Radiochemical and luminescence-based binding and functional assays for human histamine receptors using genetically engineered cells

For the discovery and detailed pharmacological characterisation of new ligands of G-protein coupled receptors simple, robust and reliable in vitro assays using whole cells are required. This thesis aimed at the development of new binding and functional assays for the human histamine H1, H2 and H4 receptor (hH1R, hH2R, hH4R), respectively. Whereas the ratiometric fura-2 based calcium assay in cuvettes has been state of the art for the determination of intracellular calcium for decades, non-ratiometric dyes like fluo-4 are preferred in high throughput screening. In order to combine the advantages of ratiometric calcium measurements with increased throughput in the microplate format, hH1R expressing U-373 MG cells were used as a model. The characterisation of the agonist histamine yielded reliable data both in 96-well and 384-well plates. The determination of antagonistic activity was most effective in the 384-well format and validated using standard H1R antagonists. Sequential reduction of the measurement time enabled the characterisation of 480 samples in one assay per day in the 384-well format. As a proof-of-concept, the applicability of this approach was confirmed by the identification of potent hH1R antagonists as lead compounds from a library of more than thousand new chemical entities. Due to the lack of appropriate wild-type cells expressing the hH2R or the hH4R, HEK293 cells were investigated for their suitability to express those receptor subtypes both on the mRNA level and by Western blot. Although a band corresponding to the hH2R was identified by mRNA analysis, the respective receptor protein was not detected in Western blots. Therefore, HEK293 cells were considered appropriate for the expression of human histamine receptor subtypes. To establish a functional assay, HEK293 cells were stably co-transfected with the hH2R and the chimeric G-protein qs5-HA, thereby redirecting the agonist-mediated receptor stimulation to a robust calcium signal. For validation, radioligand binding experiments were performed comparing the co-transfected cells (HEK293-hH2R-qs5-HA cells) with HEK293-FLAG-hH2R-His6 cells as reference. The affinities determined for the investigated standard ligands were on both cell types and on membranes from HEK293-hH2R-qs5-HA cells in the same order of magnitude and mainly in agreement with data reported in literature, except for the H2R agonist arpromidine, which showed lower affinity than reported for binding experiments performed in a different model (fusion proteins of the hH2R and the short splice variant of Gs-alpha). Generally, the binding properties of the investigated ligands were not significantly influenced by the co-expression of qs5-HA in HEK293-hH2R-qs5-HA cells. As an alternative to the use of radioligands, fluorescence-based binding assays were developed. The specific binding of a cyanine-dye-labelled H2R antagonist to HEK293-hH2R-qs5-HA cells was visualized by confocal microscopy. In addition, flow cytometric saturation binding experiments were performed on HEK293-hH2R-qs5-HA cells with this cyanine-labelled compound and with a pyrylium(Py)-dye-labelled H2R ligand. Although the fluorescent ligands showed moderate submicromolar affinities, these compounds are appropriate for binding assays, since their unspecific binding is extremely low. Affinities determined in binding experiments by displacing the Py-labelled ligand with H2R standard ligands were slightly lower for several compounds than reference data from literature, possibly due to the need for DMSO as a co-solvent in the assay. For the determination of the functional activity of hH2R ligands, fura-2 assays were established in cuvettes and in the microtitre format. Data obtained in the fura-2 assays were in agreement with results reported for calcium measurements performed with fluo-3 and were essentially in the same order of magnitude as reported for GTPase activity. Preliminary experiments aiming at the development of binding and functional assays for the hH4R were performed. Taken together, the established flow cytometric binding assay using fluorescence-labelled ligands and stably co-transfected cells represents an innovative alternative to radioligand binding assays for the determination of ligand affinity on the hH2R. The principle of stable co-expression of receptor and chimeric G-protein qs5-HA allowed the development of fluorescence-based calcium measurements, which can be performed in the microtitre format, thereby enabling reasonable throughput. The developed methods will contribute to the characterisation of compounds on histamine receptor subtypes with regard to affinity, receptor subtype selectivity, quality of action and potency