Remo Guerrini

Pharmacology of nociceptin and its receptor: a novel therapeutic target

Nociceptin (NC), alias Orphanin FQ, has been recently identified as the endogenous ligand of the opioid receptor‐like 1 receptor (OP4). This new NC/OP4 receptor system belongs to the opioid family and has been characterized pharmacologically with functional and binding assays on native (mouse, rat, guinea‐pig) and recombinant (human) receptors, by using specific and selective agonists (NC, NC(1–13)NH2) and a pure and competitive antagonist, [Nphe1]NC(1–13)NH2. The similar order of potency of agonists and affinity values of the antagonist indicate that the same receptor is present in the four species. OP4 is expressed in neurons, where it reduces activation of adenylyl cyclase and Ca2+ channels while activating K+ channels in a manner similar to opioids. In this way, OP4 mediates inhibitory effects in the autonomic nervous system, but its activities in the central nervous system can be either similar or opposite to those of opioids. In vivo experiments have demonstrated that NC modulates a variety of biological functions ranging from nociception to food intake, from memory processes to cardiovascular and renal functions, from spontaneous locomotor activity to gastrointestinal motility, from anxiety to the control of neurotransmitter release at peripheral and central sites. These actions have been demonstrated using NC and various pharmacological tools, as antisense oligonucleotides targeting OP4 or the peptide precursor genes, antibodies against NC, an OP4 receptor selective antagonist and with data obtained from animals in which the receptor or the peptide precursor genes were knocked out. These new advances have contributed to better understanding of the pathophysiological role of the NC/OP4 system, and ultimately will help to identify the therapeutic potential of new OP4 receptor ligands.

Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic

Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the ‘classical’ opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes (∼60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand–receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP–compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.

The α-to-β Conformational Transition of Alzheimer’s Aβ-(1–42) Peptide in Aqueous Media is Reversible: A Step by Step Conformational Analysis Suggests the Location of β Conformation Seeding

Current views of the role of β‐amyloid (Aβ) peptide fibrils range from regarding them as the cause of Alzheimers pathology to having a protective function. In the last few years, it has also been suggested that soluble oligomers might be the most important toxic species. In all cases, the study of the conformational properties of Aβ peptides in soluble form constitutes a basic approach to the design of molecules with “antiamyloid” activity. We have experimentally investigated the conformational path that can lead the Aβ‐(1–42) peptide from the native state, which is represented by an α helix embedded in the membrane, to the final state in the amyloid fibrils, which is characterized by β‐sheet structures. The conformational steps were monitored by using CD and NMR spectroscopy in media of varying polarities. This was achieved by changing the composition of water and hexafluoroisopropanol (HFIP). In the presence of HFIP, β conformations can be observed in solutions that have very high water content (up to 99 % water; v/v). These can be turned back to α helices simply by adding the appropriate amount of HFIP. The transition of Aβ‐(1–42) from α to β conformations occurs when the amount of water is higher than 80 % (v/v). The NMR structure solved in HFIP/H2O with high water content showed that, on going from very apolar to polar environments, the long N‐terminal helix is essentially retained, whereas the shorter C‐terminal helix is lost. The complete conformational path was investigated in detail with the aid of molecular‐dynamics simulations in explicit solvent, which led to the localization of residues that might seed β conformations. The structures obtained might help to find regions that are more affected by environmental conditions in vivo. This could in turn aid the design of molecules able to inhibit fibril deposition or revert oligomerization processes.

A new selective antagonist of the nociceptin receptor

[Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 has been tested in the electrically stimulated guinea pig ileum and mouse vas deferens, two nociceptin sensitive preparations. The new compound showed per se little or no effect in the two tissues, but it displaced to the right the concentration‐response curves of nociceptin in a concentration‐dependent manner. Schild analyses of the data indicated a competitive type of antagonism and pA2 values of 7.02 and 6.75 in the guinea‐pig ileum and the mouse vas deferens, respectively. At 10 μM [Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 does not modify either the inhibitory effect of deltorphin I (the selective δ opioid receptor agonist) in the mouse vas deferens or that of dermorphine (the selective μ opioid receptor agonist) in the guinea‐pig ileum. The present findings indicate that [Phe1Ψ(CH2‐NH)Gly2]NC(1‐13)NH2 is a selective antagonist of the nociceptin receptor.

Characterization of [Nphe1]nociceptin(1‐13)NH2, a new selective nociceptin receptor antagonist

Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a specific G‐protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identification and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe1]nociceptin(1‐13)NH2 acts as the first truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity. [Nphe1]nociceptin(1‐13)NH2 binds selectively to recombinant nociceptin receptors expressed in Chinese hamster ovary (CHO) cells (pKi 8.4) and competitively antagonizes the inhibitory effects of nociceptin (i) on cyclic AMP accumulation in CHO cells (pA2 6.0) and (ii) on electrically evoked contractions in isolated tissues of the mouse, rat and guinea‐pig with pA2 values ranging from 6.0 to 6.4. [Nphe1]nociceptin(1‐13)NH2 is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe1]nociceptin(1‐13)NH2 produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine‐induced analgesia. Collectively our data indicate that [Nphe1]nociceptin(1‐13)NH2, acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics.

[Nphe1,Arg14,Lys15]Nociceptin‐NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor

Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions by activating a specific G‐protein coupled receptor (NOP). Few molecules are available that selectively activate or block the NOP receptor. Here we describe the in vitro and in vivo pharmacological profile of a novel NOP receptor ligand, [Nphe1,Arg14,Lys15]N/OFQ‐NH2 (UFP‐101). UFP‐101 binds to the human recombinant NOP receptor expressed in Chinese hamster ovary (CHO) cells with high affinity (pKi 10.2) and shows more than 3000 fold selectivity over classical opioid receptors. UFP‐101 competitively antagonizes the effects of N/OFQ on GTPγ35S binding in CHOhNOP cell membranes (pA2 9.1) and on cyclic AMP accumulation in CHOhNOP cells (pA2 7.1), being per se inactive at concentrations up to 10 μM. In isolated peripheral tissues of mice, rats and guinea‐pigs, and in rat cerebral cortex synaptosomes preloaded with [3H]‐5‐HT, UFP‐101 competitively antagonized the effects of N/OFQ with pA2 values in the range of 7.3–7.7. In the same preparations, the peptide was inactive alone and did not modify the effects of classical opioid receptor agonists. UFP‐101 is also active in vivo where it prevented the depressant action on locomotor activity and the pronociceptive effect induced by 1 nmol N/OFQ i.c.v. in the mouse. In the tail withdrawal assay, UFP‐101 at 10 nmol produces per se a robust and long lasting antinociceptive effect. UFP‐101 is a novel, potent and selective NOP receptor antagonist which appears to be a useful tool for future investigations of the N/OFQ‐NOP receptor system.

Neuropeptide S is a stimulatory anxiolytic agent: a behavioural study in mice

Neuropeptide S (NPS) was recently identified as the endogenous ligand of an orphan receptor, now referred to as the NPS receptor. In vivo, NPS produces a unique behavioural profile by increasing wakefulness and exerting anxiolytic‐like effects. In the present study, we further evaluated the effects of in vivo supraspinal NPS in mice.

Characterization of nociceptin receptors in the periphery: in vitro and in vivo studies.

Nociceptin (NC), a series of NC fragments, naloxone as well as the pseudopeptide [Phe1Ψ(CH2-NH)Gly2]NC(1–13)NH2 ([F/G]NC(1–13)NH2) were used to characterize NC receptors in peripheral isolated organs and in vivo. Experiments on isolated organs were performed in the mouse (mVD) and rat (rVD) vas deferens (noradrenergic nerve terminals), in the guinea pig ileum (gpI; cholinergic nerves) and in the renal pelvis (gpRP; sensory nerves), and, in vivo, by measuring the blood pressure (BP) and heart rate (HR) in anaesthetised rats. NC, NCNH2 and NC(1–13)NH2 acted as full agonists with similar affinities, while shorter fragments (e.g. NC(1–12)NH2, NC(1–9)NH2, NC(1–5)NH2) were much weaker or inactive. The inhibitory effects of NC were not modified by naloxone. [F/G]NC(1–13)NH2 acted as an antagonist with similar pA2-values (6.75 mVD, 6.83 rVD, 7.26 gpI) in the three species. In addition, it blocked NC actions in the rat in vivo. Linear Schild plots with slopes near to unity indicated that [F/G]NC(1–13)NH2 is a competitive antagonist, specific for NC receptors both in vitro (since it was inactive on opioid receptors) and in vivo (since it was inactive against carbachol). [F/G]NC(1–13)NH2 showed a residual agonistic activity in vitro (α = 0.2-0.3 in the rVD and gpI) and especially in vivo (α = 0.4 BP, 0.2 HR). These pharmacological data indicate that NC and related peptides exert their inhibitory effects in peripheral organs of various species by activating the same receptor type. Moreover, [F/G]NC(1–13)NH2 appears to be a useful tool for receptor characterization and classification.

The mouse vas deferens: a pharmacological preparation sensitive to nociceptin

The newly discovered neuropeptide, nociceptin (alias orphanin FQ), was tested for its potential direct effects, as well as for its ability to modify the electrically evoked contractions in several isolated organs suspended in vitro. The electrically stimulated mouse vas deferens is a sensitive preparation on which nociceptin exerts an inhibitory effect which is not affected by naloxone. The mouse vas deferens is therefore proposed as a bioassay for nociceptin and related compounds.

Pharmacological characterization of the nociceptin receptor mediating hyperalgesia in the mouse tail withdrawal assay

1 The newly discovered neuropeptide nociceptin (NC) has recently been reported to be the endogenous ligand of the opioid‐like orphan receptor. Despite its structural similarity to opioids, when injected intracerebroventricularly (i.c.v.) in the mouse, NC exerts a direct hyperalgesic effect and reverses opioid‐induced analgesia. In the present investigation, these two effects of NC were evaluated under the same experimental conditions; in addition, a pharmacological characterization of the receptor mediating these central effects of NC was attempted. 2 NC caused a dose dependent (0.1–10 nmol/mouse), naloxone‐insensitive reduction of tail withdrawal latency with a maximal effect of about 50% of the reaction time observed in saline injected mice. In the same range of doses, NC inhibited morphine (1 nmol/mouse) induced analgesia. 3 The effects of the natural peptide were mimicked by NCNH2 and NC(1–13)NH2 (all tested at 1 nmol/mouse) while 1 nmol NC(1–9)NH2 was found to be inactive either in reducing tail withdrawal latency or in preventing morphine analgesia. 4 [Phe1ψ(CH2‐NH)Gly2]NC(1–13)NH2 ([F/G]NC(1–13)NH2), which has been shown to antagonize NC effects in the mouse vas deferens, acted as an agonist, mimicking NC effects in both the experimental paradigms. In addition, when NC and [F/G]NC(1–13)NH2 were given together, their effects were additive. 5 These results demonstrate that both the direct hyperalgesic action and the anti‐morphine effect of NC can be studied under the same experimental conditions in the mouse tail withdrawal assay. Moreover, the pharmacological characterization of the NC functional site responsible for these actions compared with the peripherally active site, indicates the existence of important differences between peripheral and central NC receptors.