The History of N/OFQ and the NOP Receptor.

The discovery of nociceptin/orphanin FQ (N/OFQ) marks the genuine start of the reverse pharmacology era, when systematic hunting for ligands of orphan receptors began. The choice of this particular target was no coincidence as the orphan receptor ORL-1 displayed high similarity to known opioid receptors, and thus its elusive ligand held promise to find more than a ligand but a missing opioid peptide. N/OFQ indeed turned out to belong to the opioid peptide family, but with significant pharmacological and functional distinctions. The quest for understanding N/OFQ's physiological functions has produced some novel insights into stress regulation and many other body functions but is still ongoing almost 25 years after its discovery. This chapter highlights the early steps of orphan receptor research and some of the protagonists who helped to advance the field.

Neuropeptide S precursor knockout mice display memory and arousal deficits.

Activation of neuropeptide S (NPS) signaling has been found to produce arousal, wakefulness, anxiolytic-like behaviors, and enhanced memory formation. In order to further study physiological functions of the NPS system, we generated NPS precursor knockout mice by homologous recombination in embryonic stem cells. NPS-/- mice were viable, fertile, and anatomically normal, when compared to their wild-type and heterozygous littermates. The total number of NPS neurons-although no longer synthesizing the peptide - was not affected by the knockout, as analyzed in NPS-/- /NPSEGFP double transgenic mice. Analysis of behavioral phenotypes revealed significant deficits in exploratory activity in NPS-/- mice. NPS precursor knockout mice displayed attenuated arousal in the hole board test, visible as reduced total nose pokes and number of holes inspected, that was not confounded by increased repetitive or stereotypic behavior. Importantly, long-term memory was significantly impaired in NPS-/- mice in the inhibitory avoidance paradigm. NPS precursor knockout mice displayed mildly increased anxiety-like behaviors in three different tests measuring responses to stress and novelty. Interestingly, heterozygous littermates often presented behavioral deficits similar to NPS-/- mice or displayed intermediate phenotype. These observations may suggest limited ligand availability in critical neural circuits. Overall, phenotypical changes in NPS-/- mice are similar to those observed in NPS receptor knockout mice and support earlier findings that suggest major functions of the NPS system in arousal, regulation of anxiety and stress, and memory formation.

G protein-coupled receptor deorphanizations.

G protein-coupled receptors (GPCRs) are major regulators of intercellular interactions. They initiate these actions by being activated by a wide variety of natural ligands. Historically, ligands were discovered first, but the advent of molecular biology reversed this trend. Most GPCRs are identified on the basis of their DNA sequences and thus are initially unmatched to known natural ligands. They are termed orphan GPCRs. Discovering their ligands-i.e., "deorphanizing" the GPCRs-gave birth to the field of reverse pharmacology. This review discusses the present status of GPCR deorphanization, presents a few examples of successes and surprises, and highlights difficulties encountered in these efforts.

Rapid elucidation of agonist-driven regulation of the neurokinin 1 receptor using a GPCR phosphorylation immunoassay.

Agonist-induced phosphorylation is a crucial step in the activation/deactivation cycle of G protein-coupled receptors (GPCRs), but direct determination of individual phosphorylation events has remained a major challenge. We have recently developed a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in 96-well plates, thus eliminating the need for western blot analysis. In the present study, we adapted this assay to three novel phosphosite-specific antibodies directed against the neurokinin 1 (NK1) receptor, namely pS338/pT339-NK1, pT344/pS347-NK1, and pT356/pT357-NK1. We found that substance P (SP) stimulated concentration-dependent phosphorylation of all three sites, which could be completely blocked in the presence of the NK1 receptor antagonist aprepitant. The other two endogenous ligands of the tachykinin family, neurokinin A (NKA) and neurokinin B (NKB), were also able to induce NK1 receptor phosphorylation, but to a much lesser extent than substance P. Interestingly, substance P promoted phosphorylation of the two distal sites more efficiently than that of the proximal site. The proximal site was identified as a substrate for phosphorylation by protein kinase C. Analysis of GPCR kinase (GRK)-knockout cells revealed that phosphorylation was mediated by all four GRK isoforms to similar extents at the T344/S347 and the T356/T357 cluster. Knockout of all GRKs resulted in abolition of all phosphorylation signals highlighting the importance of these kinases in agonist-mediated receptor phosphorylation. Thus, the 7TM phosphorylation assay technology allows for rapid and detailed analyses of GPCR phosphorylation.

Activation of neuropeptide S-expressing neurons in the locus coeruleus by corticotropin-releasing factor.

A recently discovered neurotransmitter system, consisting of neuropeptide S (NPS), NPS receptor, and NPS-expressing neurons in the brain stem, has received considerable interest due to its modulating influence on arousal, anxiety and stress responsiveness. Comparatively little is known about the properties of NPS-expressing neurons. Therefore in the present study, a transgenic mouse line expressing enhanced green fluorescent protein (EGFP) in NPS neurons was used to characterize the cellular and functional properties of NPS-expressing neurons located close to the locus coeruleus. Particular emphasis was on the influence of corticotropin-releasing factor (CRF), given previous evidence of stress-related activation of the NPS system. Upon acute immobilization stress, an increase in c-fos expression was detected immunocytochemically in brain stem NPS-EGFP neurons that also expressed the CRF receptor 1 (CRF1). NPS-EGFP neurons were readily identified in acute slice preparations and responded to CRF application with a membrane depolarization capable of triggering action potentials. CRF-induced responses displayed pharmacological properties indicative of CRF1 that were mediated by both a reduction in membrane potassium conductance and an increase in a non-specific cation conductance different from the hyperpolarization-activated cation conductance Ih, and involved protein kinase A signalling. In conclusion, stress exposure results in activation of brain stem NPS-expressing neurons, involving a CRF1-mediated membrane depolarization via at least two ionic mechanisms. These data provide evidence for a direct interaction between the CRF and the NPS system and thereby extend previous observations of NPS-modulated stress responsiveness towards a mechanistic level.

Neuropeptide S Encodes Stimulus Salience in the Paraventricular Thalamus.

Evaluation of stimulus salience is critical for any higher organism, as it allows for prioritizing of vital information, preparation of responses, and formation of valuable memory. The paraventricular nucleus of the thalamus (PVT) has recently been identified as an integrator of stimulus salience but the neurochemical basis and afferent input regarding salience signaling have remained elusive. Here we report that neuropeptide S (NPS) signaling in the PVT is necessary for stimulus salience encoding, including aversive, neutral and reinforcing sensory input. Taking advantage of a striking deficit of both NPS receptor (NPSR1) and NPS precursor knockout mice in fear extinction or novel object memory formation, we demonstrate that intra-PVT injections of NPS can rescue the phenotype in NPS precursor knockout mice by increasing the salience of otherwise low-intensity stimuli, while intra-PVT injections of NPSR1 antagonist in wild type mice partially replicates the knockout phenotype. The PVT appears to provide stimulus salience encoding in a dose- and NPS-dependent manner. PVT NPSR1 neurons recruit the nucleus accumbens shell and structures in the prefrontal cortex and amygdala, which were previously linked to the brain salience network. Overall, these results demonstrate that stimulus salience encoding is critically associated with NPS activity in the PVT.

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.

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening.

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.

Pharmacology, Physiology and Genetics of the Neuropeptide S System.

The Neuropeptide S (NPS) system is a rather 'young' transmitter system that was discovered and functionally described less than 20 years ago. This review highlights the progress that has been made in elucidating its pharmacology, anatomical distribution, and functional involvement in a variety of physiological effects, including behavior and immune functions. Early on, genetic variations of the human NPS receptor (NPSR1) have attracted attention and we summarize current hypotheses of genetic linkage with disease and human behaviors. Finally, we review the therapeutic potential of future drugs modulating NPS signaling. This review serves as an introduction to the broad collection of original research papers and reviews from experts in the field that are presented in this Special Issue.

Neuropeptide S-Mediated Modulation of Prepulse Inhibition Depends on Age, Gender, Stimulus-Timing, and Attention.

Conflicting reports about the role of neuropeptide S (NPS) in animal models of psychotic-like behavior and inconsistent results from human genetic studies seeking potential associations with schizophrenia prompted us to reevaluate the effects of NPS in the prepulse inhibition (PPI) paradigm in mice. Careful examination of NPS receptor (NPSR1) knockout mice at different ages revealed that PPI deficits are only expressed in young male knockout animals (<12 weeks of age), that can be replicated in NPS precursor knockout mice and appear strain-independent, but are absent in female mice. PPI deficits can be aggravated by MK-801 and alleviated by clozapine. Importantly, treatment of wildtype mice with a centrally-active NPSR1 antagonist was able to mimic PPI deficits. PPI impairment in young male NPSR1 and NPS knockout mice may be caused by attentional deficits that are enhanced by increasing interstimulus intervals. Our data reveal a substantial NPS-dependent developmental influence on PPI performance and confirm a significant role of attentional processes for sensory-motor gating. Through its influence on attention and arousal, NPS appears to positively modulate PPI in young animals, whereas compensatory mechanisms may alleviate NPS-dependent deficits in older mice.

Neandertal introgression and accumulation of hypomorphic mutations in the neuropeptide S (NPS) system promote attenuated functionality.

The neuropeptide S (NPS) system plays an important role in fear and fear memory processing but has also been associated with allergic and inflammatory diseases. Genes for NPS and its receptor NPSR1 are found in all tetrapods. Compared to non-human primates, several non-synonymous single-nucleotide polymorphisms (SNPs) occur in both human genes that collectively result in functional attenuation, suggesting adaptive mechanisms in a human context. To investigate historic and geographic origins of these hypomorphic mutations and explore genetic signs of selection, we analyzed ancient genomes and worldwide genotype frequencies of four prototypic SNPs in the NPS system. Neandertal and Denisovan genomes contain exclusively ancestral alleles for NPSR1 while all derived alleles occur in ancient genomes of anatomically modern humans, indicating that they arose in modern Homo sapiens. Worldwide genotype frequencies for three hypomorphic NPSR1 SNPs show significant regional homogeneity but follow a gradient towards increasing derived allele frequencies that supports an out-of-Africa scenario. Increased density of high-frequency polymorphisms around the three NPSR1 loci suggests weak or possibly balancing selection. A hypomorphic mutation in the NPS precursor, however, was detected at high frequency in Eurasian Neandertal genomes and shows genetic signatures indicating that it was introgressed into the human gene pool, particularly in Southern Europe, by interbreeding with Neandertals. We discuss potential evolutionary scenarios including behavior and immune-based natural selection.

Rapid assessment of G protein signaling of four opioid receptors using a real-time fluorescence-based membrane potential assay.

Opioids are the most powerful analgesics used clinically; however, severe side effects limit their long-term use. Various concepts involving biased intracellular signaling, partial agonism or multi-receptor targeting have been proposed to identify novel opioids with increased analgesic efficacy but reduced side effects. The search for such 'better opioids' implies screening of huge compound libraries and requires highly reliable, easy to perform and high throughput screening (HTS) assays. Here, we utilize an established membrane potential assay to monitor activation of G protein-coupled inwardly rectifying potassium (GIRK) channels, one of the main effectors of opioid receptor signaling, as readout to determine pharmacological profiles of opioids in a non-invasive manner. Specifically, in this study, we optimize assay conditions and extend the application of this assay to screen all four members of the opioid receptor family, stably expressed in AtT-20 and HEK293 cells. This ultra-sensitive system yielded EC50 values in the nano-molar range. We further validate this system for screening cells stably co-expressing two opioid receptors, which could be a valuable tool for investigating bi-functional ligands and studying interactions between receptors. Additionally, we demonstrate the utility of this assay to study antagonists as well as ligands with varying efficacies. Our results suggest that this assay could easily be up-scaled to HTS assay in order to efficiently study receptor activation and screen for novel opioids.

HA-MOP knockin mice express the canonical µ-opioid receptor but lack detectable splice variants.

G protein-coupled receptors (GPCRs) are notoriously difficult to detect in native tissues. In an effort to resolve this problem, we have developed a novel mouse model by fusing the hemagglutinin (HA)-epitope tag sequence to the amino-terminus of the µ-opioid receptor (MOP). Although HA-MOP knock-in mice exhibit reduced receptor expression, we found that this approach allowed for highly efficient immunodetection of low abundant GPCR targets. We also show that the HA-tag facilitates both high-resolution imaging and immunoisolation of MOP. Mass spectrometry (MS) confirmed post-translational modifications, most notably agonist-selective phosphorylation of carboxyl-terminal serine and threonine residues. MS also unequivocally identified the carboxyl-terminal 387LENLEAETAPLP398 motif, which is part of the canonical MOP sequence. Unexpectedly, MS analysis of brain lysates failed to detect any of the 15 MOP isoforms that have been proposed to arise from alternative splicing of the MOP carboxyl-terminus. For quantitative analysis, we performed multiple successive rounds of immunodepletion using the well-characterized rabbit monoclonal antibody UMB-3 that selectively detects the 387LENLEAETAPLP398 motif. We found that >98% of HA-tagged MOP contain the UMB-3 epitope indicating that virtually all MOP expressed in the mouse brain exhibit the canonical amino acid sequence.

Structure-Activity Relationship Studies on Oxazolo[3,4-a]pyrazine Derivatives Leading to the Discovery of a Novel Neuropeptide S Receptor Antagonist with Potent In Vivo Activity.

Neuropeptide S modulates important neurobiological functions including locomotion, anxiety, and drug abuse through interaction with its G protein-coupled receptor known as neuropeptide S receptor (NPSR). NPSR antagonists are potentially useful for the treatment of substance abuse disorders against which there is an urgent need for new effective therapeutic approaches. Potent NPSR antagonists in vitro have been discovered which, however, require further optimization of their in vivo pharmacological profile. This work describes a new series of NPSR antagonists of the oxazolo[3,4-a]pyrazine class. The guanidine derivative 16 exhibited nanomolar activity in vitro and 5-fold improved potency in vivo compared to SHA-68, a reference pharmacological tool in this field. Compound 16 can be considered a new tool for research studies on the translational potential of the NPSergic system. An in-depth molecular modeling investigation was also performed to gain new insights into the observed structure-activity relationships and provide an updated model of ligand/NPSR interactions.

Neuropeptide S stimulates dopaminergic neurotransmission in the medial prefrontal cortex.

Neuropeptide S (NPS) is known to produce anxiolytic-like effects and facilitate extinction of conditioned fear. Catecholaminergic neurotransmission in the medial prefrontal cortex (mPFC) has been suggested to be crucially involved in these brain functions. In the current study, we investigated the effect of NPS on the release of dopamine and serotonin in the mPFC by in vivo microdialysis in rats. Central administration of NPS dose-dependently enhanced extracellular levels of dopamine and its major metabolite 3,4-dihydroxyphenylacetic acid, with maximal effects lasting up to 120 min. In contrast, no effect on serotonergic neurotransmission was detected. Dopamine release in the mPFC has been previously linked to modulation of anxiety states and fear extinction. The present results may thus provide a physiological and anatomical basis for the reported effects of NPS on these behaviors.

In vitro and in vivo pharmacological characterization of the neuropeptide s receptor antagonist [D-Cys(tBu)5]neuropeptide S.

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as the NPS receptor (NPSR). In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [D-Cys(tBu)(5)]NPS was identified. [D-Cys(tBu)(5)]NPS up to 100 microM did not stimulate calcium mobilization in human embryonic kidney (HEK) 293 cells stably expressing the mouse NPSR; however, in a concentration-dependent manner, the peptide inhibited the stimulatory effects elicited by 10 and 100 nM NPS (pK(B), 6.62). In Schild analysis experiments [D-Cys(tBu)(5)]NPS (0.1-100 microM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 6.44. Ten micromolar [D-Cys(tBu)(5)]NPS did not affect signaling at seven NPSR unrelated G-protein-coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmol i.c.v. reduced the percentage of animals losing the RR in response to 15 mg/kg diazepam and their sleeping time. [d-Cys(tBu)(5)]NPS (1-10 nmol) was inactive per se but dose-dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal-promoting action of 1 nmol NPS could be detected in wild-type but not in mutant mice. In conclusion, [D-Cys(tBu)(5)]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist but with moderate potency. Moreover, using this tool together with receptor knockout mice studies, we demonstrated that the arousal-promoting action of NPS is because of the selective activation of the NPSR protein.

In-depth activation of channelrhodopsin 2-sensitized excitable cells with high spatial resolution using two-photon excitation with a near-infrared laser microbeam.

We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.

Neuropeptide S: a neuropeptide promoting arousal and anxiolytic-like effects.

Arousal and anxiety are behavioral responses that involve complex neurocircuitries and multiple neurochemical components. Here, we report that a neuropeptide, neuropeptide S (NPS), potently modulates wakefulness and could also regulate anxiety. NPS acts by activating its cognate receptor (NPSR) and inducing mobilization of intracellular Ca2+. The NPSR mRNA is widely distributed in the brain, including the amygdala and the midline thalamic nuclei. Central administration of NPS increases locomotor activity in mice and decreases paradoxical (REM) sleep and slow wave sleep in rats. NPS was further shown to produce anxiolytic-like effects in mice exposed to four different stressful paradigms. Interestingly, NPS is expressed in a previously undefined cluster of cells located between the locus coeruleus (LC) and Barrington's nucleus. These results indicate that NPS could be a new modulator of arousal and anxiety. They also show that the LC region encompasses distinct nuclei expressing different arousal-promoting neurotransmitters.

Synthesis and pharmacological in vitro and in vivo profile of 3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68), a selective antagonist of the neuropeptide S receptor.

Neuropeptide S (NPS) has been shown to modulate arousal, sleep wakefulness, anxiety-like behavior, and feeding after central administration of the peptide agonist to mice or rats. We report here the chemical synthesis and pharmacological characterization of SHA 66 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid benzylamide) and SHA 68 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide), two closely related bicyclic piperazines with antagonistic properties at the NPS receptor (NPSR). The compounds block NPS-induced Ca2+ mobilization, and SHA 68 shows displaceable binding to NPSR in the nanomolar range. The antagonistic activity of SHA 68 seems to be specific because it does not affect signaling at 14 unrelated G protein-coupled receptors. Analysis of pharmacokinetic parameters of SHA 68 demonstrates that the compound reaches pharmacologically relevant levels in plasma and brain after i.p. administration. Furthermore, peripheral administration of SHA 68 in mice (50 mg/kg i.p.) is able to antagonize NPS-induced horizontal and vertical activity as well as stereotypic behavior. Therefore, SHA 68 could be a useful tool to characterize physiological functions and pharmacological parameters of the NPS system in vitro and in vivo.

Synthesis and biological activity of human neuropeptide S analogues modified in position 2.

Neuropeptide S (NPS) has been identified as the endogenous ligand of a previously orphan receptor now named NPSR. Previous studies demonstrated that the N-terminal sequence Phe (2)-Arg(3)-Asn(4) of the peptide is crucial for biological activity. Here we report on a focused structure-activity study of Phe(2) which has been replaced with a series of coded and noncoded amino acids. Thirty-one human NPS analogues were synthesized and pharmacologically tested for intracellular calcium mobilization by using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated the following NPS position 2 structure-activity features: (i) lipophilicity but not aromaticity is crucial, (ii) both the size of the chemical moiety and its distance from the peptide backbone are important for biological activity, and (iii) this position plays a role in both receptor binding and activation, since [4,4'-biphenyl-Ala(2)]hNPS behaved as a partial agonist.