor, are also consistent with this interpretation.

A bifunctional nociceptin and mu opioid receptor agonist is analgesic without opioid side effects in nonhuman primates.

Misuse of prescription opioids, opioid addiction, and overdose underscore the urgent need for developing addiction-free effective medications for treating severe pain. Mu opioid peptide (MOP) receptor agonists provide very effective pain relief. However, severe side effects limit their use in the clinical setting. Agonists of the nociceptin/orphanin FQ peptide (NOP) receptor have been shown to modulate the antinociceptive and reinforcing effects of MOP agonists. We report the discovery and development of a bifunctional NOP/MOP receptor agonist, AT-121, which has partial agonist activity at both NOP and MOP receptors. AT-121 suppressed oxycodone's reinforcing effects and exerted morphine-like analgesic effects in nonhuman primates. AT-121 treatment did not induce side effects commonly associated with opioids, such as respiratory depression, abuse potential, opioid-induced hyperalgesia, and physical dependence. Our results in nonhuman primates suggest that bifunctional NOP/MOP agonists with the appropriate balance of NOP and MOP agonist activity may provide a dual therapeutic action for safe and effective pain relief and treating prescription opioid abuse.

Anti-Parkinsonian and anti-dyskinetic profiles of two novel potent and selective nociceptin/orphanin FQ receptor agonists.

BACKGROUND AND PURPOSE: We previously showed that nociceptin/orphanin FQ opioid peptide (NOP) receptor agonists attenuate the expression of levodopa-induced dyskinesia in animal models of Parkinson's disease. We now investigate the efficacy of two novel, potent and chemically distinct NOP receptor agonists, AT-390 and AT-403, to improve Parkinsonian disabilities and attenuate dyskinesia development and expression. EXPERIMENTAL APPROACH: Binding affinity and functional efficacy of AT-390 and AT-403 at the opioid receptors were determined in radioligand displacement assays and in GTPγS binding assays respectively, conducted in CHO cells. Their anti-Parkinsonian activity was evaluated in 6-hydroxydopamine hemi-lesioned rats whereas the anti-dyskinetic properties were assessed in 6-hydroxydopamine hemi-lesioned rats chronically treated with levodopa. The ability of AT-403 to inhibit the D1 receptor-induced phosphorylation of striatal ERK was investigated. KEY RESULTS: AT-390 and AT-403 selectively improved akinesia at low doses and disrupted global motor activity at higher doses. AT-403 palliated dyskinesia expression without causing sedation in a narrow therapeutic window, whereas AT-390 delayed the appearance of abnormal involuntary movements and increased their duration at doses causing sedation. AT-403 did not prevent the priming to levodopa, although it significantly inhibited dyskinesia on the first day of administration. AT-403 reduced the ERK phosphorylation induced by SKF38393 in vitro and by levodopa in vivo. CONCLUSIONS AND IMPLICATIONS: NOP receptor stimulation can provide significant albeit mild anti-dyskinetic effect at doses not causing sedation. The therapeutic window, however, varies across compounds. AT-403 could be a potent and selective tool to investigate the role of NOP receptors in vivo.

A Novel and Selective Nociceptin Receptor (NOP) Agonist (1-(1-((cis)-4-isopropylcyclohexyl)piperidin-4-yl)-1H-indol-2-yl)methanol (AT-312) Decreases Acquisition of Ethanol-Induced Conditioned Place Preference in Mice.

BACKGROUND: Nociceptin/orphanin FQ, the endogenous peptide agonist for the opioid receptor-like receptor (also known as NOP or the nociceptin receptor), has been shown to block the acquisition and expression of ethanol (EtOH)-induced conditioned place preference (CPP). Here, we report the characterization of a novel small-molecule NOP ligand AT-312 (1-(1-((cis)-4-isopropylcyclohexyl)piperidin-4-yl)-1H-indol-2-yl)methanol) in receptor binding and GTPγS functional assays in vitro. We then investigated the effect of AT-312 on the rewarding action of EtOH in mice using the CPP paradigm. Further, using mice lacking the NOP receptor and their wild-type controls, we also examined the involvement of NOP in the effect of AT-312. Motivational effects of AT-312 alone were also assessed in the CPP paradigm. METHODS: Female mice lacking NOP and/or their wild-type controls received conditioning in the presence or absence of the NOP agonist [AT-312 (1, 3, and 10 mg/kg) or the control NOP agonist SCH221510 (10 mg/kg)] followed by saline/EtOH for 3 consecutive days (twice daily) and tested for CPP in a drug-free state on the next day. RESULTS: Our in vitro data showed that AT-312 is a high-affinity, selective NOP full agonist with 17-fold selectivity over the mu opioid receptor and >200-fold selectivity over the kappa opioid receptor. The results of our in vivo studies showed that AT-312 reduced EtOH CPP at the lowest dose (1 mg/kg) tested but completely abolished EtOH CPP at higher doses (3 or 10 mg/kg) compared to their vehicle-treated control group. AT-312 (3 mg/kg) did not alter EtOH-induced CPP in mice lacking NOP, confirming that AT-312 reduced EtOH CPP through its action at the NOP receptor. AT-312 (3 mg/kg) did not induce reward or aversion when administered alone, showing that the novel small-molecule NOP agonist shows efficacy in blocking EtOH-induced CPP via the NOP receptor. CONCLUSIONS: Together, these data suggest that small-molecule NOP agonists have the potential to reduce alcohol reward and may be promising as medications to treat alcohol addiction.

Probing ligand recognition of the opioid pan antagonist AT-076 at nociceptin, kappa, mu, and delta opioid receptors through structure-activity relationships.

Few opioid ligands binding to the three classic opioid receptor subtypes, mu, kappa and delta, have high affinity at the fourth opioid receptor, the nociceptin/orphanin FQ receptor (NOP). We recently reported the discovery of AT-076 (1), (R)-7-hydroxy-N-((S)-1-(4-(3-hydroxyphenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, a pan antagonist with nanomolar affinity for all four subtypes. Since AT-076 binds with high affinity at all four subtypes, we conducted a structure-activity relationship (SAR) study to probe ligand recognition features important for pan opioid receptor activity, using chemical modifications of key pharmacophoric groups. SAR analysis of the resulting analogs suggests that for the NOP receptor, the entire AT-076 scaffold is crucial for high binding affinity, but the binding mode is likely different from that of NOP antagonists C-24 and SB-612111 bound in the NOP crystal structure. On the other hand, modifications of the 3-hydroxyphenyl pharmacophore, but not the 7-hydroxy Tic pharmacophore, are better tolerated at kappa and mu receptors and yield very high affinity multifunctional (e.g. 12) or highly selective (e.g. 16) kappa ligands. With the availability of the opioid receptor crystal structures, our SAR analysis of the common chemotype of AT-076 suggests rational approaches to modulate binding selectivity, enabling the design of multifunctional or selective opioid ligands from such scaffolds.

In Vitro and In Vivo Profile of PPL-101 and PPL-103: Mixed Opioid Partial Agonist Analgesics with Low Abuse Potential.

Opiates are still the most effective and widely used treatments for acute and chronic pain. However, the problems associated with morphine and other standard opioid analgesics severely limit their effectiveness in the clinic. PPL-101 and PPL-103 derived from morphine and morphinan ring systems contain a chiral N-substituent, which confers it with a unique combination of high-binding affinities and partial agonist activities at mu, delta, and kappa opioid receptors, leading to unique in vivo pharmacology compared to other conventional opioids. Acute antinociceptive and reward acquisition of PPL-101 and PPL-103 were assessed in mice using the tail flick assay and conditioned place preference (CPP) paradigm, respectively. The reinforcing effects of these compounds were assessed in rats using the self-administration paradigm. In mice, PPL-101 and PPL-103 produced antinociception reaching maximal effects that were equivalent to morphine at approximately 1/3 and 1/10 of morphine's dose, respectively. PPL-101-induced antinociception was attenuated following pretreatment with the kappa antagonist JDTic, but not the mu opioid antagonist beta-FNA. In mice, PPL-101 and PPL-103 produced dose-dependent decreases in activity, similar to other kappa agonists; however, they did not produce conditioned place aversion, and in fact elicited a trend toward CPP. In rats, neither PPL-101 nor PPL-103 were self-administered when substituted for morphine and PPL-101 attenuated morphine self-administration, when administered systemically prior to the self-administration session. Collectively, these results indicate that mixed opioid receptor partial agonists can produce potent antinociceptive activity with a lack of aversion in mice and without being self-administered in rats. Compounds with this profile could be superior analgesics with greatly reduced addiction liability and fewer side-effects compared to traditional opiates.

In vitro functional characterization of novel nociceptin/orphanin FQ receptor agonists in recombinant and native preparations.

Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [35S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65-6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127≥AT-090≥AT-035>AT-004= AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [35S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists.

High affinity α3ß4 nicotinic acetylcholine receptor ligands AT-1001 and AT-1012 attenuate cocaine-induced conditioned place preference and behavioral sensitization in mice.

Cholinergic signaling via the nicotinic acetylcholine receptors (nAChRs) in the mesolimbic circuitry is involved in the rewarding effects of abused drugs such as cocaine and opioids. In mouse studies, nonselective nAChR antagonist mecamylamine blocks cocaine-induced conditioned place preference (CPP) and behavioral sensitization. Among subtype-selective nAChR antagonists, the ß2-selective antagonist dihydrobetaerythroidine and α7 antagonist methyllycaconitine (MLA), but not MLA alone prevent behavioral sensitization to cocaine. Since the role of the α3ß4 nAChR subtype in the rewarding and behavioral effects of cocaine is unknown, the present study investigated the effect of two potent and selective α3ß4 nAChR ligands, AT-1001 and AT-1012, on the acquisition of cocaine-induced CPP and behavioral sensitization in mice. At 5-30mg/kg, cocaine produced robust CPP, whereas behavioral sensitization of locomotor activity was only observed at the higher doses (20-30mg/kg). Pretreatment with AT-1001 (1-10mg/kg) or AT-1012 (3-10mg/kg) blocked CPP induced by 5mg/kg cocaine, but not by 30mg/kg cocaine. Lower doses of AT-1001 (0.3-1mg/kg) and AT-1012 (1-3mg/kg) did not affect the increase in locomotor activity induced by 5 or 30mg/kg cocaine. But AT-1001, at these doses, blocked locomotor sensitization induced by 30mg/kg cocaine. These results indicate that the α3ß4 nAChR play a role in the rewarding and behavioral effects of cocaine, and that selective α3ß4 nAChR ligands can attenuate cocaine-induced behavioral phenomena. Since the selective α3ß4 nAChR functional antagonist AT-1001 has also been shown to block nicotine self-administration in rats, the present results suggest that α3ß4 nAChRs may be a target for the treatment of cocaine addiction as well as for cocaine-nicotine comorbid addiction.

Discovery of the first small-molecule opioid pan antagonist with nanomolar affinity at mu, delta, kappa, and nociceptin opioid receptors.

The trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine scaffold is a known pharmacophore for mu opioid (MOP), kappa opioid (KOP), and delta opioid (DOP) receptor antagonists; however, it has not been explored in nociceptin opioid (NOP/ORL-1) receptor ligands. We recently found that the selective KOP antagonist JDTic, (3R)-7-hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide, containing this opioid antagonist pharmacophore, has significant binding affinity at the NOP receptor (Ki 16.67 ± 0.76 nM), with no intrinsic activity in the [(35)S]GTPγS functional assay. Since this is the first ligand containing the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist pharmacophore to have affinity for the NOP receptor, we explored the structural determinants of its NOP binding affinity. When rational chemical modifications of JDTic were carried out, based on our previously established NOP pharmacophoric structure-activity relationship (SAR) model, most modifications led to a significant decrease in NOP and opioid binding affinity compared to JDTic. Interestingly, however, removal of the 3,4-dimethyl groups of the trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine antagonist scaffold of JDTic increased the binding affinity at NOP by 10-fold (Ki 1.75 ± 0.74 nM) while maintaining comparable affinity for KOP, MOP, and DOP receptors (Ki 1.14 ± 0.63, 1.67 ± 0.6, and 19.6 ± 1.3 nM, respectively). In vitro functional efficacy studies using the [(35)S]GTPγS assay showed that this compound AT-076 functions as an antagonist at all four opioid receptors. Detailed characterization of the antagonist activity of AT-076 shows that it has a noncompetitive antagonist profile at the NOP and KOP receptors (insurmountable antagonism), but is a potent competitive antagonist at the MOP and DOP receptors, with Ke values 3-6-fold more potent than those of JDTic. AT-076 is the first opioid pan antagonist with high affinity at all four opioid receptor subtypes. Our SAR studies show that the 3,4-dimethyl groups of the well-known trans-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonist scaffold may be removed without significant loss in binding affinity or antagonist potency to obtain an opioid pan antagonist such as AT-076.

Structure-based virtual screening of the nociceptin receptor: hybrid docking and shape-based approaches for improved hit identification.

The antagonist-bound crystal structure of the nociceptin receptor (NOP), from the opioid receptor family, was recently reported along with those of the other opioid receptors bound to opioid antagonists. We recently reported the first homology model of the 'active-state' of the NOP receptor, which when docked with 'agonist' ligands showed differences in the TM helices and residues, consistent with GPCR activation after agonist binding. In this study, we explored the use of the active-state NOP homology model for structure-based virtual screening to discover NOP ligands containing new chemical scaffolds. Several NOP agonist and antagonist ligands previously reported are based on a common piperidine scaffold. Given the structure-activity relationships for known NOP ligands, we developed a hybrid method that combines a structure-based and ligand-based approach, utilizing the active-state NOP receptor as well as the pharmacophoric features of known NOP ligands, to identify novel NOP binding scaffolds by virtual screening. Multiple conformations of the NOP active site including the flexible second extracellular loop (EL2) loop were generated by simulated annealing and ranked using enrichment factor (EF) analysis and a ligand-decoy dataset containing known NOP agonist ligands. The enrichment factors were further improved by combining shape-based screening of this ligand-decoy dataset and calculation of consensus scores. This combined structure-based and ligand-based EF analysis yielded higher enrichment factors than the individual methods, suggesting the effectiveness of the hybrid approach. Virtual screening of the CNS Permeable subset of the ZINC database was carried out using the above-mentioned hybrid approach in a tiered fashion utilizing a ligand pharmacophore-based filtering step, followed by structure-based virtual screening using the refined NOP active-state models from the enrichment analysis. Determination of the NOP receptor binding affinity of a selected set of top-scoring hits resulted in identification of several compounds with measurable binding affinity at the NOP receptor, one of which had a new chemotype for NOP receptor binding. The hybrid ligand-based and structure-based methodology demonstrates an effective approach for virtual screening that leverages existing SAR and receptor structure information for identifying novel hits for NOP receptor binding. The refined active-state NOP homology models obtained from the enrichment studies can be further used for structure-based optimization of these new chemotypes to obtain potent and selective NOP receptor ligands for therapeutic development.

Designing bifunctional NOP receptor-mu opioid receptor ligands from NOP-receptor selective scaffolds. Part II.

The nociceptin opioid receptor (NOP) and its endogenous peptide ligand nociceptin/orphanin FQ have been shown to modulate the pharmacological effects of the classical opioid receptor system. Suppression of opioid-induced reward associated with mu-opioid receptor (MOP)-mediated analgesia, without decreasing anti-nociceptive efficacy, can potentially be achieved with NOP agonists having bifunctional agonist activity at MOP, to afford 'non-addicting' analgesics. In Part II of this series, we describe a continuing structure-activity relationship (SAR) study of the NOP-selective piperidin-4-yl-1,3-dihydroindol-2-one scaffold, to obtain bifunctional activity at MOP, and a suitable ratio of NOP/MOP agonist activity that produces a non-addicting analgesic profile. The SAR reported here is focused on the influence of various piperidine nitrogen aromatic substituents on the ratio of binding affinity and intrinsic activity at both the NOP and MOP receptors.

[¹²5I]AT-1012, a new high affinity radioligand for the α3ß4 nicotinic acetylcholine receptors.

Recent genetic and pharmacological studies have implicated the α3, ß4 and α5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3ß4* nAChR subtype has been shown to co-assemble with the α5 or ß3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3ß4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [(125)I]-radiolabeled analog of a high affinity, selective small-molecule α3ß4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3ß4* nAChR. Binding of [(125)I]AT-1012 was characterized at the rat α3ß4 and α4ß2 nAChR transfected into HEK cells, as well as at the human α3ß4α5 nAChR in HEK cells. Binding affinity of [(125)I]AT-1012 at the rat α3ß4 nAChR was 1.4 nM, with a B(max) of 10.3 pmol/mg protein, similar to what was determined for unlabeled AT-1012 using [(3)H]epibatidine. Saturation isotherms suggested that [(125)I]AT-1012 binds to a single site on the α3ß4 nAChR. Similar high binding affinity was also observed for [(125)I]AT-1012 at the human α3ß4α5 nAChR transfected into HEK cells. [(125)I]AT-1012 did not bind with high affinity to membranes from α4ß2 nAChR-transfected HEK cells. Binding studies with [(3)H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for α3ß4 over α4ß2 nAChR. K(i) values determined for known nAChR compounds using [(125)I]AT-1012 as radioligand were comparable to those obtained with [(3)H]epibatidine. [(125)I]AT-1012 was also used to label α3ß4 nAChR in rat brain slices in vitro using autoradiography, which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3ß4 nAChR. We demonstrate that [(125)I]AT-1012 is an excellent tool for labeling the α3ß4 nAChR in the presence of other nAChR subtypes.

Designing bifunctional NOP receptor-mu opioid receptor ligands from NOP receptor-selective scaffolds. Part I.

The nociceptin receptor (NOP) and its endogenous agonist, nociceptin/orphanin FQ (N/OFQ), members of the opioid receptor and peptide families respectively, modulate the pharmacological effects of classical opioids, particularly opioid-induced reward and nociception. We hypothesized that compounds containing both NOP and opioid receptor activity in a single molecule may have useful pharmacological profiles as non-addicting analgesics or as drug abuse medications. We report here our forays into the structure-activity relationships for discovering 'bifunctional' NOP-mu opioid receptor (MOP) ligands, starting from our NOP-selective scaffolds. This initial SAR suggests pharmacophoric elements that may be modified to modulate/increase opioid affinity, while maintaining high affinity for the NOP receptor, to result in potent bifunctional small-molecule NOP/MOP ligands.

AT-1001: a high affinity and selective α3ß4 nicotinic acetylcholine receptor antagonist blocks nicotine self-administration in rats.

Genomic and pharmacologic data have suggested the involvement of the α3ß4 subtype of nicotinic acetylcholine receptors (nAChRs) in drug seeking to nicotine and other drugs of abuse. In order to better examine this receptor subtype, we have identified and characterized the first high affinity and selective α3ß4 nAChR antagonist, AT-1001, both in vitro and in vivo. This is the first reported compound with a Ki below 10 nM at α3ß4 nAChR and >90-fold selectivity over the other major subtypes, the α4ß2 and α7 nAChR. AT-1001 competes with epibatidine, allowing for [³H]epibatidine binding to be used for structure-activity studies, however, both receptor binding and ligand-induced Ca²âº flux are not strictly competitive because increasing ligand concentration produces an apparent decrease in receptor number and maximal Ca²âº fluorescence. AT-1001 also potently and reversibly blocks epibatidine-induced inward currents in HEK cells transfected with α3ß4 nAChR. Importantly, AT-1001 potently and dose-dependently blocks nicotine self-administration in rats, without affecting food responding. When tested in a nucleus accumbens (NAcs) synaptosomal preparation, AT-1001 inhibits nicotine-induced [³H]dopamine release poorly and at significantly higher concentrations compared with mecamylamine and conotoxin MII. These results suggest that its inhibition of nicotine self-administration in rats is not directly due to a decrease in dopamine release from the NAc, and most likely involves an indirect pathway requiring α3ß4 nAChR. In conclusion, our studies provide further evidence for the involvement of α3ß4 nAChR in nicotine self-administration. These findings suggest the utility of this receptor as a target for smoking cessation medications, and highlight the potential of AT-1001 and congeners as clinically useful compounds.

Structural determinants of opioid and NOP receptor activity in derivatives of buprenorphine.

The unique pharmacological profile of buprenorphine has led to its considerable success as an analgesic and as a treatment agent for drug abuse. Activation of nociceptin/orphanin FQ peptide (NOP) receptors has been postulated to account for certain aspects of buprenorphine's behavioral profile. In order to investigate the role of NOP activation further, a series of buprenorphine analogues has been synthesized with the aim of increasing affinity for the NOP receptor. Binding and functional assay data on these new compounds indicate that the area around C20 in the orvinols is key to NOP receptor activity, with several compounds displaying higher affinity than buprenorphine. One compound, 1b, was found to be a mu opioid receptor partial agonist of comparable efficacy to buprenorphine but with higher efficacy at NOP receptors.

Differential effects of nociceptin/orphanin FQ (NOP) receptor agonists in acute versus chronic pain: studies with bifunctional NOP/µ receptor agonists in the sciatic nerve ligation chronic pain model in mice.

1-(1-Cyclooctylpiperidin-4-yl)-indolin-2-one (SR14150) and 1-(1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)piperidinl-4-yl)-indolin-2-one (SR16835) are moderately selective nociceptin/orphanin FQ (NOP) receptor agonists. In the [(35)S]guanosine 5'-O-(3-thiotriphosphate) assay in vitro, SR14150 is a partial agonist at both the NOP and µ-opioid receptors, whereas SR16835 is a full agonist at the NOP receptor and has low efficacy at µ receptors. These compounds were tested for antinociceptive and antiallodynic activity, using mice in chronic pain, subsequent to spinal nerve ligation (SNL) surgery. When administered subcutaneously to mice after SNL surgery, SR14150 but not SR16835 increased tail-flick latency, which was blocked by the opioid antagonist naloxone, but not by the NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). In contrast, both SR14150 and SR16835 had antiallodynic activity when mechanical allodynia was measured with von Frey monofilaments. This effect was completely blocked by SB-612111 but not by naloxone. On the other hand, morphine antinociception and antiallodynia were both blocked by naloxone and potentiated by SB-612111. These results indicate that, in mice, circuitry mediating antinociceptive activity in acute and chronic pain states is different. It is possible that during a chronic pain state, an up-regulated NOP system in the spinal cord leads to NOP receptor-mediated antiallodynia, which is blocked by NOP antagonists. However, supraspinal up-regulation could lead to an attenuation of morphine antinociception and antiallodynia, which can be alleviated by an NOP receptor antagonist. Thus, although neither NOP agonists nor antagonists are effective as analgesics in acute pain, they may have efficacy as analgesics, either alone or in combination with morphine, for treatment of chronic pain.