( Key words: ...)Development of an NK2 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System

Key words: NK2 • GPCR • receptor binding assay • LEADseeker • SPA Imaging Beads

Neurokinins, also known as tackykinins, are a family of small neuropeptides. Members of this family include peptides, substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and the recently discovered hemokinin-1 (1). Neurokinins are widely distributed throughout the central and peripheral nervous system where they act via G protein-coupled receptors (NK1, NK2, and NK3 receptors) as neurotransmitters and neuromodulators. They are also considered key mediators in the communication between neurons (particularly sensory neurons) and effector cells (e.g., smooth muscle, glands, and immune cells), a role that is significant to the pathophysiology of a wide variety of diseases including gastrointestinal disorders, emesis, chronic pain, depression, and asthma (2). Members of the neurokinin family favor specific receptors. SP, for instance, shows greatest affinity for NK1 receptors, NKA for NK2, and NKB for NK3 (3). Of these receptors, NK2 receptors are thought to be expressed mainly on smooth muscle cells (4). Additionally, NKA and its truncated form, NKA (4-10), are potent contractors of human colon circular muscle, an action mediated exclusively via NK2 receptors (5).

This application note describes a 384-well NK2 neuropeptide receptor binding assay developed using LEADseeker™ Multimodality Imaging System. The miniaturized assay is robust, achieving a Z’ value of 0.62 and a stability window of 18 h. These factors make the assay suitable for adaptation to automated screening formats.

Materials
Products used
LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA)-PEI Type A RPNQ0287
PS SPA Imaging Beads

(2-[¹²⁵I]iodohistidyl¹) Neurokinin A ([¹²⁵I]Substance K) IM168

Other materials required
Human recombinant NK2 receptor membrane preparation (Euroscreen)

[β-Ala⁸]-Neurokinin A fragment (4–10) (Sigma)

Costar™ solid white 384-well microplate (Corning)

Protease-free BSA (Sigma)

Buffer: 50 mM Tris pH 7.4
5 mM MgCl₂
2 mM EDTA
0.2% BSA (w/v)

GraphPad Prism™ software v4.0 (GraphPad Software)

Protocol
Human recombinant NK2 receptor membrane preparation was used in conjunction with [¹²⁵I]NKA ligand and WGA-PEI Type A PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 5 µM NKA (4–10). The standard assay format was as follows:

1) Reagents were added in the following order: buffer, unlabeled ligand (NSB wells), labeled ligand, membrane, and bead. Total assay volume was 50 µl.

2) Each well contained 10 µl of 0.85 nM [¹²⁵I]NKA (final concentration 0.17 nM) unless otherwise stated, 2.5 µg of membrane, and 250 µg of beads. Membrane and beads were added in 10 µl volumes.

3) NSB wells contained 20 µl of 13 µM NKA (4–10) (final concentration 5 µM) in addition to the above.

4) Plates were sealed and incubated in darkness for 5 h at room temperature (20–25 °C).

5) Following incubation, plates were imaged on LEADseeker Multimodality Imaging System for 5 min using quasi-coincident averaging and 3 ×3 binning.

Results
K_d determination
Saturation binding was performed with dilutions of [¹²⁵I]NKA to give a range of concentrations from 0.01 to 1.34 nM in the wells. Figure 1 shows the saturation curve, which was fitted using non-linear regression with the data analysis software package GraphPad Prism v4.0. A K_d value of 0.4 nM (95% confidence intervals 0.3–0.4 nM) was estimated directly from the curve.

IC₅₀ determination
Competitive binding of 0.17 nM [¹²⁵I]NKA with unlabeled NKA (4–10) was assessed and the IC₅₀ calculated as shown in Figure 2. Final concentrations in the well were 0.002–10 000 nM. Over two experiments, the mean NKA (4–10) IC₅₀ value was determined to be 18.9 nM (95% confidence interval range 13.7–26.1 nM) and mean K_i value was 13.3 nM (95% confidence intervals 9.6–18.3 nM).

Time course
In addition, a time course was performed using standard reagent concentrations detailed in the protocol with an increased incubation time of 18 h. The assay appeared to be stable for the full 18 h as shown in Figure 3.

Z’ analysis
Finally, a Z’ analysis was performed using 42 replicate values for "total" and NSB wells (6). The Z’ value indicated by Figure 4 is 0.62, which was well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.

References
1. Patacchini, R. et al. Trends in Pharmacological Sciences 25, 1–3 (2004).

2. Longmore, J. et al. Canadian Journal Physiology and Pharmacology 75, 612–621 (1997).

3. Regoli, D. et al. Pharmacological Review 46, 551–599 (1994).

4. Lecci, A. et al. British Journal Pharmacology 141, 1249–1263 (2004).

5. Giuliani, S. et al. European Journal Pharmacology 203, 365–370 (1991).

6. Zhang, J. et al. Journal Biomolecular Screening 4 (2), 67–73 (1999).

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