Structure-Based SAR in the Design of Selective or Bifunctional Nociceptin (NOP) Receptor Agonists.

The nociceptin opioid receptor (NOP), the fourth member of the opioid receptor family, and its endogenous peptide ligand, nociceptin or orphanin FQ (N/OFQ), play a vital role in several central nervous system pathways regulating pain, reward, feeding, anxiety, motor control and learning/memory. Both selective NOP agonists as well as bifunctional agonists at the NOP and mu opioid receptor (MOP) have potential therapeutic applications in CNS disorders related to these processes. Using Surflex-Dock protocols, we conducted a computational structure-activity study of four scaffold classes of NOP ligands with varying NOP-MOP selectivity. By docking these compounds into the orthosteric binding sites within an active-state NOP homology model, and an active-state MOP crystal structure, the goal of this study was to use a structure-based drug design approach to modulate NOP affinity and NOP vs. MOP selectivity. We first docked four parent compounds (no side chain) to determine their binding interactions within the NOP and MOP binding pockets. Various polar sidechains were added to the heterocyclic A-pharmacophore to modulate NOP ligand affinity. The substitutions mainly contained a 1-2 carbon chain with a polar substituent such as an amine, alcohol, sulfamide, or guanidine. The SAR analysis is focused on the impact of structural changes in the sidechain, such as chain length, hydrogen bonding capability, and basic vs neutral functional groups on binding affinity and selectivity at both NOP and MOP receptors. This study highlights structural modifications that can be leveraged to rationally design both selective NOP and bifunctional NOP-MOP agonists with different ratios of functional efficacy.

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.

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.

Buprenorphine requires concomitant activation of NOP and MOP receptors to reduce cocaine consumption.

Buprenorphine's clinical use is approved for the treatment of heroin addiction; however, evidence supporting its efficacy in cocaine abuse also exists. While for heroin it has been demonstrated that the effect of buprenorphine is mediated by its ability to activate µ-opioid peptide receptor (MOP) receptors, the mechanism through which it attenuates cocaine intake remains elusive. We explored this mechanism using operant models where rodents were trained to chronically self-administer cocaine for 2 hours daily. Buprenorphine (0.3, 1.0 and 3.0 mg/kg) given intraperitoneally 90 minutes before access to cocaine significantly and dose dependently reduced its intake. Pre-treatment with naltrexone or with the selective nociceptin/orphanin FQ peptide (NOP) antagonist SB-612111 did not prevent buprenorphine-induced reduction of cocaine intake. However, when naltrexone and SB-612111 were combined, the effect of buprenorphine on cocaine was completely prevented. To confirm that co-activation of MOP and NOP receptors is the underlying mechanism through which buprenorphine reduces cocaine intake, three compounds, namely, AT-034, AT-201 and AT-202, with a range of affinity and intrinsic activity profiles for MOP and NOP receptors, but weak ability for kappa-opioid peptide receptor (KOP) transmission, were tested. Consistent with our hypothesis based on buprenorphine's effects, results demonstrated that AT-034 and AT-201, which co-activate MOP and NOP receptors, reduced cocaine self-administration like buprenorphine. AT-202, which selectively stimulates NOP receptors, was not effective. Together, these data demonstrate that for buprenorphine, co-activation of MOP and NOP receptors is essential to reduce cocaine consumption. These results open new vistas on the treatment of cocaine addiction by developing compounds with mixed MOP/NOP agonist properties.

The α3ß4 nAChR partial agonist AT-1001 attenuates stress-induced reinstatement of nicotine seeking in a rat model of relapse and induces minimal withdrawal in dependent rats.

The strong reinforcing effects of nicotine and the negative symptoms such as anxiety experienced during a quit attempt often lead to relapse and low success rates for smoking cessation. Treatments that not only block the reinforcing effects of nicotine but also attenuate the motivation to relapse are needed to improve cessation rates. Recent genetic and preclinical studies have highlighted the involvement of the α3, ß4, and α5 nicotinic acetylcholine receptor (nAChR) subunits and the α3ß4 nAChR subtype in nicotine dependence and withdrawal. However, the involvement of these nAChR in relapse is not fully understood. We previously reported that the α3ß4 nAChR partial agonist AT-1001 selectively decreases nicotine self-administration in rats without affecting food responding. In the present experiments, we examined the efficacy of AT-1001 in attenuating reinstatement of nicotine-seeking behavior in a model of stress-induced relapse. Rats extinguished from nicotine self-administration were treated with the pharmacological stressor yohimbine prior to AT-1001 treatment and reinstatement testing. We also examined whether AT-1001 produced any withdrawal-related effects when administered to nicotine-dependent rats. We found that AT-1001 dose-dependently reduced yohimbine stress-induced reinstatement of nicotine seeking. When administered to nicotine-dependent rats at the dose that significantly blocked nicotine reinstatement, AT-1001 elicited minimal somatic withdrawal signs in comparison to the nicotinic antagonist mecamylamine, which is known to produce robust withdrawal. Our data suggest that α3ß4 nAChR-targeted compounds may be a promising approach for nicotine addiction treatment because they can not only block nicotine's reinforcing effects, but also decrease motivation to relapse without producing significant withdrawal effects.

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.

Small-molecule nociceptin receptor agonist ameliorates mast cell activation and pain in sickle mice.

Treatment of pain with morphine and its congeners in sickle cell anemia is suboptimal, warranting the need for analgesics devoid of side effects, addiction and tolerance liability. Small-molecule nociceptin opioid receptor ligands show analgesic efficacy in acute and chronic pain models. We show that AT-200, a high affinity nociceptin opioid receptor agonist with low efficacy at the mu opioid receptor, ameliorated chronic and hypoxia/reoxygenation-induced mechanical, thermal and deep tissue/musculoskeletal hyperalgesia in HbSS-BERK sickle mice. The antinociceptive effect of AT-200 was antagonized by SB-612111, a nociceptin opioid receptor antagonist, but not naloxone, a non-selective mu opioid receptor antagonist. Daily 7-day treatment with AT-200 did not develop tolerance and showed a sustained anti-nociceptive effect, which improved over time and led to reduced plasma serum amyloid protein, neuropeptides, inflammatory cytokines and mast cell activation in the periphery. These data suggest that AT-200 ameliorates pain in sickle mice via the nociceptin opioid receptor by reducing inflammation and mast cell activation without causing tolerance. Thus, nociceptin opioid receptor agonists are promising drugs for treating pain in sickle cell anemia.

AT-1001: a high-affinity α3ß4 nAChR ligand with novel nicotine-suppressive pharmacology.

BACKGROUND AND PURPOSE: The α3ß4 subtype of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating nicotine reinforcement processes. AT-1001 has been recently described as a high-affinity and selective α3ß4 nAChR antagonist that blocks nicotine self-administration in rats. The aim of this study was to investigate the mechanism of action underlying the nicotine-suppressive effects of AT-1001. EXPERIMENTAL APPROACH: Effects of AT-1001 were determined using in vitro assays and rat models of nicotine addiction, and compared with varenicline. KEY RESULTS: AT-1001 and its analogue AT-1012 were functionally selective as antagonists for α3ß4 over α4ß2 nAChRs, but not to the same extent as the binding selectivity, and had partial agonist activity at α3ß4 nAChRs. In contrast, varenicline was a partial agonist at α4ß2, a weak agonist at α3ß4 and inhibited α4ß2 at a much lower concentration than it inhibited α3ß4 nAChRs. AT-1001 and varenicline also had very different in vivo properties. Firstly, AT-1001 did not exhibit reinforcing properties per se while varenicline was self-administered. Secondly, systemic treatment with AT-1001 did not induce reinstatement of nicotine seeking but rather attenuated reinstatement induced by varenicline, as well as nicotine. Finally, unlike varenicline, AT-1001 selectively blocked nicotine self-administration without altering alcohol lever pressing as assessed in an operant co-administration paradigm. CONCLUSIONS AND IMPLICATIONS: These findings describe a more complex AT-1001 in vitro profile than previously appreciated and provide further support for the potential of AT-1001 and congeners as clinically useful compounds for smoking cessation, with a mechanism of action distinct from currently available medications.

Functional characterization of AT-1001, an α3ß4 nicotinic acetylcholine receptor ligand, at human α3ß4 and α4ß2 nAChR.

INTRODUCTION: Genome-wide association studies linking the α3, ß4, and α5 nicotinic acetylcholine receptor (nAChR) subunits to nicotine dependence suggest that α3ß4* nAChR may be targets for smoking cessation pharmacotherapies. We previously reported that AT-1001, a selective α3ß4* nAChR ligand binds with high affinity to rat α3ß4 and human α3ß4α5 nAChR, antagonizes epibatidine-induced activation of rat α3ß4 nAChR in HEK cells and potently inhibits nicotine self-administration in rats. METHODS: Two-electrode voltage clamp was used for functional characterization of AT-1001 at recombinant human α3ß4 and α4ß2 nAChR expressed in Xenopus oocytes. RESULTS: Concentration-response curves show that AT-1001 is a partial agonist at human α3ß4 nAChR, evoking up to 35% of the maximal acetylcholine (ACh) response (50% effective concentration [EC50] = 0.37 µM). AT-1001 showed very little agonist activity at the α4ß2 nAChR, evoking only 6% of the ACh response (EC50 = 1.5 µM). Pre- and co-application of various concentrations of AT-1001 with 50 µM ACh revealed a complex pattern of activation-inhibition by AT-1001 at α3ß4 nAChR, which was best fitted by a 2-site equation. At α4ß2 nAChR, co-exposure of AT-1001 with ACh only showed inhibition of ACh current with a shallower curve. CONCLUSIONS: AT-1001 is a partial agonist at the human α3ß4 nAChR and causes desensitization at concentrations at which it evokes an inward current, resulting in an overall functional antagonism of α3ß4 nAChR. AT-1001 does not significantly activate or desensitize α4ß2 nAChR at the same concentrations as at the α3ß4 nAChR, but does inhibit ACh responses at α4ß2 nAChR at higher concentrations. A combination of these mechanisms may underlie the inhibition of nicotine self-administration by AT-1001, suggesting that AT-1001 and compounds from this class may have clinical potential for smoking cessation pharmacotherapy.