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Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System

Key words: H1 receptor • GPCR • receptor binding assay • LEADseeker • SPA Imaging Beads

Histamine is a biogenic amine that plays important pathophysiological roles in central and peripheral tissues. Generally released from stimulated mast cells, it contributes to several biological processes that characterize allergic responses (1).The function of histamine is mediated via four G protein-coupled receptors, H1, H2, H3 and H4 (2). H1 receptor antagonists are one of the most commonly used class of drugs used to relieve the symptoms of allergic diseases, such as allergic rhinitis, atopic dermatitis, psoriasis and allergic conjunctivitis (3). The H1 receptor is thought to be involved in mediating inflammatory responses via effects on cytokine production (4).

This application note describes a 384-well histamine H1 receptor binding assay developed using the LEADseeker™ Multimodality Imaging System.

Products used

LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA) YOx SPA RPNQ0270
Imaging Beads

[pyridinyl-5-3H]Pyrilamine TRK608

Other materials required
Human recombinant Histamine (Euroscreen, ES-390-M)
H1 receptor membrane preparation

Pyrilamine maleate salt (Sigma, P5514)

Triprolidine hydrochloride (Sigma, T6764)

Costar™ solid white 384-well (Corning, 3705)
non-treated assay plate

Protease-free BSA (Sigma, A3059)

50-mM Tris pH 7.4
5-mM MgCl2
0.2% BSA (w/v)

Graphpad Prism™ software v4.0 (GraphPad Software)

Human recombinant Histamine H1 receptor membrane preparation was used in conjunction with [pyridinyl-5-3H]Pyrilamine ligand and WGA YOx SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 10-µM pyrilamine. The standard assay format was as follows:

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

2. Diluted bead and membrane was premixed immediately prior to addition to the wells.

3. Wells contained 10 µl of 28-nM [pyridinyl-53H]Pyrilamine (final concentration 5.6 nM) unless otherwise stated. Wells also contained 5 µg of membrane and 500 µg of WGA YOx SPA Imaging Beads. Bead and membrane were premixed immediately prior to addition to the wells. Premixed bead and membrane was added in a 20-µl volume.

4. NSB wells contained 10 µl of 50-µM pyrilamine (final concentration 10 µM) in addition to the above.

5. Plates were sealed and incubated in darkness for 3 h at room temperature (20–25 ºC) unless otherwise stated.

6. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min using q uasi-coincident averaging and 3 × 3 binning.

Kd determination

Saturation binding was performed with dilutions of [pyridinyl-5-3H]Pyrilamine to give a range of concentrations from 0.13 to 33.2 nM in the wells. Plates were incubated for 5 h at room temperature and in darkness prior to imaging. Figure 1 shows the saturation curve, which was fitted using non-linear regression with the data analysis software package GraphPad Prism v4.0. Over two experiments the mean Kd value was determined to be 3.7 nM (95% confidence intervals 3.1–4.4 nM), estimated directly from the curve. Figure 1 is representative of the data obtained.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.25% (v/v) to 5% (v/v) in the wells. The results shown in Figure 2 indicate the assay was tolerant to a final concentration of 2% (v/v). However, the signal was only slightly reduced in the presence of 5% DMSO.

IC50 determination
Competitive binding of 5.6-nM [pyridinyl-5-3H]Pyrilamine with pyrilamine (Fig 3A) and triprolidine (Fig 3B) was assessed and the IC50 of each ligand calculated. Final concentrations in the well ranged from 0.005 to 10 000 nM respectively. DMSO was present at a final concentration of 2% (v/v) in each well.

Over two experiments, the mean pyrilamine IC50 value was determined to be 14.6 nM (95% confidence interval range 10.6–20.7 nM) and the mean Ki value was 5.7 nM (95% confidence intervals 4.2–7.9 nM). The mean triprolidine IC50 value was determined to be 6.9 nM (95% confidence interval range 5.3–9.4 nM) and the mean Ki value was 2.7 nM (95% confidence intervals 2.1–3.6 nM). Figures 3A and 3B are representative of the data obtained.

Time course
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 at least 18 h as demonstrated by the curve in Figure 4.

Z’ analysis
Finally, two independent Z’ analysis (5) evaluations were performed, each using 48 replicate values for "total" and NSB wells. DMSO was present at a final concentration of 2% (v/v) in each well. A mean Z’ value of 0.82 was generated. Figure 5 is representative of the data obtained. This is well within the acceptable Z’ value range (0.5–1.0) and confirmed the robustness of the assay.

The histamine H1 receptor binding assay has been successfully miniaturized to a 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 0.82 and a stability window of at least 18 h.

1. Hill, S. J. et al. Pharmacological Reviews 49, 253–278 (1997).
2. Oda, T. et al. Journal Biological Chemistry 275, 36781-36786 (2000).
3. Curran, M. P. et al. Drugs 64, 523-561 (2004).
4. Matsubara, M. et al. Biochemical Pharmacology 69, 433-449 (2004).
5.Zhang, J. et al. Journal Biomolecular Screening 4 (2), 67–73 (1999).

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