Radioligand binding and reporter gene assays for histamine H3 and H4 receptor species orthologs

G-protein coupled receptors (GPCRs) represent the most important class of drug targets for the currently available pharmacotherapeutics. In the histamine receptor (HR) field, antagonists of H1 and H2 receptors are well established drugs for many decades, whereas the more recently identified H3 (H3R) and H4 receptors (H4R) are considered promising biological targets for drug discovery programs. Translational animal models are indispensable for investigations on the (patho)physiological role of novel receptors as well as for preclinical studies of potential drug candidates. Thus, with respect to the predictivity of such studies, pharmacological differences between H3R and H4R species orthologs should be identified as early as possible, both in binding and functional assays, in particular on human (h) compared to rodent (m, r) HR subtypes. This thesis aimed at the development of cellular binding and functional assays for the hH4R, mH4R, rH4R, hH3R and rH3R. In a whole-cell radioligand binding assay, saturation binding experiments on HEK293T cells, stably expressing the hH4R, the H3,4R ligand [3H]UR-PI294 was bound with high affinity. Competition binding experiments revealed affinities of reference ligands in a highly reproducible manner. Affinities of agonists were lower than reported data from binding assays using broken cell preparations, probably due to high GTP concentrations in intact cells. Regarding the mH4R, saturation binding experiments on HEK293T cells stably transfected with the mH4R gene revealed substantially lower affinities of [3H]UR-PI294 and [3H]histamine compared to the hH4R. Therefore, it turned out to be exceedingly difficult to obtain valid and robust mH4R binding data for reference ligands. To establish functional assays for the hH4R and mH4R, using a more distal and non-radioactive readout, compared to [32P]GTPase or [35S]GTPγS assays, the aforementioned transfectants were stably co-transfected with a vector encoding the firefly luciferase (Luc), the transcription of which is under the control of the cAMP responsive element (CRE). Assay parameters were optimized, and a luciferase reporter gene assay was established in the 96-well format of a luminescence plate reader. Moreover, HEK293T cells lacking the H4R were stably transfected with the CRE-linked luciferase gene (HEK293-CRE-Luc) and established to uncover off-target effects in control experiments. To develop a reporter gene assay for the rH4R, HEK293-CRE-Luc cells were stably co-transfected with the rH4R gene. For validation, functional data of agonists, antagonists and inverse agonists were determined. In case of agonism at the hH4R, the data correlated well with data gained from [32P]GTPase or [35S]GTPγS assays. This also held for the rank order of agonists at the mH4R and rH4R, however, the potencies were up to 100-fold higher in the luciferase assay. Obviously, there was a positive effect on the distal readout by activation/amplification of or cross-talk between different signaling pathways in the established reporter gene assay. Saturation binding experiments on whole HEK293T cells, stably expressing the hH3R, revealed high-affinity binding of [3H]N(alpha)methylhistamine ([3H]NAMH). This is consistent with recognition of the active receptor population, which was predominantly present due to high constitutive activity of the hH3R expressed in HEK293T cells. Whereas affinities of agonists and antagonists determined in competition binding experiments were in agreement with published data, inverse agonists achieved lower affinities. In studies at the rH3R stably expressed in HEK293-CRE-Luc cells, [3H]UR-PI294 was able to label a distinctly higher number of rH3Rs than [3H]NAMH, presumably, due to binding to the receptor protein in both, the active and inactive state. Consequently, in competition binding experiments with [3H]UR-PI294, cytosolic GTP shifted the binding data of agonists toward lower affinities, whereas affinities of antagonists and inverse agonists were consistent with reported data from broken cell systems. For the functional characterization of H3R ligands at the human and rat H3 receptors, a luciferase assay was established. HEK293-CRE-Luc cells were stably co-transfected with a vector encoding for the hH3R. In case of the rH3R the aforementioned rH3R expressing transfectant was used. In contrast to the rH3R, the hH3R displayed exceptionally high constitutive activity. This became obvious by increased basal activity of the hH3R and was confirmed by means of inverse agonists. The established reporter gene (luciferase) assays allowed for the quantification of agonistic, inverse agonistic and antagonistic activity in a highly sensitive and reliable manner. In summary, the developed cell-based methods will improve the predictability of in vivo results by characterizing compounds on H3R and H4R species orthologs regarding affinity, subtype selectivity, quality of action and potency.