Influence of neuropathic pain on nicotinic acetylcholine receptor plasticity and behavioral responses to nicotine in rats.

Tobacco smoking is particularly evident in individuals experiencing chronic pain. This complex relationship is poorly understood at both molecular and behavioral levels. Here, we describe experiments aimed at understanding whether a chronic pain state induces neuroadaptations into the brain or peripheral nerves that involve nicotinic acetylcholine receptors (nAChRs) and whether these neuroadaptations directly lead to increased vulnerability to nicotine addiction or to the development of coping strategies to relieve pain symptoms. We found that ligation of the rat L5 spinal nerve led to a dramatic downregulation in the mRNA expression levels of all nAChR subunits examined in dorsal root ganglia and a time-dependent downregulation of discrete subunits, particularly in the cingulate cortex and the amygdala. Spinal nerve ligation and sham-operated rats showed minor or no changes in patterns of acquisition and motivation for nicotine taking. Spinal nerve ligation rats also showed similar vulnerability to nicotine seeking as sham animals when reinstatement was induced by nicotine-associated cues, but failed to reinstate lever pressing when relapse was induced by nicotine priming. Spinal nerve ligation and sham rats were equally sensitive to nicotine-induced anxiety-like behavior and antinociception; however, nicotine produced a potent and long-lasting antiallodynic effect in spinal nerve ligation rats. These results demonstrate that chronic pain leads to plasticity of nAChRs that do not directly facilitate nicotine addictive behaviors. Instead, nicotine potently decreases allodynia, an effect that could lead to increased nicotine consumption in chronic pain subjects.

Differential regulation of alcohol taking and seeking by antagonism at α4ß2 and α3ß4 nAChRs.

RATIONALE: Alcoholism is a serious public health problem throughout the world. Current pharmacotherapies for the treatment of this disorder are poorly effective. Preclinical and clinical findings point to nicotinic acetylcholine receptors (nAChRs) as a promising target for the development of novel and effective medications. Assuage Pharmaceuticals, in collaboration with Torrey Pines Institute for Molecular Studies, has discovered a new class of potent and selective α4ß2 nAChR antagonists. OBJECTIVE: Here, it was hypothesized that α4ß2 nAChR antagonism is a viable approach for treatment of alcohol use disorders. RESULTS: When tested in rats, one lead compound, AP-202, attenuated both operant alcohol and nicotine self-administration in a paradigm in which the two reinforcers were concurrently available. The conotoxin TP2212-59, a selective α3ß4 nAChR antagonist, was only effective in reducing nicotine self-administration. AP-202 also reduced alcohol but not food responding when alcohol was presented as the only reinforcer, whereas the commercially available α4ß2 nAChR antagonist dihydro-ß-erythroidine failed to alter alcohol self-administration. AP-202 did not block relapse-like behavior induced by previously alcohol-associated stimuli or yohimbine stress. In a reinstatement paradigm, in which alcohol seeking was triggered by a nicotine challenge, a behavior successfully inhibited by the nonselective nAChR antagonist mecamylamine, AP-202 was not effective, while pretreatment with TP2212-59 abolished nicotine-induced reinstatement of alcohol seeking. CONCLUSIONS: These findings suggest differential roles for α4ß2 and α3ß4 nAChR on alcohol taking and seeking with selective blockade of α4ß2 nAChR being more implicated in modulating alcohol taking while selective blockade of α3ß4 nAChR is involved in nicotine-induced alcohol seeking.

Highly Selective and Potent α4ß2 nAChR Antagonist Inhibits Nicotine Self-Administration and Reinstatement in Rats.

The α4ß2 nAChR is the most predominant subtype in the brain and is a well-known culprit for nicotine addiction. Previously we presented a series of α4ß2 nAChR selective compounds that were discovered from a mixture-based positional-scanning combinatorial library. Here we report further optimization identified highly potent and selective α4ß2 nAChR antagonists 5 (AP-202) and 13 (AP-211). Both compounds are devoid of in vitro agonist activity and are potent inhibitors of epibatidine-induced changes in membrane potential in cells containing α4ß2 nAChR, with IC50 values of approximately 10 nM, but are weak agonists in cells containing α3ß4 nAChR. In vivo studies show that 5 can significantly reduce operant nicotine self-administration and nicotine relapse-like behavior in rats at doses of 0.3 and 1 mg/kg. The pharmacokinetic data also indicate that 5, via sc administration, is rapidly absorbed into the blood, reaching maximal concentration within 10 min with a half-life of less than 1 h.

A key role for the N/OFQ-NOP receptor system in modulating nicotine taking in a model of nicotine and alcohol co-administration.

Alcohol and nicotine are often co-abused. Although the N/OFQ-NOP receptor system is considered a potential target for development of drug abuse pharmacotherapies, especially for alcoholism, little is known about the role of this system in nicotine dependence. Furthermore, the effect of prior history of nicotine dependence on subsequent nicotine and alcohol taking is understudied. Using an operant co-administration paradigm, in which rats concurrently self-administer nicotine and alcohol, we found that nicotine dependent rats increased nicotine self-administration over time as compared to non-dependent animals, while patterns of alcohol lever pressing did not change between groups. Pretreatment with the potent NOP receptor agonist AT-202 (0.3-3 mg/kg) increased nicotine lever pressing of both dependent and non-dependent groups, whereas the selective antagonist SB612111 (1-10 mg/kg) elicited a clear reduction of nicotine responses, in both dependent and non-dependent rats. In parallel, AT-202 only produced minor changes on alcohol responses and SB612111 reduced alcohol taking at a dose that also reduced locomotor behavior. Results indicate that a history of nicotine dependence affects subsequent nicotine- but not alcohol-maintained responding, and that NOP receptor antagonism, rather than agonism, blocks nicotine self-administration, which strongly suggests a critical role for the endogenous N/OFQ in the modulation of nicotine reinforcement processes.

Varenicline decreases nicotine but not alcohol self-administration in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats.

BACKGROUND: Alcohol and nicotine are largely co-abused. Here, we investigated whether concurrent exposure to both addictive drugs influences each other's consumption and whether varenicline attenuates alcohol consumption in the presence of nicotine. METHODS: Marchigian Sardinian alcohol-preferring (msP) rats trained to simultaneously self-administer oral alcohol (10% v/v) and intravenous nicotine (30µg/kg/inf) were used. Additional groups of rats were trained to self-administer either alcohol or nicotine. Further, msP rats were also trained to self-administer nicotine followed by 22-h/day access to alcohol and water in a two bottle free choice paradigm or water alone. The effects of varenicline (0.0, 0.3, 1.0, 3.0mg/kg, p.o.) on alcohol and nicotine consumption were tested. RESULTS: In a self-administration paradigm, msP rats showed a significantly high level of alcohol and nicotine intake when the drugs were administered alone. However, when access to both drugs occurred concomitantly, the number of nicotine infusions self-administered was significantly decreased. Nicotine self-administration was markedly reduced by varenicline regardless of whether it was self-administered alone or concurrently with alcohol. In a two bottle choice test, varenicline significantly decreased nicotine self-administration but had no influence on alcohol consumption. CONCLUSION: Varenicline is highly efficacious in decreasing nicotine self-administration either alone or in combination with alcohol. However, varenicline failed to influence both operant responding for alcohol and home-cage alcohol drinking in msP animals. Taken together, our findings suggest that the effects of varenicline could be specific to nicotine under conditions where excessive alcohol drinking is facilitated by genetic factors as in msP rats.

Activation of ß2-adrenergic receptor by (R,R')-4'-methoxy-1-naphthylfenoterol inhibits proliferation and motility of melanoma cells.

(R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a highly-selective ß2 adrenergic receptor (ß2-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R')-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R')-MNF positively correlated with the ß2-AR expression levels across tested cell lines. The anti-motility activity of (R,R')-MNF was inhibited by the ß2-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20-1724 mimicked the ability of (R,R')-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R')-MNF caused significant inhibition of cell growth in ß2-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon ß2-AR activation with (R,R')-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified ß2-AR as a novel potential target for melanoma management, and (R,R')-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in ß2-AR-expressing lesions.

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.

Scaffold ranking and positional scanning utilized in the discovery of nAChR-selective compounds suitable for optimization studies.

Nicotine binds to nicotinic acetylcholine receptors (nAChR), which can exist as many different subtypes. The α4ß2 nAChR is the most prevalent subtype in the brain and possesses the most evidence linking it to nicotine seeking behavior. Herein we report the use of mixture based combinatorial libraries for the rapid discovery of a series of α4ß2 nAChR selective compounds. Further chemistry optimization provided compound 301, which was characterized as a selective α4ß2 nAChR antagonist. This compound displayed no agonist activity but blocked nicotine-induced depolarization of HEK cells with an IC50 of approximately 430 nM. 301 demonstrated nearly 500-fold selectivity for binding and 40-fold functional selectivity for α4ß2 over α3ß4 nAChR. In total over 5 million compounds were assessed through the use of just 170 samples in order to identify a series of structural analogues suitable for future optimization toward the goal of developing clinically relevant smoking cessation medications.

Oxidative folding and preparation of α-conotoxins for use in high-throughput structure-activity relationship studies.

α-Conotoxins are peptide neurotoxins that selectively inhibit various subtypes of nicotinic acetylcholine receptors. They are important research tools for studying numerous pharmacological disorders, with profound potential for developing drug leads for treating pain, tobacco addiction, and other conditions. They are characterized by the presence of two disulfide bonds connected in a globular arrangement, which stabilizes a bioactive helical conformation. Despite extensive structure-activity relationship studies that have produced α-conotoxin analogs with increased potency and selectivity towards specific nicotinic acetylcholine receptor subtypes, the efficient production of diversity-oriented α-conotoxin combinatorial libraries has been limited by inefficient folding and purification procedures. We have investigated the optimized conditions for the reliable folding of α-conotoxins using simplified oxidation procedures for use in the accelerated production of synthetic combinatorial libraries of α-conotoxins. To this end, the effect of co-solvent, redox reagents, pH, and temperature on the proportion of disulfide bond isomers was determined for α-conotoxins exhibiting commonly known Cys loop spacing frameworks. In addition, we have developed high-throughput 'semi-purification' methods for the quick and efficient parallel preparation of α-conotoxin libraries for use in accelerated structure-activity relationship studies. Our simplified procedures represent an effective strategy for the preparation of large arrays of correctly folded α-conotoxin analogs and permit the rapid identification of active hits directly from high-throughput pharmacological screening assays.

Cannabinoid receptor activation correlates with the proapoptotic action of the ß2-adrenergic agonist (R,R')-4-methoxy-1-naphthylfenoterol in HepG2 hepatocarcinoma cells.

Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with ß(2)-adrenergic receptor (ß(2)-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the ß(2)-AR agonists (R,R')-fenoterol (Fen) and (R,R')-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [(3)H]thymidine incorporation assays. Despite the expression of ß(2)-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a ß(2)-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of ß(2)-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gα(i)/Gα(o)-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB(1)R and CB(2)R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The ß(2)-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting ß(2)-AR-CBR ligand.

Nociceptin/orphanin FQ inhibition with SB612111 ameliorates dextran sodium sulfate-induced colitis.

Inflammatory bowel diseases, primarily Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract with unknown etiology. The majority of current therapeutic agents focus on controlling proinflammatory molecules. The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been described as a potential immunomodulator for inflammatory bowel diseases. In this study, we asked whether the small molecule N/OFQ antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB612111) would inhibit the development of dextran sodium sulfate-induced colitis in C57BL/6 mice. Inhibition of the N/OFQ receptor (NOP) by SB612111 significantly ameliorated the clinical disease course in these animals, as indicated by reduced fecal bleeding, improved recovery from diarrhea and weight loss, and a reduction in histopathological alterations. In addition, the inflammatory response in the colon was diminished, as demonstrated by reduced cytokine protein and messenger RNA expression for CXCL1/keratinocyte-derived chemokine, interferon-γ, interleukin-1ß, interleukin-6, and tumor necrosis factor-α, some of which are known targets for the treatment of this devastating disease. Our results strongly support a role for the receptor-ligand pair NOP-N/OFQ in the pathogenesis of colitis. We conclude that inhibition of NOP receptors with small molecule inhibitors may constitute a novel, urgently needed approach for the treatment of inflammatory bowel diseases.

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.

Peptides derived from the prohormone proNPQ/spexin are potent central modulators of cardiovascular and renal function and nociception.

Computational methods have led two groups to predict the endogenous presence of a highly conserved, amidated, 14-aa neuropeptide called either spexin or NPQ. NPQ/spexin is part of a larger prohormone that contains 3 sets of RR residues, suggesting that it could yield more than one bioactive peptide; however, no in vivo activity has been demonstrated for any peptide processed from this precursor. Here we demonstrate biological activity for two peptides present within proNPQ/spexin. NPQ/spexin (NWTPQAMLYLKGAQ-NH(2)) and NPQ 53-70 (FISDQSRRKDLSDRPLPE) have differing renal and cardiovascular effects when administered intracerebroventricularly or intravenously into rats. Intracerebroventricular injection of NPQ/spexin produced a 13 ± 2 mmHg increase in mean arterial pressure, a 38 ± 8 bpm decrease in heart rate, and a profound decrease in urine flow rate. Intracerebroventricular administration of NPQ 53-70 produced a 26 ± 9 bpm decrease in heart rate with no change in mean arterial pressure, and a marked increase in urine flow rate. Intraventricular NPQ/spexin and NPQ 53-70 also produced antinociceptive activity in the warm water tail withdrawal assay in mice (ED(50)<30 and 10 nmol for NPQ/spexin and NPQ 53-70, respectively). We conclude that newly identified peptides derived from the NPQ/spexin precursor contribute to CNS-mediated control of arterial blood pressure and salt and water balance and modulate nociceptive responses.

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.

Effect of fenoterol stereochemistry on the ß2 adrenergic receptor system: ligand-directed chiral recognition.

The ß(2) adrenergic receptor (ß(2)-AR) is a model system for studying the ligand recognition process in G protein-coupled receptors. Fenoterol (FEN) is a ß(2)-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van't Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x')-isomers is almost entirely enthalpy controlled whereas binding of (R,x')-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R')-FEN was shown to selectively activate G(s) protein signaling while the (S,R')-isomer activated both G(i) and G(s) protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site, and the global mechanism of ß(2)-AR activation. Differences in thermodynamic parameters and nonuniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x')-FEN stereoisomers with a different receptor conformation than the one with which the (S,x')-isomer interacts.

Orphanin FQ/nociceptin activates nuclear factor kappa B.

Endogenous neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, nociceptin orphanin FQ peptide receptor (NOPr), play a modulatory role throughout the body including nociceptive sensitivity, motor function, spatial learning, and the immune system. NOPr is an inhibitory G protein coupled receptor (GPCR) that modulates expression and release of inflammatory mediators from immune cells and in the CNS. Inhibitory GPCRs have been shown to activate the immune and central nervous system regulator, nuclear factor kappa B (NFκB), whose family consists of several subunits. When activated, NFκB translocates to the nucleus and can modify transcription. To determine if OFQ/N modulates NFκB activity, SH-SY5Y human neuroblastoma cells were treated with OFQ/N and assessed for changes in nuclear accumulation, DNA binding, and transcriptional activation. For the first time, we show that OFQ/N increases the nuclear accumulation (1.9-2.8-fold) and the DNA binding of NFκB (2.9-fold) by 2 h as determined by immunoblotting and electromobility shift assay, respectively. OFQ/N induction of NFκB binding to DNA is protein kinase C-dependent and NOPr-specific. OFQ/N stimulated binding of both NFκB p50 and p65 subunits to their consensus binding site on DNA. OFQ/N also induces transcriptional activation of an NFκB reporter gene 2.2-fold by 2 h with an EC(50) of 6.3 nM. This activation of NFκB by OFQ/N suggests a likely mechanism for its modulation of the central nervous and immune systems.

Evolutionary sequence modeling for discovery of peptide hormones.

There are currently a large number of "orphan" G-protein-coupled receptors (GPCRs) whose endogenous ligands (peptide hormones) are unknown. Identification of these peptide hormones is a difficult and important problem. We describe a computational framework that models spatial structure along the genomic sequence simultaneously with the temporal evolutionary path structure across species and show how such models can be used to discover new functional molecules, in particular peptide hormones, via cross-genomic sequence comparisons. The computational framework incorporates a priori high-level knowledge of structural and evolutionary constraints into a hierarchical grammar of evolutionary probabilistic models. This computational method was used for identifying novel prohormones and the processed peptide sites by producing sequence alignments across many species at the functional-element level. Experimental results with an initial implementation of the algorithm were used to identify potential prohormones by comparing the human and non-human proteins in the Swiss-Prot database of known annotated proteins. In this proof of concept, we identified 45 out of 54 prohormones with only 44 false positives. The comparison of known and hypothetical human and mouse proteins resulted in the identification of a novel putative prohormone with at least four potential neuropeptides. Finally, in order to validate the computational methodology, we present the basic molecular biological characterization of the novel putative peptide hormone, including its identification and regional localization in the brain. This species comparison, HMM-based computational approach succeeded in identifying a previously undiscovered neuropeptide from whole genome protein sequences. This novel putative peptide hormone is found in discreet brain regions as well as other organs. The success of this approach will have a great impact on our understanding of GPCRs and associated pathways and help to identify new targets for drug development.

Regulation of the prepronociceptin gene and its effect on neuronal differentiation.

Nociceptin/orphanin FQ (NOP/OFQ) is the endogenous ligand for the NOP receptor and is processed from a precursor protein in the family of opioid peptides. Prepronociceptin (ppN/OFQ) mRNA has been shown to be upregulated by an increase in cAMP, a treatment that leads to differentiation of NS20Y neuroblastoma cells. Although a large increase in endogenous ppN/OFQ mRNA upon cAMP stimulation can be shown in cellular systems, a similar increase cannot be expressed in pGL3 luciferase vector containing 1.3 kb proximal promoter, suggesting that a larger portion of the sequence or a different chromatin structure is necessary for a fully functional promoter. The induction of ppN/OFQ mRNA by cAMP appears to be mediated by a cAMP-response element. Chromatin immunoprecipitation (ChIP) assays show that CREB is recruited to the promoter region upon treatment of NS20Y cells with dibutyryl cAMP. In addition, the production of ppN/OFQ mRNA is regulated by histone acetylation, also through CREB, as the histone deacetylase (HDAC) inhibitor trichostatin A increases both CREB binding to the promoter and ppN/OFQ mRNA expression. In rat progenitor and mouse neuroblastoma cell lines, agents that increase ppN/OFQ mRNA expression also induce neurite outgrowth, suggesting a close relationship between ppN/OFQ and cellular differentiation.

Regulation of transcription of the human prepronociceptin gene by Sp1.

Nociceptin/orphanin FQ is a recently discovered neuropeptide and the endogenous ligand for opioid receptor-like-1. The promoter region of the precursor protein prepronociceptin (ppN/OFQ) has been cloned and sequenced. We have previously shown that a stretch of 110 bases immediately 5' to the first intron 23 bp upstream of the ATG start codon is responsible for significant enhancement of transcription of the human ppN/OFQ gene. We performed electromobility shift assays (EMSAs) using oligonucleotides spanning portions of the promoter region close to the intron to determine which DNA elements were important for transcriptional regulation. EMSAs using Sp1 antibody revealed a cis-acting regulatory element from bases 35-67 that appeared to bind Sp1 transcription factor and cause a shift to higher molecular weight. Deletion of this 30-bp region of DNA from the 1.2 kb promoter caused a significant loss of transcription as measured by luciferase reporter assays. Mutation of four bases at the Sp1 binding site also induced a significant loss of transcription compared to wildtype constructs. Finally, an Sp1- but not Etf-binding consensus oligonucleotide was able to compete with the interaction of the oligo with the NS20Y nuclear extract. Combined with the data from the supershift EMSAs, it appears that Sp1 is the transcription factor binding to the GC region close to the intron to regulate transcription of the human ppN/OFQ gene.