Dimeric histamine H2 receptor agonists as molecular tools and genetically engineered HEK293T cells as an assay platform to unravel signaling pathways of hH1R and hH2R

Previously, our workgroup applied the bivalent ligand approach for the H2R by linking two acylguanidine moieties. This led to highly potent and selective H2R agonists. However, the acylguandines turned out to decompose upon long-term storage in aqueous solution, in particular under alkaline conditions. Based on this preceding work, the medicinal chemistry part of this thesis deals with the synthesis of stable bivalent H2R agonists in which the acylguanidine group was replaced by a bioisosteric carbamoylguanidine moiety. With respect to studying the mode and stoichiometry of binding of such bivalent compounds, the synthesis of a chain-branched dimeric ligand, enabling radio- and fluorescence labeling was considered. The prepared compounds were investigated in GTPγS binding assays on recombinant human and guinea pig H2R and by radioligand competition binding on human H1, H2, H3 and H4 receptors expressed in Sf9 insect cells. Moreover, representative compounds were studied on human monocytes, with regard to cAMP formation and the inhibition of fMLP induced reactive oxygen species. The bivalent carbamoylguanidine-type H2R ligands proved to be high affinity H2R agonists, equipotent with the corresponding carba analogs, the acylguanidines. Achieving up to 2500 times the potency of histamine, these compounds are among the most potent H2R agonists known so far. Unlike the imidazole-type compounds, the bioisosteric aminothiazoles proved to be selective for the H2R over the other histamine receptor subtypes. Although the “cold” version of a synthesized propionamide derivative proved to be a potent and selective H2R agonist, the corresponding tritiated analog, due to a high degree of unspecific binding, was not applicable to saturation binding studies, making the determination of the number of binding sites impossible. Unfortunately, the fluorescently labeled ligands entered the cells in an H2R-independent manner. In human monocytes the investigated compounds induced cAMP accumulation and inhibited the fMLP-induced ROS production in a highly potent manner so that such compounds represent a good starting point for the development of selective H2R drugs for the treatment of AML. The bioisosteric and bivalent approach led to highly potent, selective and stable H2R agonists that proved to be useful pharmacological tools for functional studies on recombinant receptors and native cells. Regarding the bioanalytical and toxicological properties of the bivalent ligands, compounds bearing a short spacer should be preferred for future in-vivo studies. The second part of this work aimed at the establishment of cell-based functional assays for the discrimination between alternative signaling pathways activated by HxR stimulation. For this purpose, HEK293T CRE Luc cells were stably transfected with the cDNA encoding the hH1R or the hH2R, respectively. The transfectants were investigated in a luciferase reporter gene assay, in a fura-2 assay and in an aequorin assay. Moreover, a TR-FRET based cAMP assay was applied. The activation of the H1R led to an increase in the transcription of luciferase. In the presence of the selective Gαq inhibitor UBO-QIC the luciferase expression was reduced, whereas in combination with UBO-QIC and PKA inhibitors (Rp-cAMP-S or Rp-8-Br-cAMP-S) the luciferase activity was almost completely inhibited. The data obtained from the luciferase reporter gene assay suggested that the luciferase expression, mediated by the H1R, was at least in part mediated by cAMP. Furthermore, it was shown that cells preincubated with UBO-QIC were no longer capable of triggering a Ca2+ signal in the aequorin assay, upon activation of the H1R. Detection of a cAMP signal failed most probably due to a low signal-to-noise ratio, hence it should be taken into consideration that the activation of the hH1R is associated with additional pathways. The H2R revealed high constitutive activity in the luciferase reporter gene assay, probably due to a high expression level of the receptor in the cells. In the presence of UBO-QIC the luciferase activity was reduced. Additionally, it was shown in both Ca2+ assays that the activation of the H2R led to a Gαq mediated Ca2+ response. The data indicate that, regardless of preferential coupling of hH1R and hH2R to Gαq and Gαs, respectively, both receptors are capable of triggering differential signaling pathways, at least in genetically modified cells. In general, the stably co-transfected HEK293T CRE Luc cells represent a powerful pharmacological toolkit for: - The identification of alternative signaling pathways. - The investigation of GPCRs and the pharmacological characterization of functionally selective ligands. - The deconvolution of signals from label-free assays