Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acid peptide, Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. The receptor is highly expressed in brain areas that have been implicated in modulation of arousal, stress and anxiety. Central administration of NPS in mice produces an unusual profile of activity by inducing wakefulness and arousal, while at the same time suppressing anxiety. Therefore, NPSR may represent a novel drug target for the treatment of sleep and anxiety disorders. The relative importance of Gs and Gq G protein-coupled signaling to NPSR action is not known. The goal of the project is to generate a series of compounds that can selectively antagonize these signaling pathways. ML079 (CID-3719993) is one compound from a series of congeners satisfies this criteria and can be used to dissect the role these pathways play in NPSR biology.
Assigned Assay Grant #: X01-DA026210-01
Screening Center Name & PI: NIH Chemical Genomics Center, Christopher Austin
Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher Austin
Assay Submitter & Institution: Markus Heilig, National Institute on Alcohol Abuse and Alcoholism
PubChem Summary Bioassay Identifier (AID): AID-1461
Recommendations for the scientific use of this probe
This probe exhibits functional antagonism equally for cAMP and intraceullar calcium signaling pathways stimulated by NPS in the cell-based assays, as opposed to the only other reported antagonist of NPS, SHA68, which selectively inhibits calcium signaling. The complete blockade of both cAMP and calcium signaling pathways may be necessary to fully antagonize NPSR1 function in animal models. As such, this probe should be used as a research tool to further study the functions of NPS receptor ex vivo and in animal models. In addition, this probe could serve as the starting point of drug development for treatment of sleep or anxiety disorders such as post-traumatic stress disorder, addictive disorders, and primarily alcoholism. The goal of the project was to generate a selective inhibitor of NPS, with a potency better than 1 micromolar in a displacement assay against the target; CID-3719993 satisfies these criteria. CID-3719993 was the most potent molecule tested in this displacement assay.
Throughout this probe report, MLSMR and NCGC identification numbers are used to identify compounds. A complete listing of corresponding PubChem CIDs can be found in table 9.
1. Scientific Rationale for Project
Neuropeptide S receptor (NPSR), previously known as GPR154, is a recently de-orphanized G protein coupled receptor. Its endogenous ligand is the 20 amino acid peptide Neuropeptide S (NPS). Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. NPS and its receptor are found in various tissues. The receptor is highly expressed in brain areas that have been implicated in modulation of arousal, stress and anxiety. Central administration of NPS in mice produces an unusual profile of activity by inducing wakefulness and arousal, while at the same time suppressing anxiety. Therefore, NPSR may represent a novel drug target for the treatment of sleep and anxiety disorders.
Recent work from Dr. Heilig’s laboratory has indicated that a class of stress-related neuropeptides constitutes a novel, promising category of candidate targets for the treatment of alcoholism (1,2). Corticotropin-releasing Hormone (CRH) and neuropeptide Y (NPY) are key mediators that belong to this category. New, unpublished data from the Heilig laboratory indicates that NPS may also belong to this circuitry. For example, cerebroventricular administration of NPS mimics stress and CRH in inducing relapse-like behavior in an animal model of alcohol seeking behavior.
Prompted by these data, the main hypothesis is that NPS receptors may be candidate targets for relapse prevention in alcoholism. A major roadblock for research aimed at establishing this role of the NPS system is the lack of pharmacological tools. Development of non-peptide NPS antagonists through this project will provide the pharmacological tools necessary to establish the role of endogenous NPS transmission in relapse to alcohol seeking behavior, and allow validation of the NPS receptor as a candidate treatment target.
During the process of our probe discovery for the NPS receptor, one structural series of antagonist were reported (3,4), represented by SHA68, 1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylicacid4-fluoro-benzylamide, CAS#= 847555-75-3). We have found that this compound selectively inhibited the intracellular calcium signaling stimulated by NPS (25nM IC50) compared with that of cAMP signaling pathway (583nM IC50), yielding a >20-fold functional selectivity for this compound.
2. Project Description
The process of NPSR antagonist discovery is summarized and shown in Figure 1.
a. Overall Goal of Project
To identify antagonists for neuropeptide S receptor as research probes for further studies on the function of NPS receptor in animal models. In addition, the antagonists of NPS receptor identified from this project would also serve as the starting point of drug development to treat anxiety disorders, including post-traumatic stress disorder, and addictive disorders, and primarily alcoholism. An additional focus is to obtain structurally distinct probes with new structural features, emphasizing appropriate LogP and low polar surface area (tPSA≤ 95) for increasing the probability of crossing the blood-brain-barrier. Finally, the molecule should have an association constant below 1 µM in the NPS binding assay.
b. Assay implementation and screening
qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction [AID-1461; Primary DR]
Assay Rationale & Description
It is known that NPS stimulates the cAMP signaling pathway and increases the cAMP level in cells expressing NPS receptor. This change in intracellular cAMP level can be detected by a cAMP HTRF assay (Fig 2), which is based on TR-FRET (time resolved fluorescence resonance energy transfer) between a cAMP-specific antibody labeled with europium (Eu) cryptate (Ab-cAMP-Eu), and a cAMP analog conjugated to the fluorescent dye d2 (cAMP-d2). Light pulse at 320 nm excites the europium of Ab-cAMP-Eu and the energy emitted is transferred to the cAMP-d2 bound to the antibody, generating a TR-FRET signal at 665 nm. Residual energy from the Eu-cryptate will produce a light at 620 nm. The native unlabeled cAMP from cell lysates competes with the cAMP-d2 for Ab-cAMP-Eu binding and reversely reduces the emission signal of cAMP-d2 by interrupting FRET between the two labeled molecules. Both emission signals from the FRET donor (620 nm) and the acceptor (665 nm) can be measured by a plate reader. Expression of result in fluorescence ratio (665 nm/620 nm) helps to normalize differences due to cell density and reagent dispensing.
All the reagents used for the NPSR cAMP assay and their resources are listed in Table 2. A schematic illustration of the assay principle is shown in figure 2. The assay protocol is described below, and in table 3.
A Chinese hamster ovary cell line stably expressing the NPSR was generated in Dr. Heilig’s lab. The cells were maintained in F12 medium containing 10 % FBS, 100 units/ml Penicillin, 100 μg/ml Streptomycin, and 200 μg/ml Geneticin at 37 ºC, 5% CO2.
Suspended CHO-NPSR cells were seeded into 1536-well tissue culture-treated white plates at a density of 1800 cells/well in 4 μl media without Geneticin and incubated at 37 ºC, 5 % CO2 for overnight. After 1μl of stimulation buffer (1X PBS buffer, 0.1% BSA, 0.05% Tween-20, 500 μM Ro 20-1724, EC80 of NPS) was added to each well, cells were incubated at 37 ºC, 5 % CO2 for 30 min. 1.25 μl of d2 conjugated cAMP and 1 μl of cryptate conjugated anti-cAMP antibody were then added. D2 conjugated cAMP and cryptate conjugated anti-cAMP antibody were both prepared in cell lysis buffer according to the manufacturer’s instruction. After 30 minutes, plates were then read in Viewlux plate reader (Perkin Elmer) using the TRF detection mode optimized for HTRF.
Summary of Results
NCGC tested 1284 1536-well plates in the primary screen. The average Z’ for the screen was 0.68 +/− 0.10, excluding 34 plates which failed visual QC. Results are reported for 221,370 samples. 2,309 compounds gave significant concentration-responses in the primary screen. Compounds were deprioritized for confirmation if they were suspected to interfere with the readout of the assay platform, or to affect GPCR signal transduction at a point in the pathway beyond the neuropeptide S receptor using internal NCGC SAR data from other related assays.
Confirmation Concentration-Response Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction [AID-1491; Confirmatory DR]
Select samples active in the primary screen were obtained in DMSO solution from the MLSMR and/or as powders from compound vendors to confirm activity in the original assay.
The assay protocol and reagents are identical to: qHTS Assay for Antagonists of the Neuropeptide S Receptor: cAMP Signal Transduction [AID-1651; Primary]
Summary of Results
Prioritized compounds had a high confirmation rate, but the most potent compounds from the MLSMR were only in the micromolar range using the HTRF assay format.
qHTS Assay for Antagonists of the Neuropeptide S Receptor: Calcium Mobilization [AID-1489; DR]
The fluorescent calcium dyes Fluo-4 AM has been widely used in GPCR studies to visualize and measure changes in intracellular calcium. It is able to enter the cell by passive diffuse and is de-esterified by endogenous esterases in the cytosol that is not fluorescent. The dye becomes fluorescent upon binding of calcium, resulting in fluorescent signals proportional to the cytosol free calcium concentration that is dramatically increased by the stimulation of certain GPCRs. Because intracellular calcium responses are transient with a fast kinetic (a half-life in the range of seconds to minutes), its detection requires special instruments that combine both the automated reagent dispenser and the kinetic fluorescence reader. A FDSS-7000 system (FDSS, Hamamatsu, Hamamatsu City, Japan) was used at NCGC to measure the intracellular calcium changes in response to agonist stimulation on GPCRs.
The CHO-NPSR cell line used in the cAMP assay was also used in this calcium mobilization assay. The suspended cells were plated at 3 μl/well with 2000 cells in the black, tissue culture treated, clear bottom 1536-well plates. After overnight incubation at 37 ºC, 5% CO2, 3 μl of the calcium dye (no wash High Performance PBX Calcium Assay Kit, BD Biosciences) was loaded to each well and the plates were incubated at 37 ºC, 5 % CO2 for 1 hour followed by 10-min incubation with 23 nl compound prepared in DMSO solution. The assay plates were then placed onto the FDSS-7000 kinetic fluorescence plate reader for measuring the changes of intracellular free calcium. The basal fluorescence signal was recorded for 6 sec at 1 Hz followed by an addition of 1 μl of NPS stimulation buffer (1X PBS buffer, 0.1% BSA, 0.05% Tween-20, 500 μM Ro 20-1724, EC80 of NPS) and 4-minute continuously recording at 1 Hz.
Summary of Results
Most compounds tested were more potent against the calcium mobilization assay than the cAMP assay, indicating potential antagonist functional selectivity for the Gs and Gq signaling pathways. The literature compound SHA 68 is a potent antagonist of Gq signaling.
Counterscreen for Antagonists of the Neuropeptide S Receptor: Muscarinic Acetylcholine M1 Receptor Antagonism [AID-1492; Counterscreen]
Muscarinic acetylcholine M1 receptor is a G protein-coupled receptor found in the plasma membranes of certain neurons and other cells. Because this receptor is coupled to the same G proteins as NPSR, it provides a good counterscreen for compounds that antagonize signal transduction through receptor-independent mechanisms (such as direct modulators of cellular cAMP levels).
All the reagents used for this assay and their resources are listed in Table 6. The assay protocol is described below, and in table 7. A Chinese hamster ovary (CHO) cell line stably expressing muscarinic acetylcholine M1 receptor (CHO-M1) was obtained from ATCC and maintained in F-12 Kaighn’s media (Invitrogen, Carlsbad, CA, 21127) supplemented with 10 % FBS, 100 units/ml penicillin, 100 ug/ml streptomycin and 250 ug/ml geneticin at 37C, 5% CO2 in a humidified atmosphere. Before the assay, aliquots of cells were frozen and stored at −135C. The assay was performed on a FDSS-7000 kinetic plate reader in 1536-well format. The maximums of kinetic fluorescence responses were converted into text files using the instrument’s software data export utility. Data for antagonist response were normalized to the controls for basal activity (DMSO only) and 100% inhibition. AC50 values were determined from concentration-response data modeled with the standard Hill equation.
Frozen CHO-M1 cells were thawed, washed once with fresh media and resuspended in F-12 Kaighn’s media supplemented with 10 % FBS, 100 units/ml penicillin and 100 ug/ml streptomycin. Cells were plated at 3 ul/well (1200 cells) to black, clear-bottom, tissue-culture treated 1536-well plates, and then cultured at 37C, 5 % CO2 for 16 to 30 hours. 3 ul of calcium dye (from High Performance PBX Calcium Assay Kit, BD Biosciences) was added. The calcium dye was prepared according to the manufactory’s instruction.
Plates were incubated at 37C, 5 % CO2 for 1 hour. Add 23 nl/well of compound in DMSO solution was added. The final titration for each compound was between 0.6 nM and 46 µM. Plates were then loaded onto the FDSS-7000. The following steps were performed on the FDSS-7000. (1) Record fluorescent background (Ex 480 nm, Em 520–560 nm) for 10 s. (2) Add 2 μl of stimulation buffer (1X HBSS buffer, 0.1% BSA, 60 nM carbachol). (3) Record antagonist response (Ex 480 nm, Em 520–560 nm) for 180 s.
Summary of Results
Validated molecules did not antagonize M1 stimulation by carbachol, indicating some selectivity of the molecules for NPSR.
qHTS Assay for Antagonists of the Neuropeptide S Receptor: Radioactive Ligand Displacement [AID-1493; Secondary]
Select samples was tested in a direct ligand binding assay, measuring [125I] Y10-hNPS displacement as previously described (5).
All the reagents used for this assay and their resources are listed in Table 8. The assay was carried out as described (5) with minor modification. Y10-NPS labeled with 125I was bought from NEN Perkin Elmer (Boston, MA). CHO cells stably expressing human NPSR were seeded into 24-well plates and cultured until reaching 90–95% confluency. Cells were washed with 1ml PBS once and then incubated with radioligand with or without compounds or in DMEM medium containing 0.1% bovine serum albumin at 20C for 1.5 hr. Increasing concentrations of compounds or unlabeled human NPS were used to compete with 0.15 nM [125I] Y10-NPS. Nonspecific binding was determined in the presence of 1 µM unlabeled human NPS. Cells were washed twice with cold PBS and lysed with 1 N NaOH. Bound radioactivity was counted in a liquid scintillation counter.
Summary of Results
Validated molecules displaced radioactive NPS ligand, typically at concentrations well below that required for antagonism of the receptor in the cell-based assays. It is possible that molecules which did not displace radioactive NPS ligand bind allosterically to the receptor, but the project team had no assay with which to verify this possibility.
b. Probe optimization (Naphthopyranopyrimidine)
Initial results of lead series
MLS-000558527 antagonized an NPS response with an IC50 of 2μM in the primary screening; in the confirmatory cAMP and calcium assays, it exhibited 250nM activity in a direct binding, displacement assay, and was the most potent molecule identified during screening. It did not exhibit any agonism towards the NPS receptor (not shown; data given in PubChem AID-1491). The activity of MLS-000558527 was confirmed for the both a sample re-ordered from the MLSMR repository, as well as for a sample synthesized in-house.
Figure 3Structure of MLS-000558527
Development of lead series
During the process of this RO3 application, two small molecule antagonists of NPS receptor were reported (3, 4, 6). They are SHA66 (3-oxo-1,1-diphenyl-tetrahydro-ox-azolo[3,4-a]pyrazine-7-carboxylicacidbenzylamide, CAS# 847553-81-5) and SHA68, 1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylicacid4-fluoro-benzylamide, CAS# 847555-75-3), two closely related bi-cyclicpiperazines with antagonistic properties at the NPS receptor (NPSR).
Figure 4Structure of SHA-66 and SHA-68
This report changed our initial SAR strategy, which was directed towards systematic substitution. We decided to use molecular modeling to compare the energy minimized structures of the two series. Chemical Computing Group’s MOE was used to flexibly align the molecules, with MMFF94x parameterization and without hydrogen bonding penalty. Figures 5 and 6 show two different ways of superimposing the minimized structures. The stereochemistry of the active enantiomer of MLS-000558527 is not known. Alternate stereochemistries were used to generate alternate models shown in figure 5 (R) and 6 (S).
Both models present a good overlapping of the core cyclic rings as well as the adjacent aromatic regions. The main difference between these two series is the conformation and structure of the ureic benzyl amine functional group. This group sticking out from the main core of SHA68, and it is absence in the NCGC series. In a recent publication, it has been shown that the introduction of the ureic benzyl amine functional group in the main core is fundamental for the activity of the series. For these reasons, we decided to develop several exploratory series introducing an aromatic ring at different regions and distances of our series. Figure 7 shows some of our analogues synthesized for this strategy.
As it can be seen on Table 9, the potency of the series did not improve with this first round of chemistry. However, all the compounds in this round were synthesized without the methoxy substituents in the top phenyl ring, which were thought to be beneficial for binding. Interestingly, NCGC-00181361 exhibited activity within experimental error of MLS-000558527, and along with NCGC-00182492 and NCGC-00182493, give us insight into the relationship between the SHA68 series and this one (Figure 8).
The next round of synthesis explored the need di-methoxy substitution on the phenyl ring. Figure 9 shows some of the analogues initially synthesized for evaluating the di-methoxy functional groups. Table 9 shows that the analogue with meta and para di-methoxy substitution is five times more active than the unsubstituted phenyl ring. Although mono-para-methoxy substitution showed some effect improving the activity, it is the meta-methoxy substituent the one that provide the main increment on activity, showing that the mono-meta-methoxy substitution is equally potent that the para-meta di-methoxy substitution. Focusing on the imidine substitution comparing NCGC00181382 and NCGC00183143, it can be seen that the phenyl substitution is close to five times better than the ethyl morpholino substitution.
Activation of NPSR induces transient increases in intracellular calcium and cAMP, suggesting coupling of this receptor to both Gs and Gq G proteins. Appendix 3 shows the values of MLS-000558527 in both functional assays. It can be seen that our probe is equally potent antagonizing both biological pathways. In contrast, SHA68 has a 20 times selectivity towards calcium activation that cAMP activation.
The activity of all these analogues and others can be seen in the Appendix. MLS-000558527 is the most thoroughly characterized and among the most potent compounds from this series, and has been selected as the probe molecule. Gram scale synthesis of the compounds has been completed to facilitate ex vivo and in vivo characterization of this compound’s activity by other investigators. Potencies of series analogs are presented in Table 9.
a. Chemical name
10-(2-morpholinoethyl)-11-imino-12-(m,p-dimethoxyphenyl)-10,11-dihydro-12H-naphtho[1',2':5,6]-pyrano [2,3-d]pyrimidine (MLS-000558527) [ML079]
b. Probe chemical structure
Sample is an isomeric mixture.
c. Structural Verification Information of probe SID
Structural verification and initial purity quantification was performed by 1H NMR analysis using a Varian spectrometer dissolving the sample in Cl3CD. In addition, further analysis was carried out by LC/MS using an Agilent system in the following conditions:
- Column: 3 x 75 mm Luna C18, 3 micron
- Run time: 4.5 minutes
- Gradient: 4% to 100% over 2.8 minutes
- Mobile phase: acetonitrile (0.025% TFA), water (0.05% TFA)
- Flow rate: 0.8 to 1.0 mL/min
- Temperature: 50 C
- UV Wavelength: 220 nm, 254 nm
The retention time of MLS-000558527 in those conditions was of 2.98 minutes. The purity of the Mass spectra was recorded in the positive ionization mode using an electrospray (API-ES) ionizing source with nitrogen as drying gas. Both NMR and LC/MS analysis showed purity greater than 99% for those batches of MLS-000558527 use in the biological evaluation.
d. PubChem CID (corresponding to the SID)
e. Availability from vendor
MLS-000558527 can be purchased from the following vendors
f. Mode of action for biological activity of probe
Neuropeptide S receptor antagonist
g. Detailed synthetic pathway for making probe
h. Summary of probe properties
MLS-000558527 is a very lypophilic compound ease to dissolve in organic solvents such us MeOH, Cl2CH2 or acetone. As pure compound, MLS-000558527 is a white powder at room temperature, chemically stable and with no apparent reactivity with air.
i. Properties Computed from Structure
Chemical Formula: C29H30N4O4
Molecular Weight: 498.57
Log P: 4.17
Number of hydrogen donors: 1
Number of hydrogen acceptors: 7
a. Summary of activities of SHA 68 and MLS-000558527
SHA 68, while more potent in the radioligand experiment, only partially blocked NPS-induced activity when injected at 50 mg/kg i.p. (3) This dosing yielded single-digit micromolar concentrations in brain. This is consistent with the hypothesis that antagonism of NPS stimulation requires functional antagonism of either cAMP or both cAMP and calcium signaling. MLS-000558527 provides a means of testing this hypothesis because this series does not appear to be functionally selective for one pathway or the other.
b. Concentration-Response data for MLS-000558527
c. Comparative data on probe, similar compound structures (establishing SAR) and prior probes
i. HTRF cAMP Assay
Calcium Mobilization Assay
ii. [125I]NPS Displacement
d. Performance of NPS standard in these assays
i. Calcium mobilization assay
ii. cAMP assay
iii. Radioligand displacement
See radioligand data plot above.
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- Okamura N, Habay SA, Zeng J, Chamberlin AR, Reinscheid RK. 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. J Pharmacol Exp Ther. 2008;325 (3):893–901. [PMC free article: PMC2583099] [PubMed: 18337476]
- Fukatsu K, Nakayama Y, Tarui N, Mori M, Matsumoto H, Kurasawa O, Banno H, inventors. WO/2005/021555. Mar 10, 2005.
- Xu YL, Reinscheid RK, Huitron-Resendiz S, Clark SD, Wang Z, Lin SH, Brucher FA, Zeng J, Ly NK, Henriksen SJ, de Lecea L, Civelli O. Neuropeptide S: a neuropeptide promoting arousal and anxiolytic-like effects. Neuron. 2004;43 (4):487–97. [PubMed: 15312648]
- Zhang Y, Gilmour BP, Navarro HA, Runyon SP. Identifying structural features on 1,1-diphenyl-hexahydro-oxazolo[3,4-a]pyrazin-3-ones critical for Neuropeptide S antagonist activity. Bioorg Med Chem Lett. 2008;18 (14):4064–7. [PubMed: 18555684]
Juan Marugan,1 Ke Liu,1 Wei Zheng,1 Robert Eskay,2 Noel Southall,1 Markus Heilig,2 James Inglese,1 and Christopher Austin1.
Received: February 13, 2009; Last Update: September 2, 2010.
National Center for Biotechnology Information (US), Bethesda (MD)
Marugan J, Liu K, Zheng W, et al. Identification of Functionally Selective Small Molecule Antagonists of the Neuropeptide-S Receptor: Naphthopyranopyrimidines. 2009 Feb 13 [Updated 2010 Sep 2]. In: Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.