( Key words: ...)The development of a serotonin 5-HT2C receptor-binding assay using the LEADseeker Multimodality Imaging System

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

In mammals, serotonin (5-hydroxytryptamine, 5-HT) is a regulator of neural transmission which induces its biological effect by binding to serotonin receptors.

The serotonin 5-HT_2C receptor is a subtype of the serotonin receptor that belongs to the rhodopsin-like seven transmembrane superfamily of G protein-coupled receptors (1). 5-HT_2C receptors are widely expressed in the brain and spinal cord and are particularly enriched in the choroid plexus (2). They are reported to play a significant role in disorders such as obesity (3, 4) and hyperactivity (4). It has also been demonstrated that the 5-HT_2C receptors bind a wide range of drugs used in the treatment of psychiatric disorders such as depression and schizophrenia (5, 6).

This application note describes a 384-well serotonin 5-HT_2C receptor-binding assay which has been developed using the LEADseeker™ Multimodality Imaging System.

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

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

[N⁶-methyl-³H]Mesulergine TRK845

Other material required
Human recombinant 5-HT_2C (Euroscreen ES-315-M)
edited form, receptor membrane preparation

Mianserin hydrochloride (Sigma M2525)

Ketanserin tartrate (Sigma S006)

384-well white flat-bottom polystyrene (Corning 3705)
not-treated microplate, non-sterile

Binding buffer: 50-mM Tris pH 7.4, 0.1 %
(w/v) ascorbic acid, 10-µM pargyline.

GraphPad Prism™ software (GraphPad Software)

Protocol
Human recombinant serotonin 5-HT_2C, edited form, receptor membrane preparation was used in conjunction with [³H]Mesulergine ligand and WGA PEI type A PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 4-µM mianserin. The standard assay format was as follows.

1. Reagents were added in the following order: buffer or DMSO solution, unlabeled ligand (NSB wells), labeled ligand, and premixed bead and membrane. Total assay volume was 80 µl.

2. Diluted membrane and bead were premixed at room temperature immediately prior to assay addition.

3. Wells contained 10 µl of 6.08-nM [³H]Mesulergine (final concentration 0.76 nM) unless otherwise stated. Wells also contained 0.125 mg of bead and 1.25 µg of membrane, which were added together in a 20-µl volume.

4. NSB wells contained 10 µl of 32-µM mianserin (final concentration 4 µM) in addition to the above.

5. For DMSO tolerance studies, DMSO was diluted in binding buffer to give final concentrations per well as shown in the results.

6. Plates were sealed and incubated in darkness for 8 h at room temperature (20–25 ºC) unless otherwise stated.

7. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min with quasi-coincident averaging and 3 x 3 binning.

Results
K_d determination
Saturation binding was performed with dilutions of [³H]Mesulergine to give a range of concentrations from 30 nM to 1.18 pM in the wells. The saturation curve (Fig 1) was fitted using non-linear regression with the data analysis software package GraphPad Prism v4.0. A K_d value of 3.2 nM (95% confidence intervals 2.8 to 3.6 nM) was estimated directly from the curve.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.002% (v/v) to 30% (v/v) in the wells. From the results shown in Figure 2 it can be seen that the assay was tolerant to DMSO up to a final concentration of 2% (v/v).

IC₅₀ determination
Competitive binding of 7.6-nM [³H]Mesulergine with unlabeled ketanserin and mianserin was assessed and the IC₅₀ values calculated as shown in Figure 3. DMSO was included at an assay concentration of 2% (v/v). Final concentrations in the well for unlabeled mianserin and ketanserin were 0.3 nM to 5 µM.

The IC₅₀ value for mianserin was determined to be 6.3 nM (95% confidence interval range 5.6 to 7.1 nM) and the K_i value was 1.8 nM (95% confidence intervals 1.6 to 2.0 nM). The IC₅₀ value for ketanserin was 77.5 nM (95% confidence interval range 67.3 to 89.2 nM) and the K_i value was 22 nM (95% confidence intervals 19 to 25.3 nM).

Time course
A time course was performed using standard reagent concentrations as detailed in the protocol and DMSO included at an assay concentration of 2% (v/v). Readings were taken over an increased incubation time of 18 h. The assay reached equilibrium after approximately 5 h and appeared to be stable for at least a further 13 h (Fig 4).

Z analysis
Finally, a Z analysis was performed using 48 replicate values each for "total" and NSB wells, Z was determined to be 0.67 (Fig 5), which confirmed the robustness of the assay (7).

Conclusions
The serotonin 5-HT_2C receptor-binding assay has been successfully miniaturized to 384-well format using the LEADseeker Multimodality Imaging System and is suitable for adaptation to automated screening formats. The assay is tolerant to DMSO up to a concentration of 2% (v/v), robust; achieving a Z’ value of 0.67, and has a stability window of at least 13 h.

References
1. Nonogaki, K., et al. Diabetes, 52, 315–320 (2003).

2. Nguyen, M. G., et al. Neuroscience, 35, 577–591 (1990).

3. Visiers, I., et al. Protein Engineering, 14 (6), 409–414 ( 2001).

4. Gibson, W. T., et al. Canadian Journal of Physiology and Pharmacology, 82 (6), 426–429 (2004).

5. Lykouras, L., et al. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 25 (3), 507–518 (2001).

6. Moreau, J. F., et al. European Journal Neuro-Psychopharmacology, 8 (3), 161–168 (1998).

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

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