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.

Figure 2

Schematic illustration of the assay principle of the HTRF cAMP assay.

Assay Protocol

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.

Table 2

Reagents and resources for the HTRP cAMP assay.

Table 3

cAMP assay protocol for the CHO-NPSR cells in 1536-well plate format.

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.

Assay Description

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.

Assay Protocol

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.

Assay Description

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.

Assay Protocol

All the reagents used for this assay and their resources are listed in Table 4. The assay protocol is described below, and in table 5.

Table 4

Reagents and resources for the calcium mobilization assay.

Table 5

Calcium mobilization assay protocol for the CHO-NPSR cells in 1536-well plate format.

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.

Assay Description

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).

Assay Protocol

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.

Table 6

Reagents and resources for the calcium mobilization assay.

Table 7

Calcium mobilization assay protocol for the CHO-M1 cells in 1536-well plate format.

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.

Assay Description

Select samples was tested in a direct ligand binding assay, measuring [¹²⁵I] Y¹⁰-hNPS displacement as previously described (5).

Assay Protocol

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.

Table 8

Reagents and resources for the radioactive ligand binding assay.

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.