Pharmacological profile of the neuropeptide S receptor: Dynamic mass redistribution studies

Abstract Neuropeptide S (NPS) is the endogenous ligand of the neuropeptide S receptor (NPSR). NPS modulates several biological functions including anxiety, wakefulness, pain, and drug abuse. The aim of this study was the investigation of the pharmacological profile of NPSR using the dynamic mass redistribution (DMR) assay. DMR is a label‐free assay that offers a holistic view of cellular responses after receptor activation. HEK293 cells stably transfected with the murine NPSR (HEK293mNPSR) have been used. To investigate the nature of the NPS‐evoked DMR signaling, FR900359 (Gq inhibitor), pertussis toxin (Gi inhibitor), and rolipram (phosphodiesterase inhibitor) were used. To determine the pharmacology of NPSR, several selective ligands (agonists, partial agonists, antagonists) have been tested. NPS, through selective NPSR activation, evoked a robust DMR signal with potency in the nanomolar range. This signal was predominantly, but not completely, blocked by FR900359, suggesting the involvement of the Gq‐dependent signaling cascade. NPSR ligands (agonists and antagonists) displayed potency values in DMR experiments similar, but not identical, to those reported in the literature. Furthermore, partial agonists produced a higher efficacy in DMR than in calcium experiments. DMR can be successfully used to study the pharmacology and signaling properties of novel NPSR ligands. This innovative approach will likely increase the translational value of in vitro pharmacological studies.


| INTRODUCTION
Neuropeptide S (NPS, primary sequence in humans: SFRNGVGTG MKKTSFQRAKS) was identified in 2002 as the endogenous ligand of the previously orphaned G protein-coupled receptor (GPCR) GPR154, now referred to as neuropeptide S receptor (NPSR), using the reverse pharmacological approach. In 2004, an elegant study by Xu et al described, for the first time, some functional features of the NPS/ NPSR system. 1 NPSR is a GPCR showing moderate homology to other members of the GPCR family. 2 The in vitro pharmacology of the human and mouse NPSR has been mainly studied in heterologous expression systems. These studies showed that NPS increases both intracellular calcium levels and cAMP accumulation with EC 50 values in the low nanomolar range. This indicates that NPSR can signal via both Gq and Gs pathways to increase cellular excitability. 1,3 In vivo, NPS has been shown to modulate several biological functions in rodents including stress, anxiety, social behavior, locomotor activity, wakefulness, food intake and gastrointestinal functions, memory processes, pain, and drug abuse (for reviews see 4 and 5).
Up to now, NPSR ligands have been characterized in vitro using single end-point assays, that is, calcium mobilization and cAMP accumulation. This approach might be reductionist providing incomplete pharmacological profiles and eventually biasing the translatability from medicinal chemistry to the biological level. Label-free assays now offer the possibility to have, in a noninvasive manner, a holistic view of cellular responses after receptor activation. Label-free assays use special biosensors (electron-conducting or light-diffracting plates) to translate the receptor-dependent holistic cellular response to physical parameters such as variations of impedance or modulations of wavelength shift of an incident light in real time. The dynamic mass redistribution (DMR) assay is a label-free approach based on an optical biosensor technology. 6,7 Using a resonant waveguide grating, DMR measures changes in the refractive index of the bottom portion of the cell layer. Several intracellular events can lead to changes in the cells refractive index, that is, protein recruitment, receptor internalization and recycling, second messenger alternation, cytoskeletal remodeling, and cell adhesion changes. 8 DMR has been already applied to study the pharmacological properties of new ligands acting at various GPCRs, such as histamine H 1 , 9,10 β 2 adrenergic, 11,12 muscarinic M 3 , 13 purinergic P2Y, 14 formyl peptide, 15 and protease-activated 16,17 receptors. Classical opioid 18,19 and the nociceptin/orphanin FQ peptide (NOP) 20 receptors were also investigated in DMR studies. Additionally, the DMR assay, together with different biochemical tools, has been successfully used for GPCRs signaling deconvolution studies. 7,9,21,22 The present study investigates the pharmacological profile and signaling of the murine NPSR expressed in human embryonic kidney 293 (HEK293) cells using the DMR assay. Importantly, this isoform of the receptor and these cells have been chosen based on the fact that all the compounds investigated in the present study have been previously characterized in calcium mobilization studies performed on HEK293 mNPSR cells, thus making possible a direct comparison of the DMR and calcium mobilization results.

| Cells
HEK293 cells stably expressing the murine NPSR receptor (HEK293 mNPSR ) were described previously. 3 Wild-type HEK293 cells were used as a control. Cells were maintained in Dulbecco's Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin and, 2 mmol L −1 L-glutamine. The medium was complemented with 100 mg/L hygromycin for HEK293 mNPSR . Cells were cultured at 37°C in 5% CO 2 humidified air. antagonists were added manually 25 minute before reading the 5minute baseline. After baseline establishment, NPS was injected and DMR signal was recorded for 60 minute. Antagonist properties of ligands were measured by assessing the concentration-response curve to NPS in the absence and in presence of a fixed concentration of compound. FR900359 was added 60 minute before NPS, rolipram was incubated for 90 minute before NPS, while PTX was added 24 hours before NPS. Maximum picometer (pm) modifications (peak measured at 60 minute time point) were used to determine agonist response after baseline normalization.

| Data analysis and terminology
All data were analyzed using Graph Pad Prism 6.0 (La Jolla, CA, USA). Concentration-response curves were fitted using the four parameters log logistic equation. Data are expressed as mean ± SEM of n experiments performed in duplicate and were analyzed using one-or two-way analysis of variance (ANOVA) followed by Dunnett's or Tukey's test for multiple comparisons wherever appropriate.
Agonist potency was expressed as pEC 50 , which is the negative logarithm to base 10 of the agonist molar concentration that produces 50% of the maximal possible effect of that agonist. Antagonists potencies were assayed at single concentrations against the concentration-response curve to NPS and their pA 2 was derived using the following equation: , where CR is the ratio between agonist potency (EC 50 ) in the presence and in absence of antagonist and [A] is the molar concentration of antagonist.

| DMR effects of NPS
In HEK293 mNPSR cells, NPS evoked a robust concentration-dependent DMR response, with pEC 50 of 8.78 (8.22-9.34) and maximal effect of 690 ± 39 pm ( Figure 1A). A representative trace of the NPS DMR signal is shown in Figure 1B. NPS was completely inactive in wild-type HEK293 cells (Table 1). To analyze the biochemical nature of the NPS response, the peptide was tested in the absence and presence of the Figure 2A), the Gi inhibitor PTX (100 ng mL −1 , Figure 2B), and, with the aim to investigate the Gs dependent pathway, the phosphodiesterase inhibitor rolipram (10 μmol L −1 , Figure 2C). FR900359 strongly, but not completely, reduced NPS effects in the DMR assay. Importantly, in parallel experiments performed in the same cells, FR900359 completely abolished the NPS stimulated calcium mobilization ( Figure 2D). Two-way ANOVA (treatment × assay) followed by the Tukey's multiple comparisons test, revealed that NPS 1 μmol L −1 + FR900359 evoked a significant response in the DMR but not in the calcium assay (treatment  results were obtained with rolipram that did not change NPS effects neither when tested alone nor in presence of FR900359. Of note, at this concentration, rolipram was able to significantly enhance the DMR response of the adenosine receptor agonist NECA 10 μmol L −1 from 229 ± 18 to 464 ± 58 pm (P ˂ 0.05, according to Student's t test) in HEK293 mNPSR cells.
Finally, [Ala 7 ]NPS was able to evoke a DMR response in HEK293 mNPSR with maximal effects similar to those of NPS but demonstrating~20 fold loss in potency (Figure 4). Of note, the shape of the DMR responses promoted by the above-mentioned NPSR ligands was similar to that recorded in response to NPS (right panels of Figures 3 and 4).

| DISCUSSION AND CONCLUSION
Preclinical studies suggest NPSR as a promising therapeutic target for the treatment of anxiety disorders, cognitive deficits, pain, and  This difference can be explained considering that ligand efficacy is a strongly system-dependent pharmacological parameter, that increases proportionally with increasing efficiency of the stimulus/response coupling. 39 Most probably in the DMR assay, signal amplification phenomena due to integration of all cellular events that follow receptor activation, make the stimulus/response coupling particularly efficient therefore leading to a relative overestimation of ligand efficacy. Of note, similar considerations can be done for the compound [D-Cys( t Bu) 5 ]NPS, that behaves as a pure NPSR antagonist in the calcium mobilization assay, 26 while in this study displayed robust residual agonist activity behaving as NPSR partial agonist.
Regarding NPSR agonists, another point that must be addressed is the pharmacological behavior of PWT1-NPS. This is a tetrabranched derivative of NPS that behaved in calcium experiments as an NPSR full agonist 3-fold more potent than the natural peptide.
The high in vitro potency of PWT1-NPS was associated with a high in vivo potency and long-lasting duration of action. 31 20 and in calcium mobilization studies performed in cells expressing chimeric G proteins. [42][43][44] Further studies are clearly needed to corroborate these initial findings and eventually to investigate the reasons underlying this tendency of the DMR assay to underestimate antagonist potency.
In conclusion, the results obtained in this study further corroborate the usefulness of the DMR assay for the investigation of the pharmacological profile of GPCRs, as well as their signaling properties. In particular, information from the present DMR studies complements data from previous calcium mobilization studies regarding pharmacological features that is, efficacy, potency, and selectivity of action of a large panel of NPSR ligands. The label-free nature of the DMR assay associated with its high sensitivity will likely allow in the near future to perform studies in cell lines and eventually in primary culture cells expressing the native NPSR, thus substantially increasing the translational value of in vitro pharmacological studies.

AUTHOR CONTRIBU TI ONS
Participated in research design: CR and GC.
Conducted experiments: CR and FF.
Contributed new reagents or analytic tools: RG, EM, RKR, and DP.
Performed data analysis: CR and FF.
Wrote or contributed to the writing of the manuscript: CR, GC, RG, and RKR. The author thank Mark Bird (University of Leicester) for proofreading the article.

CONFLI CT OF INTEREST
All the authors declare no conflicts of interest.